THE ELABORATION OF EXTRACELLULAR CAPSULAR …
Transcript of THE ELABORATION OF EXTRACELLULAR CAPSULAR …
THE ELABORATION OF EXTRACELLULAR CAPSULAR POLYSACCHARIDE BY
KLEBSIELLA PNEUMONIAE AND ITS RELATIONSHIP TO VIRULENCE
By
PHILIP DOMENICO, B.A.
DISSERTATION
IN
MICROBIOLOGY
Presented to the Graduate Faculty of Texas Tech University Health Sciences Center
in Partial Fulfillment of the Requirements for
the Degree of
DOCTOR OF PHILOSOPHY
Approved
Acc'epted
December, 1983
f^C-'^'^ . ACKNOWLEDGEMENTS
I extend my gratitude to all who have made this dissertation
possible:
To my supervising professor, Dr. David C. Straus, who
encouraged me when I was frustrated and frustrated me when I was
encouraged.
To Dr. Dana L. Diedrich, who enriched my scientific repertoire
with his insight into microbial biochemistry and physiology, and
who burned the late-night candle with me on numerous occassions.
To Dr. Charles W. Garner for his guidance and his knowledge of
chemical phenomena.
To Dr. Rial D. Rolfe and Dr. David J. Hentges for their
assistance and constructive criticisms.
To Cathy Portnoy Duran who put up with my mess for two years.
To Linda Froelich, who inspired me and helped me through a
variety of difficulties during this period.
And finally to my parents who taught me the gift of
perseverence.
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I-V"*.
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS i i
LIST OF TABLES vi
LIST OF FIGURES x
LIST OF ABBREVIATIONS xiii
I. INTRODUCTION AND LITERATURE REVIEW 1
II. MATERIALS AND METHODS 11
Bacterial Strains 11
Media and Growth Conditions 11
Purification of the Extracellular Polysaccharides
of K_. pneumoniae 12
Preparation of Rabbit Anti-Type-Specific Antiserum.. 16
Rocket Immunoelectrophoresis 17
Opsonophagocytic Assay and Serum Sensitivity 18
Assay for Virulence of l<. pneumoniae in a Mouse
Model 20
Assay for the Production of Lobar Pneumonia in a
Rat Model 21
Assay for Characterization of Outer Membrane
Proteins of j<. pneumoniae 23
Electrodialysis of Extracellular
Polysaccharides from l<. pneumoniae 24
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Determination of Capsule Size of J<. pneumoniae 26
In Vitro Quantitation of Extracellular
Polysaccharides Produced by K. pneumoniae 26
Electron Microscopy 27
Gel Diffusion Method for Immunological Analysis 27
Saponification of K. pneumoniae Polysaccharides
and Quantitation of Fatty Acids 28
Hydrofluoric Acid Treatment 29
Statistical Analysis 29
III. RESULTS 30
Strain Variation and the Production of Apparent
Isogenic Sets 30
The Establishment of a Chronic Lobar Pneumonia by J<.
pneumoniae in a Rat Model 32
J<. pneumoniae Virulence in a Mouse Model 59
In Vitro Quantitation of Extracellular
Polysaccharides Produced by J<. pneumoniae 61
Serum Sensitivities and Opsonophagocytic Assays
for J<. pneumoniae 75
Purification of the EPS of J<. pneumoniae 80
Effect of Purified Extracellular Products from K_.
pneumoniae on Virulence in a Mouse Model 126
Structural Studies on the EPS Produced by
J<. pneumoniae 146
Gel Immunodiffusion Studies for Identification and
Quantitation of ECPS Produced by K_. pneumoniae.. 186
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Survey of the Outer Membrane Proteins of
J<. pneumoniae 194
IV. DISCUSSION 200
LITERATURE CITED 225
LIST OF TABLES
Page
1. Capsule size of K. pneumoniae 31
2. Establishment of a chronic Lobar Pneumonia in Rats 37
3. Effect of Dosage on the Ability of KPl to Produce
Pneumonia in Rats 51
4. Establishment of a Chronic Lobar Pneumonia in Rats
Emp 1 oyi ng KP1 -0 54
5. KPl-T in the Rat Lung Model 55
6. KP2-0 in the Rat Lung Model 57
7. KP2 2-70 in the Rat Lung Model 58
8. LDcn Values in Mice and ID^n Values in Rats for Strains
50 50
of J<. pneumoniae 60
9. ECPS Production by Strains of K_. pneumoniae Serotype 1 at
Various Intervals of Incubation 62
10. ECPS Production by Strains of j<. pneumoniae Serotype 2 at
Various Intervals of Incubation 68
11. Production of ELPS by K. pneumoniae Serotypes 1 and 2
after 48h of Culture in Defined Medium 71
12. Comparison of ECPS, ELPS, Capsule Size and Virulence of
J<. pneumoniae Serotypes 1 and 2 73
13. Correlations Between Polysaccharide Production and
Virulence in the Mouse Model 74
14. Serum Sensitivity of K. pneumoniae 76
15. Opsonophagocytic Assay 78
16. Effect of the Addition of EPS on the OPA 79
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Page
17. Elution Ionic Strength and Apparent Molecular Weights of
the ECPS from Various Strains of KPl and KP2 82
18. Elution Volumes for Dextran Calibration Standards on
Sepharose 2B (S-2B) 103
19. The Extracellular Products Found in Ethanol Fractionated
Supernatants of K_. pneumoniae 104
20. Comparison of the ECPS and ELPS Content in the Neutral
(N) and Acidic (A) Fractions from DEAE-Sephacel 106
21. The Extracellular Products Found in KPl and KP2 EPS after
Purification 107
22. Percent Yield Obtained from ECPS Purification for Two KPl
Strains 109
23. Pur i f i ca t ion of KPl-0 (EtOH) EPS by ED, cetavlon and Gel
Fi 1 t r a t i on I l l
24. Pur i f i ca t ion of KPl-0 (EtOH) EPS by ED, Cetavlon and Gel
F i l t r a t i o n : Percent Contamination wi th ELPS and
Protei n 115
25. Purification of KP2-0 EPS by ED and Cetavlon 121
26. Purification of KP2 2-70 EPS by ED and Cetavlon 123
27. Effect of KPl EPS on KPl-T Virulence in the Mouse Model.. 127
28. Probability Matrix Comparing the Virulence Enhancing
Potentials for all EPS Fractions Co-injected with
the KPl-T Strain in the Mouse Model 129
29. Effect of KP2 EPS on KPl-T Virulence in the Mouse Model.. 132
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30. Effect of KPl or KP2 EPS on the Virulence of KP2-0 in
the Mouse Model 134
31. Effect of ED on the Virulence Enhancement of KPl-T by
KP2 2-70 EPS in the Mouse Model 137
32. Effect of ED on the Virulence Enhancement of KPl-T by
KP2-0 EPS in the Mouse Model 138
33. Effect of Saponification on the Virulence Enhancement
of KPl-T and KP2-0 by KPl-0 (N) EPS
in the Mouse Model 140
34. Virulence Enhancement of KPl-T in the Mouse Model:
Comparison to the Dosage of ELPS in KPl EPS
Sampl es 141
35. Virulence Enhancement of KPl-T in the Mouse Model:
Comparison to the Dosage of ELPS in KP2 EPS
Sampl es 142
36. Effect of an Al ternat ive Pur i f i ca t ion of KPl-0 EPS on
the Virulence Enhancement of KPl-T in the
Mouse Model 145
37. The pH D i f fe ren t ia l of the Anode and Cathode Chambers
During ED 161
38. Quantitation of Ions Retrieved from the Cathode and the
Anode Chambers During ED of KP2 2-70 (EtOH) EPS 163
39. Effect of ED on the Quantity of Divalent Cations Found
in KP2 EPS 164
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40. Effect of ED on the Quantity of Phosphate Found in the
KP2 EPS 165
41. Quantitation of Fatty Acid Methyl Ester (FAME) Released
from EPS after Saponification 181
42. Quantitation of FAME Released from the EPS of KPl-0
and KPl-T Obtained from Gel Filtration 182
43. Standard Curve for the Rocket Immunoelectrophoresis
(RIE) of KPl-0 HMW and KPl-0 LMW EPS 187
44. RIE of Standard Concentrations of KP2 2-70 ECPS and
Serum from an Infected Rat 189
45. Quantitation of KP2 2-70 EPS in the Serum of an
Infected Rat by Radial Immunodiffusion (RID) 193
IX
LIST OF FIGURES
Page
l a . Transmission Electron Micrograph (TEM) of KP2-0 and i t s
Capsule 34
36 lb. TEM of KP2-T and its Capsule
2. Photomicrocrographs of H&E Stained Lung Tissue Sections
During J<. pneumoniae Infection in Rats 39
2a. Normal Rat Lung Section 41
2b. Rat Lung Section at 24h Post-inoculation 44
2c. Rat Lung Section at 3 days Post-inoculation 46
2d. Rat Lung Section at 6 days Post-inoculation 48
2e. Rat Lung Section at 9 days Post-inoculation 50
3. Comparison of the Rate of Production of ECPS by KPl-0 and
KPl-T at Various Intervals of Incubation 64
4. TEM of KPl 2-70: Example of Capsule Sloughing
5. Comparison of the Rate of Production of ECPS by KP2-0
and KP2 2-70 at Various Intervals of Incubation.
66
6.
7.
8.
9.
10.
11.
12.
13.
Elution
Elution
Elution
Elution
Elution
^ Elution
Elution
Elution
Profi
Profi
Profi
Profi
Profi
Profi
Profi
Profi
le fo r KPl-T EPS on DEAE-Sephacel
le fo r KPl-0 (A) EPS on Sepharose 2B (S-2B)
le fo r KPl-0 (N) EPS on S-2B
le fo r KPl-T (A) EPS on S-2B
le fo r KPl-T (N) EPS on S-2B
le fo r KP2-0 (A) EPS on S-2B
le fo r KP2-0 (N) EPS on S-2B
le for KP2 2-70 (A) EPS on S-2B
70
84
87
89
91
93
95
97
99
Page
14. Elution Profile for Dextran Calibration Standards
on S-2B 101
15. Elut ion Pro f i le fo r KPl-0 F r I I on S-2B 114
16. Elut ion Pro f i le for KPl-0 Fr I I I on P-300 118
17. Elut ion Pro f i le fo r KP2-0 F r I I on S-2B 120
18. Effect of an Al ternat ive Pur i f i ca t ion on the Elut ion
Pro f i le for KP2 2-70 EPS on S-2B 125
19. Effect of ED on the Elut ion Pro f i le for KP2 2-70 EPS on
BGA-150m 148
20. Effect of ED on the RID pro f i les of KP2 2-70 EPS 151
21. Effect of ED on the Elution Pro f i le fo r KP2 2-70 LMW
EPS on BGA-150m 154
22. Effect of ED on the Elution Pro f i le for KP2-0 EPS
on S-2B 157
23. Effect of ED on the Elution Profile for KP2-0 EPS on
S-2B: Effect of a Small Sample Volume 159
24. Histogram of the Effect of ED on the [PO^"^] in the
KP2 2-70 EPS 167
25. Effect of Sodium Dodecyl Sulfate (SDS) on the Elut ion
Pro f i le fo r Electrodialyzed KP2-0 EPS on S-2B 171
26. Effect of ED on the Elut ion Pro f i le for KPl-0 EPS
on S-2B 173
27. Effect of Hydrofluoric acid (HF) on the Elut ion
Pro f i l e fo r KPl-0 EPS on S-2B 177
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28. Effect of Saponification on the Elution Profi le for
KPl-0 EPS on S-2B 179
29. Elution Profile for KP2 2-70 (EtOH) EPS Obtained from
Growth in DMH on BGA-150m 185
30. Standard Curve for RIE of KP2 2-70 ECPS 192
31. Outer Membrane Protein Profiles for KPl Strains 196
32. Outer Membrane Protein Profiles for KP2 Strains 198
33. Hypothetical Model for the Electrophilic Associations
Between Strands of KP2 2-70 ECPS 219
x n
LIST OF ABBREVIATIONS
AB antibody/antiserum
BGA-150m BioGel A 150m gel filtration resin
BSA bovine serum albumin
Ca calcium
CET cetavlon
CFU colony forming units
CPS capsular polysaccharide
DEAE diethyl amino ethyl ion exchange resin
DHpO deionized water
DMH defined medium with Hepes buffer
DW defined medium with phosphate buffer
ECPS extracellular capsular polysaccharide(s)
ED electrodialysis
ELPS extracellular lipopolysaccharide(s)
EPS extracellular polysaccharide(s)
EtOH ethanol
FAME fatty acid methyl ester
Fr I fraction I
Fr II fraction II
Fr III fraction III
GLC gas liquid chromatography
H&E hematoxylin and eosin
HIAB heat-inactivated antibody
• • •
xm
HIRS heat-inactivated rabbit serum
HMW high molecular weight
ID^Q 50% infectious dose
IP intraperitoneal
KDO ketodeoxyoctanate
KPl Klebsiella pneumoniae serotype 1
KPl-0 J<. pneumoniae serotype 1 (ATCC 8047), opaque
variant
KPl-Or KPl-0 revertant
KPl-T l<. pneumoniae serotype 1 (ATCC 8047),
translucent variant
KPl-Tr KPl-T revertant
KPl 2-70 K.. pneumoniae serotype 1 CDC 2-70
KP2 K.. pneumoniae serotype 2
KP2-0 K.. pneumoniae serotype 2 (ATCC 29011), opaque
variant
KP2-T K,. pneumoniae serotype 2 (ATCC 29011),
translucent variant
J<. pneumoniae serotype 2 (CDC 2-70)
K. pneumoniae serotype 2 (ATCC 8052)
Limulus amoebocyte lysate
50% lethal dose
low molecular weight
lipopolysaccharide
milliamperage
minimal essential medium
xiv
KP2
KP2
LAL
LD50
LMW
LPS
MA
MEM
2-70
8052
1
Mg magnesium
MW molecular weight
ND none detected
NP not performed
OD optical density
OPA ppsonophagocytic assay
PBS phosphate-buffered saline
PMN polymorphonuclear neutrophil -3
PO. phosphate
PPT precipitate
RID radial immunodiffusion
RIE rocket immunoelectrophoresis
RS rabbit serum
S-2B Sepharose 2B gel filtration resin
SCD surface charge density
SDS sodium dodecyl sulfate
SUPE supernatant
TBC total bacterial count
TD transverse diameter(s)
TEM transmission electron micrograph
TSA Trypticase Soy Agar
TSB Trypticase Soy Broth V volts
WBC white blood cells
XV
CHAPTER I
INTRODUCTION AND LITERATURE REVIEW
The prolonged survival of chronically and critically ill
patients, due to the increased quality of medical care in this
country, has been paralleled by a striking increase in the
occurrence of gram-negative bacterial infections (65). This has
been especially true in the last three decades, when
hospital-acquired infections due to gram-negative bacilli have been
quite devastating (65,77). Hospital-acquired pneumonias caused by
these organisms have increased to where they now comprise nearly 50
percent of all nosocomial pneumonias (38).
One gram negative rod, Klebsiella pneumoniae, accounts for 25
to 43 per cent of gram-negative nosocomial pneumonias, thus making
it the most common agent in this disease process (77). Pneumonia
caused by K. pneumoniae is particularly dangerous, because once it
is established, it is difficult to control (38,65) and mortality
rates may reach or exceed 50 percent, even in treated cases (34,
41, 53). K.. pneumoniae pneumonias differ from most other
pneumonias in that lung tissue destruction seen in this disease
process is often extensive (70). Little is known about why such
extensive tissue necrosis is seen in this form of pneumonia.
The association between the ability of bacteria, such as J<.
pneumoniae and Streptococcus pneumoniae (serotype 3), to produce
large quantities of capsular polysaccharide (CPS) and to cause a
destructive lobar pneumonia is highly suggestive of a relationship
between these two parameters. Undoubtedly the rate of production
1
of CPS for these organisms is intimately associated with their
pathogenicity (28,45,55), and generally differentiates these
species from other medically important bacteria. Many theories
have been proposed as to how the CPS functions as a virulence
factor, most of which regard the CPS as an antiphagocytic substance
(45). Fukutome et al. (33) were able to show that j<. pneumoniae
could not be phagocytosed by polymorphonuclear leukocytes (PMN) nor
by alveolar macrophages in the presence of normal serum, unless
anti-capsular antibody (AB) was present. Escherichia coli strains
possessing CPS have also been shown to be resistant to phagocytosis
by PMN in normal serum, in contrast to £. coli strains without CPS
(78). These investigators presented evidence that the decreased
phagocytosis of encapsulated strains was caused by a low rate of
complement activation of the strains, as shown by the absence of
C3b or C3d fixation to the cell wall of the bacteria. Verbrugh et
al. (79) showed that encapsulation of several bacterial species
interfered with the process of C3 fixation in normal human serum.
Coonrod et al. (15) showed that systemic decomplementation of-rats
did not affect the severity of J<. pneumoniae pneumonia.
It is apparent, then, that the bactericidal and opsonic
properties of normal serum are ineffective against certain
encapsulated gram-negative organisms. The capsule is thought by
some to provide a cover for certain bacterial structures that are
known to be reactive with bactericidal and opsonic components found
in normal serum. For example, it was demonstrated that complement
component CI directly interacts with bacterial lipopolysaccharides
(LPS) and lipid A, independent of AB, while retaining its
esterolytically active form (52). Moreover, pure or soluble
polysaccharides are generally poor immunogens (31), and it may be
that the capsule also functions to make the exterior surface of
encapsulated bacteria comparatively unreactive, immunologically
speaking.
Although the presence of a certain amount of cell associated
capsule is regarded as necessary for the virulence of J<.
pneumoniae, the presence of additional cell wall-associated capsule
may not necessarily make the organism more virulent. For example,
Mizuta et al. (57) showed that, of 9 Klebsiella 01:K2 strains, 7
were highly virulent, whereas the other 2 strains were avirulent,
even though they were encapsulated to the same extent as the
virulent strains. One possible explanation for the role of the
capsule in pathogenicity has to do with the density rather than the
size of the capsule (20,82). It is possible that a more dense
capsular network could better inhibit nonspecific defense
mechanisms, such as C3 binding, from gaining access to the cell
wall of the bacillus. Recent studies by Wilkinson et al. (47,82),
and others (84), have suggested that the capsule of many bacteria
is readily penetrable by high molecular weight proteins, such as AB
and complement. Whether the same holds true for the virulent
strains of K.. pneumoniae has yet to be determined.
Another possible role for the capsule in virulence deals with
what can be referred to as the surface charge density (SCD). The
SCD can be construed as the net negative potential of the
polysaccharide that can interact with the environment. It may be
that the negative charge on the surface of phagocytic cells and the
negatively charged polysaccharide polymers of encapsulated bacteria
tend to repel one another, thereby explaining the antiphagocytic
nature of bacterial capsules. Perhaps because acidic
polysaccharides are able to avidly bind divalent cations (9, 16),
the CPS may create a microscopic zone around the organisms where
defense mechanisms dependent on the presence of these cations
(i.e., complement activation and initiation of phagocytosis) are
unable to function. In l<. pneumoniae, glucuronic acid is the
component accounting for most of the negative charge of the
capsule, and it is found in the repeating unit structure of the
polymer in most of the l<. pneumoniae serotypes (39,59). Appendix 1
shows the repeating unit structure found in the capsule
polysaccharides from K_. pneumoniae serotypes 1 and 2. Since most
serotypes possess uronic acids in more or less the same ratio (31
and 26 percent of the repeat unit weight for KPl and KP2,
respectively), this alone cannot explain the broad range of
virulence seen among, and especially within, the 72 serotypes of K.
pneumoniae. Virulent strains of Cryptococcus neoformans however,
have been shown to produce CPS having a greater uronic acid content
and a larger molecular size than relatively nonvirulent variants
within the same population (48). Additional negative potential may
be imparted to the Klebsiella capsule by covalently linked
non-carbohydrate substituents, such as pyruvate, acetate and
formate (35,76). These organic acids are detected in some, but
5
not all strains of a given serotype in various quantities. The KPl
ECPS has been shown to have an additional pyruvyl group linked to
its repeat unit structure (30) whereas only some KP2 ECPS have
these organic acids.
A third explanation for the role of the CPS in virulence is
related to the large quantities of these substances that are
produced and exuded into the medium by J<. pneumoniae (28). This
extracellular capsular polysaccharide (ECPS) may serve as an
antiphagocytic structure for K_. pneumoniae in a variety of ways.
The production of large amounts of ECPS provides an increasing
viscosity to solutions. It may be that the high producers of ECPS
are protected in vivo from intruding immune defense cells by
slowing the flow of particles in their iminediate environment. A
zoogleal mass, such as this could surround a microcolony of
bacteria, which may leave it relatively impenetrable to phagocytic
cells. Another virulence enhancing mechanism for the ECPS could be
to compete with the cell-associated capsular material for AB
produced against the capsular polysaccharide. Circulating
cell-free CPS in the blood of a patient infected with K. pneumonaie
could conceivably neutralize any previously or newly synthesized
antibodies before opsonization of the bacterium occurred (66).
Another explanation for the role of ECPS as a virulence factor
is its ability to paralyze the immune system against challenge with
the homologous (type-specific) bacterium or against heterologous
immunogens. Batshon et al. (2) induced immunological paralysis
against homologous challenge IP in mice with the ECPS isolated from
a KP2 strain. In the groups of animals receiving 2.5 yg of KP2
ECPS, there was a greater proportion of survivors than in groups
given 750 yg of ECPS, especially within the first 20 days after
ECPS administration. Protective AB were detected within 5 days in
the serum of mice given 2.5 yg, whereas in the serum of animals
given 750 yg of KP2 ECPS, such AB were not evident before 60 days.
Nakashima et al. (58) were able to show similar phenomena using the
ECPS from KPl strains. They found that anti-ECPS titers on day 11
post-administration were highest when mice were injected IP with 1
or 10 yg of KPl ECPS, but when 100 or 1000 yg of ECPS were
injected, virtually no AB titers were evident at this time period.
These same authors were able to show an increased response to
bovine serum albumin (BSA) when the mice were pre-injected with
these low ECPS doses, but a suppressed response compared to
controls occurred at the higher doses. Therefore, the effects of
KPl or KP2 ECPS on the immune system of mice appear to vary with
the amount introduced, with an adjuvant effect seen at low dosages,
and a suppression or tolerance phenomenon seen at higher dosages.
Pollack (66) demonstrated that the presence of detectable CPS in
the serum of human patients infected with J<. pneumoniae appeared to
correlate with the severity of infection, with persistence of
active foci (i.e., lung infections), and with a poorer prognosis
than in those patients who had no detectable circulating CPS. This
phenomenon was also observed in a study with S_. pneumoniae (19) and
group C meningococcemia (42) in humans, and for the type III group
B Streptococcus in a mouse model (20).
A series of publications by Yokochi et al. (85, 86)
demonstrated that minute quantities of KPl ECPS (0.05 mg/ml)
inhibits the maturation and functional capacity of macrophages.
They also showed that co-injecting a Salmonella strain IP in mice
with 200 yg of their KPl ECPS preparation markedly increased the
virulence of the Salmonella strain over controls without ECPS
treatment. Electron micrographs of peritoneal fluids showed that
the Salmonella were being phagocytosed in both the control and the
experimental groups, but the peritoneal macrophages were seen to be
killing and digesting the bacteria in the control group, whereas no
evidence for bactericidal or digestive processes were observed in
the treated group.
The production of CPS by J<. pneumoniae, both in the form of
cell-associated and soluble, cell-free (ECPS) polysaccharide, has
been studied extensively (20,21,26,27,46). Most of these studies,
however, did not relate the production of CPS or ECPS to virulence.
Early studies with the pneumococcus and Klebsiella demonstrated the
importance of the rate of capsule production in virulence. In a
classic study, MacLeod and Krauss (55) demonstrated the
relationship of virulence of pneumococcal strains for mice to the
quantity of CPS formed in vitro. Among several strains within
three different pneumococcal serotypes, they found that the
virulent strains formed more CPS than moderately virulent or
avirulent strains. Ehrenworth and Baer (28) reported a similar
phenomenon with a Klebsiella pneumoniae isolate. In both studies
the cell-associated CPS and the ECPS were measured, and virulence
8
was correlated with total CPS production. These investigators also
found a direct relationship between total CPS production and
virulence. Virulence was correlated with capsule size as
determined by packed-cell volume, and with soluble CPS as measured
by quantitative precipitin tests. One KP2 strain and three
variants of this strain were examined both for total CPS production
and virulence as determined by IP injections in mice. It was found
that the parent strain had nearly twice the packed-cell volume and
two times the antibody-precipi table soluble substance as did two of
the three variants at 3 h culture, yet no difference in virulence
was seen among these populations. Even at 24 h of incubation the
parent strain produced nearly twice the packed-cell volume and 1.5
times the ECPS as the two other variants with the same virulence
potential. Finally the remaining variant was shown to possess a
much smaller capsule and produced a somewhat smaller amount of
soluble antigen than the two other KP2 variants at 3 h of
incubation, but by 24 h of culture this strain increased production
of both capsular and soluble CPS to equal the total CPS production
of the two other variants. This last KP2 sub-strain was then shown
to be at least 5 log-jQ units less virulent in the mouse model than
all the other KP2 strains used. The authors concluded that the
slower rate of production of CPS by this latter KP2 variant was
slower than the other strains in the earlier stages of culture; it
was this slower rate which made it less virulent. Assuming that
the rather crude and outdated methodology used by these
investigators was approximating the actual total CPS production.
one is still not able to draw the conclusion that the rate of
production of CPS was the determining factor for virulence. Their
data would have been more convincing had they been able to show
differences in virulence between the parent strain, which produced
by far the most CPS at all intervals, and all the other variants
derived from this strain.
It was the intent of this proposal to further clarify the role
of the capsular substance; especially that of the ECPS, in the
pathogenicity of j<. pneumoniae. Moreover, careful consideration
was also given for other cell wall substances, such as LPS and
outer membrane proteins, for their possible functioning in these
virulence phenomena, and in their structural association with the
CPS of J<. pneumoniae. The present study addresses, to a
considerable extent, a number of structural issues heretofore
not reported in the literature, while attempting for the most part
to relate these structural phenomena to pathogenesis. One of the
inherent difficulties in working with the CPS of gram-negative
bacteria is the difficulty in obtaining CPS free from LPS. One
important question that is addressed in this study is an
examination of the nature of the association between extracellular
CPS and LPS and their relative roles as virulence factors.
Although Salmonella and £. coli LPS have been shown to possess a
wealth of biological activities, little is known of the LPS
produced by j<. pneumoniae. Reports have indicated that the LPS of
j<. pneumoniae is a more powerful adjuvant than the £. coli LPS (58,
10
87). The following, then, is an examination of the cell wall
associated structures of l<. pneumoniae and their relationship to
the virulence of the organism for mice.
CHAPTER II
MATERIALS AND METHODS
Bacterial Strains
Two strains of Klebsiella pneumoniae serotype 1 (KPl) and two
strains of l<. pneumoniae serotype 2 (KP2) were utilized in these
studies. The KPl strains are as follows: KPl ATCC 8047, lung
isolate, and KPl CDC 2-70 (Difco Labs, Detroit, MI). KPl ATCC 8047
was found to have two predominant variants seen on Trypticase Soy
Agar (TSA) (BBL, Division Beckton Dickinson Co., Cockeysville,
MD.), one opaque and the other translucent. These were isolated
and designated KPl-0 (opaque) and KPl-T (translucent). The KP2
serotypes employed in these studies were KP2 ATCC 29011, blood
isolate, and KP2 CDC 2-70 (Difco). KP2 ATCC 29011 was also found
to contain two variants, one opaque and one translucent. These
were subsequently isolated and designated KP2-0 and KP2-T. For the
fatty acid analyses the KP2 ATCC 8052 strain (bronchus isolate) was
utilized, but not included elsewhere in these studies. All strains
were maintained frozen in Trypticase Soy Broth (TSB) (BBL, Division
Becton, Dickinson and Co., Cockeysville, MD) with 20% glycerol at
-70°C. Prior to use, the stock cultures were added to liquid
medium (1 ml of stock for each 100 ml medium) and incubated at 37°C
in a shaking water bath (200 rpm) until the medium was slightly
turbid (ODggQ = 0.20).
Media and Growth Conditions
Several liquid media were used in the course of these studies.
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12
TSB was prepared and sterilized according to manufacturers'
instructions and was mainly used for growing the organisms for
virulence studies and for use in the phagocytic and serum
sensitivity assays.
A chemically defined medium (DW) was prepared according to the
procedure of Duguid and Wilkinson (21) with one modification,
namely that zinc sulfate was added to a concentration of 0.0085 mM.
This medium was mainly used for growing the organisms for
quantitating and purifying the extracellular capsular
polysaccharide. In one study the phosphate buffer in this defined
medium was replaced with a 10 mM Hepes buffer, pH 7.3 (DMH).
For experimental procedures utilizing DW or TSB, all cultures
were grown at 37 C at 200 rpm in various volumes. Starter cultures
were grown to early logarithmic phase (ODccn = 0-2) in the
appropriate medium and 1 ml was inoculated per 100 ml of broth.
Growth was monitored by measuring the absorbance in a Bausch and
Lomb Spectronic 20 at 550 nm. Plate counts of bacteria on TSA were
routinely performed for each study utilizing DW medium. Cultures
were grown in TSB to early logarithmic phase (OD^^Q =0.2) and the
cells were harvested at this time. Cultures in DW medium were
grown for 48 hours before the cells were harvested unless otherwise
indicated. To terminate growth, cultures were immediately placed
in ice before centrifugation.
Purification of the Extracellular Polysaccharides
of K. pneumoniae
Cultures were grown in DW medium as described in Materials and
Methods, part B. Usually one liter of medium was used for each
13
organism. Two hundred ml samples were removed from the culture
after 18, 24, 36 and 48 h of growth. The total number of viable
organisms was monitored at each time period by examining colony
forming units (CFU) on TSA at appropriate dilutions. Samples were
centrifuged in a Beckman Model J2-21 refrigerated centrifuge
(Beckman Instruments, Inc., Palo Alto, CA) at 4°C at 17,700 x g for
60 min in a JA-10 rotor. The supernatant was collected and 2
volumes of cold ethanol (95%): methanol (19:1) were added to
precipitate the extracellular polysaccharides (EPS). These
solutions were left overnight stirring at 4°C or statically at
-20 C. The supernatant was then discarded or, if not clear, was
centrifuged for 30 min as above. Twenty ml of deionized water
(DH2O) was then added to the precipitate per 100 ml of original
culture volume which allowed the precipitate to go back into
solution. The crude EPS was then dialyzed with three changes
against 8 liters of DHpO at 4°C overnight. Samples were then
shell-frozen and lyophilized on a Virtis Freezemobile 6 lyophilizer
(Virtis Co., Inc., Gardiner, N.Y.).
Dried samples were dessicated over Drierite (W.A. Hammond
Drierite Co., Xenia, Ohio) overnight before being weighed out.
Various quantities of crude, dry EPS were weighed on a Mettler H51
balance (Mettler Instrument Corp., Hightstown, N.J.) and suspended
in an appropriate buffered solution. Early studies incorporated
DEAE-Sephacel ion exchange chromatography (2.5 x 25 cm column)
(Pharmacia Fine Chemicals AB, Uppsala, Sweden) as the next
purification step. The ethanol extracted, dried EPS was brought up
14
in 0.02 M (NH^)2C03 (Sigma Chemical Co., St. Louis, MO). The
DEAE-Sephacel column was equilibrated with the 0.02 M (NH4)2C03
solution and the sample (usually 10 ml) was placed on the column.
After allowing approximately 200 ml to elute from the column a
gradient was applied, which was normally from 0.02 M to 1.0 M
(NH^)2C02. The EPS elution profile was monitored by three
different methods: 1) a capillary precipitin reaction using rabbit
antiserum against whole, formalin killed cells of KPl or KP2; 2)
the anthrone assay (51) for total hexose and; 3) the uronic acid
assay of Blumenkrantz and Asboe-Hansen (8). Fractions were pooled
and dialyzed 3 times against 8L DH^O at 4°C. The final
purification step was gel filtration on Sepharose 2B (S-2B) or
Sepharose 4B (S-4B), (Pharmacia), or BioGel A 150m (Bio-Rad Labs,
Richmond, CA) in 1 meter x 2.5 cm columns. Initial studies
utilized 0.5 M NaCl, both for the column buffer and for
resuspending samples. It was later determined that 0.01 M Tris, pH
12 (Sigma Chemical Co., St. Louis, MO) was ideal for the column
buffer and for bringing up samples of EPS to be placed on these
columns. Tris buffer (0.01 M, pH 12) was also used for subsequent
ion exchange chromatography. Various molecular weight (MW)
fractions were pooled separately and dialyzed against three 8L
changes of DH2O at 4°C while stirring, and lyophilized to dryness."
The various fractions were dessicated over CaSO. under a
vacuum, weighed and were then suspended in deionized water and
analyzed for their hexose and uronic acid content as well as for
the amount of protein present, as determined by the method of Lowry
15
(54), and for their lipopolysaccharide (LPS) content by the method
of Osborn et al. (60) using the LPS of Escherichia coli 055:B5
(Difco) as the standard. Phosphate content, as measured by Chen et
al. (13), as well as the calcium and magnesium content, assayed for
by atomic absorption spectrophotometry on a Perkin Elmer AAS model
303, were also determined.
In order to obtain a better purification procedure for the KPl
EPS, the following protocol was used. One hundred fifty mg of
ethanol extracted material from 48h supernatants of KPl-0 were
electrodialyzed at 2000 V, as described in Section J of Materials
and Methods. The electrodialyzed sample was then extracted with
10% cetavlon (docecyl trimethyl ammonium bromide, Sigma) to 1%
total cetavlon as described by Scott (72). The precipitate was
pelleted by centrifugation at 12,700 x g for 10 min and the
supernatant separated. Three ml of DH2O were then added to the
cetavlon fractionated precipitate (Fr I) and a 4M CaCl2 solution
was used to bring the solution to IM. This 4M CaCl^ solution was
also added to the cetavlon fractionated supernatant (Fr II) to
bring this solution to IM. Ninety-five percent ethanol was then
added to both Fr I and Fr II to 80% by volume and placed at -20°C
for 30 min. The precipitate from both fractions was centrifuged as
above and washed 2 times with 20 ml of 95% ethanol at -20°C for 30
min. The fractions were then resuspended in 10 ml of DH2O and
lyophilized. Both fractions were assayed for uronic acid and KDO
to quantitate LPS (60). Fr II was boiled for 5 min in 0.1% SDS and
placed on a Bio Gel P-300 gel filtration column equilibrated with
16
0.1 M ammonium acetate, pH 8.1, and 0.1% SDS. The fractions from
P-300 containing hexose were collected separately, dialyzed and
precipitated with 3 vol of acetone at -20°C for one hour. The
precipitates were collected by centrifugation as above and washed
once with 95% ethanol overnight at -20°C. Again the precipitates
were collected and resuspended in a small quantity of DH^O and
lyophilized. All fractions were assayed for uronic acid and KDO as
before. A 20 mg dry weight sample of Fr I was placed on S-2B, the
fractions collected as above and tested for uronic acid and KDO.
Several of the KP2 EPS preparations were also partially purified in
a similar manner.
Preparation of Rabbit Anti-Type-Specific
Antiserum
Antisera were obtained against KPl-0, KPl-T, KP2-0 and KP2
2-70 by the procedure of Edmondson and Cooke (24). A fresh, early o
log phase (1 x 10 CFU) suspension of these organisms was killed in
10% formalin and inoculated separately into rabbits intravenously
once e^ery three days for 13 days with increasing doses of the
organism, starting with 0.25 ml on the first day, 0.5 ml on the
fourth day, 1.0 ml on the seventh day, and 1.5 ml on the tenth and
thirteenth day. Rabbits were exsanguinated 5 days later by cardiac
puncture. Approximately 100 ml of rabbit serum was obtained from
each rabbit, and frozen at -70°C in 10 ml aliquots.
Type-specificity of the antiserum was tested in capillary
precipitin reactions against purified capsular material and in
double immunodiffusion assays (62).
17
Rocket Immunoelectrophoresis
A method similar to that described by Weeke (81) was used in
this procedure. Gels were prepared using 0.2% agarose (Sigma) in
0.075 M Gelman High Resolution Buffer (Tris-Barbital), pH 8.8. The
agarose was dissolved by heating to boiling and cooling to 50°C.
Anti-type 1 or anti-type 2 rabbit antiserum (2 ml) was mixed with
23.0 ml of agarose and poured onto a 4 x 6 in. sheet of Gel Bond
film (FMC Corp., Rockland, Maine). This volume gave a gel
thickness of approximately 2 mm.
Wells (4 mm) were punched in the agarose and 15 yl quantities
of EPS samples in DH2O were added. Standards included five 2-fold
serial dilutions (2 mg/ml-0.125 mg/ml in dry weight) of low
molecular-weight EPS from KPl-0. Samples were electrophoresed on a
Pharmacia FBG 3000 apparatus coupled to an Bio Rad Model 500/200
Power Supply at 5 V/cm in the same Gelman High Resolution Buffer
for 3 h. The gels were then washed 3 times in saline at 4°C and
once in DH2O at 4°C. The gels were then pressed lightly with
several layers of filter paper until dry. The gels were then
stained with Coomassie Blue and destained as described by Weeke
(81).
For the experiments on testing for antigenemia in infected
rats and mice, the sera of these animals were first electrodialyzed
as described in Materials and Methods, Section J. The height of
the rockets produced by the sera of these animals was then compared
to the standard curve and the levels of ECPS in the sera of these
animals was determined in yg/ml.
18
Opsonophagocytic Assay and Serum Sensitivity
The OPA was similar to the one described by Edwards et al.
(25). Bacteria were grown in 50 ml TSB to early log phase
(0DcrQ=0.2). Ten ml were removed and the organisms were pelleted
by centrifugation at 17,400 x g for 20 min on a Beckman J2-21
centrifuge using a JA-10 rotor. The pellet was resuspended in 10
ml sterile, cold PBS (FTA Hemagglutination Buffer, BBL), pH 7.2,
and centrifuged at 17,4000 x g for 20 min as above. The pellet was
then resuspended in 10 ml cold normal rabbit serum (Gibco
Laboratories, Grant Island, N.Y.) that was heat inactivated at 56 C
for 30 min (HIRS) and placed on ice. Plate counts were obtained in
duplicate on TSA at appropriate dilutions. The bacteria were
further diluted to achieve a ratio of bacteria to white blood cells
(WBC) of 3-4:1 in the opsonic reaction mixture.
The WBC suspension was prepared by drawing 10-12 ml of
peripheral venous blood from normal volunteers in a non-heparinized
plastic syringe. The blood was added to 50 ml Corning centrifuge
tubes (Corning Glass Works, Corning, N.Y.) containing 4.0 ml of 6%
Dextran in DH2O (Dextran MW 80,700, Sigma) and 3.0 ml citrate
solution (16 g citric acid and 59 g sodium citrate per liter;
sodium citrate from Fischer Scientific Co., Fairlawn, N.J.; citric
acid from Sargent-Welch Scientific Co., Skokie, IL.). This mixture
was incubated at 37°C for 45 min to sediment the erythrocytes. The
WBC-rich plasma supernatant fluid was removed and washed once in
minimal essential medium (MEM) (Gibco) plus 1% bovine serum albumin
(BSA) (Gibco). Lysis of erythrocytes was accomplished by adding 5
19
ml of 0.84% ammonium chloride for 20 min at room temperature. The
WBC were then washed twice in HIRS. The WBC were then resuspended
in 3 ml HIRS and placed on ice. A 1:10 dilution of the WBC
suspension was made in sterile, cold PBS for counting purposes
performed on a hemocytometer (American Optical Corp., Buffalo,
N.Y.). The cells were adjusted to yield 1 x 10^ WBC/ml with cold
HIRS. The OPA was performed in 1.5 ml polypropylene micro test
tubes (Bio-Rad). The reaction mixture contained a total volume of
0.4 ml, consisting of 0.1 ml WBC suspension, 0.1 ml bacterial
suspension, 0.1 ml of serum [as either HIRS, rabbit antiserum (AB)
or heat-inactivated rabbit serum (HIAB) against the homologous
strain being tested, or normal rabbit serum (NRS)], and 0.1 ml of
either PBS or a suspension of EPS from the same serotype in PBS.
Control tubes were included in each experiment that were lacking in
WBC, antibodies, complement, or EPS. For serum sensitivity assays,
0.1 ml of the bacterial suspension was inoculated into 0.9 ml
rabbit serum which contained no WBC. Tubes were incubated at 37°C
for 60 min on an Ames aliquot mixer (Miles Laboratories, Inc.,
Elkhart, IN). In the OPA samples (0.01 ml) were removed
post-incubation and added to 0.99 ml sterile, cold DH2O to lyse the
WBC. Additional dilutions were prepared in sterile, cold PBS and
0.1 ml of the appropriate dilutions were streaked on TSA. After
overnight incubation at 37°C, colonies were counted and the net
growth for both the OPA and serum sensitivity assays were
calculated as follows:
CFU 60 min Log 10
CFU 0 min
20
Assay for Virulence of K. pneumoniae
in a Mouse Model
A standard virulence assay was performed, adapted from the
procedure of Baltimore et al. (1). Bacteria were grown to an early
log phase (ODncn = 0-20) in TSB and prepared as previously
described (Section B). Ten-fold dilutions in sterile, cold PBS
were prepared and groups of 3, 4, or 5 mice (Swiss Webster, males,
20-25 g) (Laboratory Supply, Indianapolis, IN) injected intra
peritoneal ly (IP) with 1.0 ml of the appropriate dilution. Dead
mice were counted and removed from their cages at 24 h intervals
for 96 h. Virulence was expressed as the 50% lethal dose ( L D ^ Q ) ,
which was calculated by the method of Reed and Muench (68).
In the studies involving the effects of crude or partially
purified EPS on virulence, bacteria were grown and inoculated as
above, and various dilutions of EPS in PBS were injected IP
simultaneously in 0.1 ml volumes to all groups of mice. The amount
of EPS for all mice in each study was held constant while the
number of organisms differed ten-fold from group to group. A
control study was performed each time a virulence assay was set up.
Controls received the bacteria in the same manner as the
experimental and received 0.1 ml IP of sterile PBS simultaneously
in place of EPS. An EPS toxicity control was also performed by
injecting the EPS in 0.1 ml amounts IP, without administering
bacteria. The EPS controls employed EPS in concentrations from
twice to one-half that of the amount used in the virulence studies.
In these control studies, four mice were injected with each
two-fold dilution of the EPS solution.
21
Assay for the Production of Lobar Pneumonia
in a Rat Model
Male Sprague-Dawley rats (Laboratory Supply) weighing 200-250
g were used in these studies. The rats were housed in plastic
cages in groups of 4 and had access to commercial chow and water ad
libitum. Before inoculation, the rats were lightly anesthetized
with ether and the ventral cervical region was cleaned with a 95
per cent alcohol rub. A 1 cm medial longitudinal incision was made
in the animal to expose the trachea. The trachea was incised and
0.05 ml of a washed suspension of varying concentrations of
log-phase organisms in PBS was placed into the left diaphragmatic
lobe of the lung via a bead-tipped, curved inoculating needle. The
incisions healed rapidly without evidence of infection.
At various intervals after inoculation, groups of rats were
exsanguinated by cardiac puncture under ether, the thoracic cavity
opened and the lungs aseptically removed. The lungs were weighed
and then used for either bacterial quantitation or for histological
examination. Small samples of lung tissue were excised from
affected areas and fixed immediately in 10 per cent formalin.
Samples were cut at 4 ym and stained with hematoxylin and eosin
(H&E) on Brown and Haup stains. The sections were examined
microscopically and photomicrographs were made from representative
areas.
For bacterial quantitation, lungs were homogenized in 5 ml of
sterile PBS at 4°C using a Brinkman polytron homogenizer (Brinkman
Inst. Houston, TX). Serial ten-fold dilutions were made from the
homogenate and 0.1 ml of selected dilutions were plated out on TSA
22
and incubated overnight at 37°C. The following day, colonies were
counted and concentrations of J<. pneumoniae in lung specimens were
determined. Lung bacterial counts were calculated as the total
number of bacteria present in an entire lung specimen and were
reported as the total bacterial count (TBC) per set of lungs. For
convenience the TBC was expressed in logarithmic units to the base
ten (log^Q TBC).
To determine the fifty percent infectious dose (IDCQ) of
organisms used in these studies, a rat with a lung TBC value equal
to or exceeding 5'x 10 CFU (log^g TBC = 4.70) was considered
infected. Infected rats were counted at day 6 post-inoculation and
the loq-, IDrn was calculated by the method of Reed and Muench IU oU
(68). All rats succumbing to the infection before day 6 were
considered infected.
Chronicity studies were also performed with KPl ATCC 8047
before it was separated into its two subvariants, KPl-0 and KPl-T.
In these studies eight experimental (receiving 0.05 ml containing 5
x 10^ CFU of early log phase KPl transtracheal ly in PBS) and two
control rats (0.05 ml sterile PBS) were sacrificed on day 1, 3, 6,
and 9 post-inoculation. Lungs from 4 of the 8 experimental rats
and from one of the two control rats on each day of sacrifice were
processed for histology, while the remaining 4 experimental and one
control rat had their lungs removed for bacterial quantitation.
Four experimental and one control rat were also sacrificed on days
7, 14, 21 and 28 post-inoculation, having also received 5 x 10 CFU
23
of KPl, and all of these rats were processed for bacterial
quantitation.
Rat sera were obtained by allowing the blood to clot at 4°C
overnight, and the serum obtained by centrifugation at room
temperature at high speed in a clinical table-top centrifuge
(International Equipment Co., Needham Hts., MA). Lysozyme levels
in serum were measured by the lyso-plate method of Osserman and
Lawlor (61) using human urine lysozyme (Kallestad) or hen egg white
lysozyme (Difco) as the standard. Serum zinc determination was
performed by atomic absorption spectrophotometry on a Perkin-Elmer
model 2380 AAS using zinc chloride as the standard.
Assay for Characterization of Outer Membrane
Proteins of K. pneumoniae
The method for preparing the outer membranes for gel
electrophoresis was that of Diedrich et al. (18). One liter
cultures were grown in DW medium as already described. At 18, 24,
36, and 48 h intervals, 200 ml samples were taken and the cells
pelleted by centrifugation. The supernatant was decanted and the
cells were frozen at -70°C until further use. The frozen pellets
were broght up in 8-10 ml of Hepes buffer, pH 7.4 (Sigma) and
fractionated in a Franch Pressure Cell (Amico, Silver Spring, MD)
at 1.8-2.0 X 10^ pounds/square inch (PSI). The fractionated
material was then centrifuged at 3020 x g for 10 min in a JA-20
rotor (Beckman) to remove cell debris. The supernatant was then
placed in ultracentrifuge tubes (DuPont Instruments, Newtown, CN)
and centrifuged in an OTD-75 centrifuge (Sorvall, DuPont) at
24
50,000 X g for 45 min in a T-865 rotor (Sorvall). The supernatant
was decanted and the pellet resuspended in 10.8 ml of Hepes. A
volume of 0.12 ml of a 0.1 M MgCl2 solution was added, the tube
inverted several times and 1.0 ml 20% Triton X-100 in Hepes was
added and inverted several times again. This was allowed to stand
for 20 min at room temperature. The solution was again centrifuged
at 50,000 X g for 45 min, the supernatant decanted and the pellet
resuspended in DH2O to one-thousandth of the original culture
volume. The samples (10-30 yg protein) were then electrophoresed
at 30 V to 60 V in 12% polyacrylamide slab gels according to the
method of Pugsley et al. (67) and stained with Coomassie blue.
Electrodialysis of Extracellular
Polysaccharides from K. pneumoniae
This procedure was adapted from the method of Galanos et al.
(33). The electrodialysis apparatus was built from a 2 1/2 x 3 1/2
x 2 1/2 in. plastic tray with cover. Three chambers were assembled
inside the tray by two plastic slide frames held fast and made
leak-proof with silicone cement. Holes were drilled in the cover
over the two outside chambers and electrodes were fastened to the
cover. Dialysis tubing (Fisher Scientific Co., Pittsburgh, PA)
with a 10,000 molecular weight cut off was inserted within the two
slide frames. The chambers were filled with cold DH2O (resistivity
equal to 15-18 megohms) and the apparatus was placed on ice.
Initially, a 1000 V potential from an ISCO Model H92 Power Supply
(ISCO, Lincoln, Nebraska) was placed across the terminals of the
apparatus without sample to remove any contaminating ions. When
25
the ammeter approached 25 mA the power was shut off, the DH2O
decanted and refilled with cold DH2O. This was performed in the
absence of sample until no increase in mA was noted. The EPS
sample was placed within a benzoylated dialysis bag (Sigma) with a
molecular weight cutoff of either 1000 or 2000, and the bag was
placed in the center chamber of the electrodialysis apparatus.
Initially the voltmeter was set at 400 V and the ammeter was
allowed to increase up to 25 mA. At this time the power supply was
shut off and one half of the DH2O (25 ml of 50 ml) was collected
separately from both the cathode and the anode chambers. The
apparatus containing the sample was washed extensively in DHpO and
refilled with cold DH2O. The process was repeated at 400 V up to
25 mA and half the liquid in the anode and cathode chambers
collected and pooled with prior trials until the increase in
amperage at 400 V was negligible over a 30 min period. At this
time the dialysis bag containing the sample was opened and an
aliquot removed. The bag was then closed and placed back into the
electrodialysis apparatus.
The voltage was then increased to 1000 V and electrodialysis
continued. The liquid from the cathode and anode chambers was
pooled seperately from each other and separately from the preceding
runs at 400 V. Electrodialysis was complete when no further
increase in amperage was noted over a 30 min period. The sample
was removed and placed at 4°C. The cathode and anode pools were
lyophilized to dryness and brought back up in DH2O at approximately
one-tenth the original volume. From electrodialysis was obtained a
26
sequential set of electrodialysed samples, and the material from
both the cathode and the anode chambers from one electrodialysed
sample to the next. These solutions were then tested for hexose,
uronic acid, protein and LPS as described above, for phosphate,
according to the procedure of Chen et al. (13), and for calcium and
magnesium as determined on a Perkin-Elmer model 303 Atomic
Absorption Spectrophotemeter (Perkin-Elmer Corp., Norwalk., CN)
according to the Perkin-Elmer manual.
Determination of Capsule Size of K. pneumoniae
Capsule size of bacteria from DW medium was determined by the
method of Duguid (20) using India ink preparations. Capsule
production was expressed as the transverse diameter (TD), which is
a measurement of both the width of the bacillus and the width of
the capsule on either side of the bacillus. One hundred bacilli
were randomly selected under oil immersion, measured with an ocular
micrometer, and the average capsule size was calculated.
In Vitro Quantitation of Extracellular
Polysaccharides Produced by K. pneumoniae
Bacteria were grown in a defined medium as described in
Section B of Materials and Methods. Samples (100 ml) of growing
cultures were taken at 18, 24, 36 and 48 h and the organisms
pelleted by centrifugation at 12,700 x g for 30 min. The
supernatant obtained was dialyzed 3 times in 8L cold DH2O overnight
while stirring, and assayed for uronic acid. Colony forming units
per ml culture at these time periods were also determined on TSA.
The production of ECPS for each organism was expressed as yg ECPS
27
ml cell (xlO ). ECPS was calculated by dividing the uronic
acid determinations by 0.3098 (for KPl) or by 0.2643 (for KP2),
which reflects the proportion of ECPS which is uronic acid (30,64).
The production of extracellular lipopolysaccharide (ELPS) for each
strain was also monitored at these time periods by the method of
Osborn et al. (60). The ELPS data were expressed as the yg ECPS
ml cell (x 10 ). ELPS data were further characterized in some
of the studies by the Limulus Amoebocyte Lysate (LAL) Assay
(Pyrotell Associates of Cape Cod, Inc., Woods Hole, Mass.) as
described by Levin (50). Both assays utilized the purified LPS
from Escherichia coli 055:B5 as the standard and the ELPS units are
expressed as yg of E.. coli LPS equivalents.
Electron Microscopy
Electromicroscopy for the visualization of the capsular
substances of J<. pneumoniae was performed according to the method
of Cassone and Garaci (12) on early log phase cultures grown in DW
medium. Preparations were observed and photographed with a Hitachi
H-600 Transmission Electron Microscope. These studies were kindly
performed by Dr. Jack Yee of the Department of Anatomy, Texas Tech
University Health Sciences Center, Lubbock, Texas.
Gel Diffusion Method for Immunological Analysis
The immunological characterization of the various fractions of
EPS from KPl and KP2 strains were performed by the method of
Ouchterlony (62). Ion agar (0.5 to 1.0% solutions) (Difco) in DH^O
were brought to boiling to dissolve the agar and cooled to about
50°C. Twenty-five ml were then poured on 3 1 / 4 x 4 inch glass
28
plates and allowed to solidify. Holes of 2 mm in diameter and 0.5
cm apart in a circular fashion were made in the gel and 5 yl of EPS
at various concentrations were placed in the outside wells.
Another well was cut in the middle of the circular wells and 5 yl
of KPl or KP2 rabbit antiserum was added to this middle well. The
reaction took place at room temperature overnight in a humidifying
chamber. The gels were then washed three times in 200 ml
physiological saline at 4°C and once with 200 ml DH2O at 4°C. The
gels were then stained with 0.2% Coomassive blue in methanol,
acetic acid and DH2O (5:1:5, by volume), and then destained in the
same solution in the absence of stain.
Radial immunodiffusion studies were also performed to
•quantitate the ECPS found in the serum of infected rats, and to
follow the effect of electrodialysis on EPS samples. A 0.5%
agarose solution in DH2O was heated to boiling and allowed to cool
to 50°C. One to two ml of type-specific antiserum was then added
to 23 or 24 ml of the agarose slurry, and poured onto 3 1 / 4 x 4
inch glass plates or in 150 mm petri plates and allowed to
solidify. Hole were made in the gel (2 mm) and 5 to 10 yl of
sample was placed in the wells. The gel plates were incubated for
18-24 h at room temperature. Zone diameters of the precipitin
reaction within the gel were measured and compared to known
quantities of ECPS tested in the same manner.
Saponification of K. pneumoniae
Polysaccharides and Quantitation
of Fatty Acids
In some studies the EPS, that was partially purified up to the
29
by EPS after saponification. No attempt was made to identify the
various methyl ester fractions.
Hydrofluoric Acid Treatment
Various fractions of EPS were weighed out (20 mg) and placed
in 1 ml 60% hydrofluoric acid (HF) for 3 h at 0°C with occasional
mixing. NaOH (4M) was then added to pH 12 and the sample was
centrifuged at 12,700 x g for 10 min to remove debris. The
supernatanat fluid was collected and tested for serological
activity with the appropriate antiserum. The supernatant was then
placed on a gel filtration column to characterize the effect of HF
on the molecular weight fractions of EPS.
Statistical Analysis
All statistical analyses performed in these studies utilized
the student's t test for unpaired samples (73).
CHAPTER III
RESULTS
Strain Variation and the Production of
Apparent Isogenic Sets
India ink preparations of a number of strains of K,. pneumoniae
revealed that not all bacilli of the same strain possessed a
similar size capsule. Two predominant capsule sizes co-existed
within many of the strains. A closer inspection of isolated
colonies on TSA revealed that the two basic colony types within a
given strain corresponded to the capsule size differences seen
under India ink. In particular, for KPl ATCC 8047 and for KP2 ATCC
29011, there existed an opaque (0) and a translucent (T) colony
type. Thus the co-variants in the KPl population were labelled
KPl-0 and KPl-T, and those in KP2 were designated KP2-0 and KP2-T.
In both cases the opaque variant possessed the larger capsule.
KPl-0 possessed a capsule with an average transverse diameter (TD)
of 5.6 ym, while KPl-T exhibited a TD of 2.5 ym. The capsules of
KP2-0 and KP2-T had TD of 2.5 ym and 1.5 ym respectively. The data
for capsule sizes of all the strains used in this study are given
in Table 1. Biochemical and serological typing were performed on
all variants to confirm species and serotype. All strains of K,.
pneumoniae used in these studies had an API 20E (Analytab) code of
5215773 except for KPl 2-70 which coded out as 5005773.
It was much more difficult to obtain large and small capsule
variants for the KPl CDC 2-70. Under India ink KPl 2-70 had a TD
of 2.2 ym. A large encapsulated organism (TD=5.6 ym) was
occassionally seen under India ink, though these were rare. The
30
31
Table 1 . Capsule Size of K. pneumoniae
OiaMlim.^ Tp^(ym) Range(ym)^
KPl-0 5.6 3.9 - 8.6
KPl-T 2.5 2.2 - 3.0
KPl 2-70 2.2 1.8 - 2.5
KP2-0 2.5 1.9 - 3.0
KP2-T 1.5 1 . 4 - 1 . 6
KP2 2-70 3.0 2.5 - 5.0
Bacteria were grown in defined medium for 18h.
TD; Transverse diameter as measured under India ink, calculated as the average (mode) of 100 random determinations.
^The range in TD re f lec ts the smallest and largest TD seen in a s ingle preparat ion.
32
large capsuled variant was finally isolated by enriching the
population by passage through mice.
After several attempts, a sub-population of KP2 CDC 2-70
differing in capsule size could not be isolated. Under India ink
the average TD for KP2 2-70 was calculated to be 3.0 ym. However
the variance in capsule size in this strain ranged from 2.5 to 5.0
ym. A population rich in large encapsulated variants of KP2 2-70
was obtained by passage through mice. However, upon subculture,
the majority of large variants were no longer present in the
population. Furthermore there appeared to be an increase in the
presence of the larger encapsulated variants during the stationary
phase of growth, with an average TD of 2.75 ym at 24h growth, 3.0
ym at 36h growth and 3.3 ym at 48h. This is the only strain among
those utilized in these studies that had the propensity to change
its average capsular diameter at various stages of culture. Figure
la and lb show transmission electron micrographs (TEM) of KP2-0 and
KP2-T, respectively, as examples of the variance in the dimension
of capsule size within a given population.
The Establishment of a Chronic Lobar
Pneumonia by K. pneumoniae
in a Rat Model
Early studies employed KPl ATCC 8047 in chronicity studies, as
described in Materials and Methods. Results of lung bacterial
concentration are shown in Table 2. The total viable bacterial
count (TBC) is expressed as the log-iQ of the average total lung
bacterial concentration for the experimental animals in each group.
As can be seen, the log-j^TBC remained elevated (range of 4.11 to
33
34
36
37
Table 2. Establishment of Chronic KPl Pneumonia in Rats
Group Number of animals Day
dead/total Sacrificed
Log 10
TBC" (CFU)
(Range)
1
2
3
4
5
6
7
8
Control
0/4
0/4
0/4
0/4
0/4
0/4
1/4
2/4
0/8
1
3
6
9
7
14
21
28
3, 6, 7, 9,
14, 21, 28
6.51
7.02
6.32
6.91
9.47
6.20
3.84
3.19
ND^
(5.96-6.90)
(5.67-8.46)
(4.11-8.51)
(4.60-8.95)
(9.05-10.16)
(5.86-6.65)
(2.00-5.00)
(ND^-6.38)
^Rats were inoculated transtracheally with 5 x 10 CFU of KPl in 0.05 ml sterile PBS and sacrificed on the days indicated. Controls received 0.05 ml sterile PBS in the same manner. All surviving rats (except the four used for histological processing on days 1, 3, 6 and 9) were used for bacterial quantitation of lung tissue.
^TBC; viable bacterial count per whole lung expressed in log.|Q units from surviving rats.
^ND; none detected at 10' dilution of lung homogenate.
38
10.16) throughout the first fourteen days of the study, while no
KPl were detected in the lungs of the control animals. After day
14 it became difficult to obtain a statistically sound estimate of
the TBC in the rat lungs due to deaths occurring in the 21 and 28
day groups. Only one of the eight experimental rats in the 21 and
28 day groups cleared KPl from its lungs, while the four remaining
animals had log^^TBC of between 2.00 and 6.38. Mortality for the
entire population was 5 per cent (3/60), but for those rats that
were sacrificed on or after 21 days, 37.5% (3/8) of the animals
died before the time of sacrifice.
By day 2 post-infection virtually all experimental rats
appeared acutely ill. Mucous secretions exuded from their eyes and
most exhibited short and rapid breathing. As the infection
progressed, their coats became shabby and considerable weight loss
was obvious. Gross examination of the lungs showed involvement of
one or more lobes, often affecting the entire lobe in a typical
lobar distribution. The involvement was characteristically massive
and voluminous, presenting as dull, greyish regions that released
copious amounts of purulent exudate upon sectioning.
Histological examination also supported the establishment of a
lobar pneumonia in this rat lung model. Figure 2 (a-e) are
photomicrographs of H&E stained sections of rat lung tissue showing
the progressive development of a confluent pneumonia. Figure 2a
depicts normal lung tissue, representative of all control animals.
The integrity of the alveolar and bronchiolar structures can be
easily visualized. In marked contrast, lung tissue typical of 24h,
39
post-exposure rats (Fig. 2b) shows a phagocytic infiltrate
consisting primarily of polymorphonuclear leukocytes (PMN) filling
the alveolar spaces. By day 3 post-infection (Fig. 2c) a confluent
pneumonia had developed. The structural integrity of the
bronchiolar, columnar epithelium had been compromised, and signs of
necrosis and early abscess formation were evident. Large abscesses
and liquefication of structural walls were characteristic of
infection by day 6 (Fig. 2d). By day 9, foci of chronic abscess
formation were evident (Fig. 2e) with collagen fibers visibly
forming a wall to contain the abscess. This process of progressive
destruction of lung tissue continued up to day 28 when the study
was terminated.
The next set of experiments was performed to determine the 50
per cent infective dose (IDCQ) in the rat model for the KPl 8047
strain, before this strain was separated into its capsular o
variants. KPl was grown to a concentration of 1.55 x 10 CFU/ml in
TSB, washed twice and resuspended in cold PBS. Serial 10-fold
dilutions were then made in cold, sterile PBS and the organisms
were kept on ice until inoculation. Thirty rats were employed in
these studies and were divided into five groups of six animals
each. Group 1 received 0.05 ml of the undiluted KPl suspension
(7.76 X 10^) transtracheally into the left lower lobe of the lung.
Group 2 received the same volume of the first 10-fold dilution r 2
(7.76 X 10 CFU) and so on to group 5 which received 7.76 x 10 CFU
of KPl. All rats were sacrificed on the sixth day after KPl
administration. Table 3 summarizes the results obtained. Twenty
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52
per cent (6/30) of the rats died during the course of this
experiment with half of these belonging to group 1. Lung weight
was determined to document the marked increase in lung size in
infected rats. Serum lysozyme levels were also examined in this
study because these values have been shown to covary with the
extent of infection (4). As can be seen in Table 3, the serum
lysozyme levels of all groups of animals receiving KPl were
elevated with respect to control values, achieving significance at
the p < 0.01 level in two of the groups. Due to the marked
swelling during the infectious process, the weight of the lungs
increased up to more than three times that of normal. The average
lung weight for the infected rats in this study was 5.0 grams (3.1
grams above the control mean lung weight). Rats were considered
infected if they either succumbed to the KPl-induced pneumonia or
if a TBC of at least 5 x 10^ Oog^Q TBC=4.7) was found in the whole
lung of those rats harboring the organism. An ID^Q for KPl of 1.55
X 10 CFU was thus obtained.
In order to test the effect of the medium in which the
bacteria were grown as contributing to the virulence of the
organism, a defined medium was used and the effect of dosage of KPl
was repeated in the same manner as above. The ID^Q obtained using 5
the defined medium was found to be 2.22 x 10 CFU, which does not
differ significantly from the ID^Q value obtained using TSB.
Therefore, the comparative effect of growing l<. pneumoniae in two
different media on the pathogenicity of KPl in the rat lung model
appears to be negligible.
53
The remainder of the bacterial strains and their subvariants,
with the exception of the KP2-T and KPl CDC 2-70 strains were then
examined in the rat lung model. All organisms were grown in
defined medium and harvested as described in materials and methods.
Table 4 shows the results obtained when various concentrations of
KPl-0 were inoculated transtracheally into the lungs of normal
rats. With an initial inoculum of 5.0 x 10^ CFU all rats became
infected, three died, and the one remaining rat harbored a TBC of
1.43 X 10^ CFU at the time of sacrifice. All rats receiving 5.01 x
10^ CFU or 5.01 x 10^ CFU of KPl-0 also became infected. One rat
died in each of these two groups, while lung weight and serum
lysozyme were elevated. In groups 4 and 5, which received 5.01 x
10^ and 5.01 x 10^ CFU of KPl-0 respectively, three of the four
rats in each group were infected, one rat in each group died and
serum lysozyme as well as lung weight was elevated. Finally, a
dose of 5.01 X 10^ CFU of KPl-0 or less did not result in an
infection of any rats.
Table 5 shows the results obtained when various concentrations
of KPl-T were inoculated transtracehally into the lungs of healthy
rats. An initial inoculum of 7.07 x 10^ CFU of KPl-T resulted in
the death of three of the four rats in the first group. The one
remaining rat effectively cleared this massive inoculum of KPl-T
organisms placed in its lungs and showed no overt signs of
pathology. Only one of the four rats in the second group, which
received 7.07 x 10^ CFU of KPl-T, succumbed to the infection, while
the remaining three rats showed no signs of infection at the time
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56
of sacrifice. One of four rats in group 3, which received 7.07 x
5 A 10 CFU of KPl-T, showed signs of infection (greater than 5 x 10
CFU per whole lung). Finally, all rats receiving 7.07 x 10^ CFU or
less of KPl-T (groups 4-6) did not become infected.
Table 6 shows the results obtained when various concentrations
of KP2-0 were inoculated transtracheally into the lungs of normal
rats. Doses of 7.2 x 10^ CFU did not result in infection in three
of the four animals in the first group. The fourth rat had a TBC 4
of 5.25 X 10 CFU with a lung weight of 3.2 grams and was
considered infected. No rats in group 2, which received 7.2 x 10
CFU of KP2-0 were considered infected using our criteria. One of 5
the four rats in group 3, which received 7.2 x 10 CFU of KP2-0 was
infected, but all other rats in this group and in the ensuing
groups (groups 4-6) had cleared K_. pneumoniae from their lungs.
Serum lysozyme was not significantly elevated in any group within
either the KPl-T or the KP2-0 study.
Table 7 shows the effect of various doses of KP2 2-70 4
inoculated into the lungs of rats. Doses of 6.5 x 10 CFU per rat
resulted in the infection of 6 of 8 animals at 7 days 3
post-inoculation. Group 2 received 6.5 x 10 CFU and showed 3 of 8
rats infected. Three animals from group 3 died and 1 of the
remaining 5 were infected. Group 4 rats which received 6.5 x 10
CFU revealed 5 of 8 animals having a TBC above the threshold for
infection. Finally group 5 rats, which received 6.5 x 10 CFU per
rat, showed 3 of the rats infected by day 7 post-inoculation. Both
lung weight and serum lysozyme were elevated in these rats, but
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59
showed a gradual decline to near control values with increasing
10-fold dilutions of inoculum. Seven rats inoculated with 6.46 x 3
10 CFU KP2 2-70 (as in group 2) were sacrificed at 14 days
post-inoculation. Of these rats one died on day 3 and one had a
9 A TBC of 4.7 X 10 . Three rats had a TBC of above 1 x 10 but not
above the threshold of 5 x 10 . The remaining two rats had TBC of
3 3
1.6 X 10 and 3.6 x 10 . Therefore none of the rats had cleared
KP2 2-70 from their lungs though only 2 of 7 rats exhibited all the
signs of infection at day 14 post-inoculation.
The IDcQ in rats for each of the five J<. pneumoniae strains
employed in these studies can be seen in Table 8 along with the
LDcQ values for each strain in mice. The data are analysed in the
following section.
K. pneumoniae Virulence in a Mouse Model
All J<. pneumoniae serotype 1 and serotype 2 strains and their
variants were employed in standard virulence assays as described in
Materials and Methods. Result of these studies are compiled in
Table 8 together with the data obtained from studies of virulence
in the rat lung model. These data show that KPl-0, which exhibited
the largest capsule, was more virulent than its covariant, KPl-T,
by 4 or more log-jQ units. Similarly KP2-0 exhibited a larger
capsule than its covariant, KP2-T, and proved to be more virulent
in the mouse model. The TD of KPl-T was slightly larger than the
TD of KPl 2-70 and was more virulent than KPl 2-70 by more than 2
log-ip, units. Therefore, a direct correlation between two distinct
60
Table 8. LDHQ Values in Mice and ID^Q Values in Rats for Strains of l<. pneumoniae Serotypes 1 and 2.
Organism LD^Q (CFU)^ ID^Q (CFU)*^
KPl (mixed) 1.92 x 10 ^ 1.55 x 10^
KPl-0 4.99 X 10^^ 3.41 x 10^
KPl-T 6.03 X 10^^ 1.53 x 10^
KP2 2-70 1.00 X 10° 4.70 x 10^
KP2 (mixed) 4.29 x 10^ NP'
KP2-0 1.78 X 10^ >7.3 x 10^
KP2-T >6.2 X 10^ NP^
^Five groups of f i ve mice each were inoculated IP with ser ia l 10-fold d i l u t i ons of the appropriate K,. pneumoniae s t ra in in 1.0 ml of s t e r i l e PBS and observed for a 72 h period. LD^Q values were calculated by the method of Reed and Muench (65) ana represent at least two determinations for each organism.
^Rats were considered to be infected i f they succumbed to the i r pneumonia or i f the TBC was 5 x 10 CFU or greater.
^The IDrr. values for KPl-0 and KPl-T were shown to be s ign i f i can t l y d i f f e ren t (p<0.025).
°NP; not performed.
61
populations within a serotype regarding the relationship between
virulence and capsule size appears to exist.
In Vitro Quantitation of Extracellular
Polysaccharides Produced by
K. pneumoniae
Bacteria were grown in a defined medium (DW) and harvested as
described in Materials and Methods. The ECPS production by KPl-0,
KPl-T and KPl 2-70 are summarized in Table 9. With regard to the
apparent isogenic covariants, the results indicate that KPl-0
produces ECPS to a much greater extent than KPl-T at all stages of
culture. -The expression of ECPS by both strains is linear with
respect to time over the entire period of analysis. The KPl-0
organism produced 0.37 yg ECPS ml" cell" (x 10 ) per hour of
culture, while the KPl-T strain produced 0.067 yg ECPS ml" cell"
(x 10^) per hour in this same period between 18 and 48h of growth
in DW. The overall ratio of the rate of production of ECPS between
KPl-0 and KPl-T is estimated to be 5.52:1. These data are
illustrated in Fig 3. The difference between the means of the
production of ECPS by these two bacteria is significant at p <
0.005. The production of ECPS by KPl 2-70 was intermediate between
that of KPl-0 and KPl-T and a rate of ECPS production of 0.15 yg
ECPS* ml"^ cell"^ x 10'^ per hour was characteristic of this
organism. Figure 4 shows a TEM of KPl 2-70, depicting both the
cell-associated and the extracellular capsular material.
62
Table 9. ECPS Production by Strains of 1<. pneumoniae Serotype 1 at Various Intervals of Incubation
Organisms Incubation Period (h) ECPS^ CFU/ml
5.50+3.68 1.17x10^
8.49+3.71 1.17x10^
10.75+3.70 5.64x10^
18.24+1.32 2.00x10^
0.67+0.03 3.7x10^
1.09+0.11 3.7x10^
1.63+0.03 2.67x10^
2.56+0.09 1.82x10'' p<0.001^
0.52+0.24 3.8x10^
2.12+1.55 4.0x10^
9.49+p.OO 3.0x10^
10.01+.2.48 1.1x10^ p<0.01^
p<0.05^
^Cultures were grown at 37°C in defined medium at 200 rpm.
^ECPS; in yg ml"^ c e l l " ^ (xlO^) as quantitated by the method of Blumencrantz and Asboe-Hansen (7) .
^S ta t i s t i ca l analysis comparing ECPS ml"^ ce l l " ^ production of KPl-0 to KPl-T or to KPl 2-70 at 48 h incubation.
^Comparison of KPl-T to KPl 2-70 as in footnote C.
KPl-0
KPl-0
KPl-0
KPl-0
KPl-T
KPl-T
KPl-T
KPl-T
KPl 2-
KPl 2-
KPl 2-
KPl 2-
•70
•70
•70
•70
18
24
36
48
18
24
36
48
18
24
36
48
63
64
UJ
(Q.OI^)J|a3J^-Scd03 E'h'
65
66
67
Table 10 summarizes the ECPS production of the various KP2
serotypes. As can be seen, KP2-0 produces more ECPS than its
co-variant, KP2-T, which produces ECPS at near non-detectable
levels in the supernatant fluid at all intervals of incubation. A
comparison of ECPS production between KP2-0 and KP2 2-70 shows a
difference in kinetics, as illustrated in Figure 5. KP2-0 begins
to produce ECPS much earlier in culture than KP2 2-70, but by 36h,
KP2 2-70 has surpassed KP2-0 in total ECPS production and has
produced twice as much ECPS by 48 h. These differences are
reflected in the rate of ECPS production within this time period.
At up to 18h of culture, the rate of production for KP2 2-70 is
essentially negligible, but it increased significantly at
approximately this time period. Production is linear between 18
and 36 h for both strains, but the rates of production differ
markedly within this time frame, with KP2 2-70 producing 0.078 yg
ECPS ml"^ cell"^ (xlO"^) per hour and KP2-0 producing 0.027 yg ECPS
ml" cell" (x 10" ) per hour. Therefore, in this particular
medium, KP2 2-70 produces nearly three times as much of ECPS as
does KP2-0 within the linear phase of production.
Extracellular LPS (ELPS) production was also examined during
these time periods and the data are shown in Table 11. This table
shows the amounts of ELPS produced by all strains at 48 h of growth
in DW as measured by both the KDO and the LAL assays. Colony
forming units at the highest concentration during cultural growth
are also listed in Table 11. The ELPS is expressed as the yg ELPS
ml' cell' (x 10" ). It can be seen from this table that the
68
Table 10. ECPS Production by Strains of J<. pneumoniae Serotype 2 at Various Intervals of Incubation
Organisms Incubation Period (h) ECPS CFU/ml
KP2-0
KP2-0
KP2-0
KP2-0
18
24
36
48
0.48+0.20
0.59+0.11
0.90+0.001
1.30+0.13
9.22x10'
1.1x10^
9.2x10^
8.6x10^
8
KP2-T
KP2-T
KP2-T
KP2-T
18
24
36
48
0.002'
ND'
1.1x10-
0.003+0.002 9.9x10 8
1.1x10"
0.005+0.0002 1.0x10-
KP2 2-70
KP2 2-70
KP2 2-70
KP2 2-70
18
24
36
48
0.06+0.03
1.66+0.16
2.13+0.11
4.0x10^ p<0.20^
8 0.38+0.02 4.2x10 p<0.10
3.9x10^ p<0.025
3.9x10^ p<0.020
^Cul tures were grown a t 37°C i n def ined medium at 200 rpm.
^ECPS; i n yg ml""" c e l l " ^ ( x l O ' ^ ) .
^ND; none de tec ted .
^ S t a t i s t i c a l ana lys is comparing ECPS ml"^ c e l l " product ion of KP2-0 and KP2 2-70 a t the same stage of incuba t ion .
^Only one determinat ion made.
69
70
24 TIME(h)
71
_ Dtypes 1 and ; at 48h of Culture in Defined Medium
Table 11. Production of ELPS^ by J<. pneumoniae Serotypes 1 and 2
Organism
KPl-0
KPl-T
KPl 2-70
KP2-0
KP2-T
KP2 2-70
CFU/ml
1.17x10^
3.70x10^
4.00x10^
9.48x10^
1.05x10^
4.25x10^
(xlO"^)(KDO)^
1.43+0.04
0.38+0.14
0.54+0.03
0.03+p.OOl
0.01+0.002
0.07+0.007
yg ELPS mr^cell"^ yg ELPS ml'^ceir^
(xlO"^)(LAL)^
1.37 - 2.74
0.22 - 0.43
0.21 - 0.42
0.008-0.017
0.004-0.008
0.04 - 0.08
^Ext racel lu lar l ipopolysaccharide (N=2).
KDO; Thiobarbi tur ic acid assay for ketodeoxyoctanate (57).
^LAL; Limulus Amoebocyte Lysate assay. Values are presented as the range in which the quant i ty of ELPS in the samples is l im i ted (47).
72
KPl-0 organism produces the greatest quantity of ELPS per ml per
cell of all the strains, whereas the KP2 2-70 organism is the
highest ELPS producer among the KP2 strains.
Comparisons of ECPS, ELPS and capsule size at 48h growth for
all serotype 1 and 2 strains as well as virulence in both the rat
lung model and the mouse model can be seen in Table 12. First of
all, there exists a strong postive correlation (r=0.97) between the
production of ECPS and ELPS, per ml per cell. The two serotypes
differ in this regard in that the KPl strains produced
approximately 59 yg of ELPS for each mg of ECPS produced, while the
KP2 strains produced about 30 yg of ELPS per mg of ECPS. Therefore
the correlation between the production of ECPS and ELPS is
strongest within serotypes.
The relationship between ECPS and the capsule size of all
strains shows a direct positive correlation (r = 0.95), which is
even of greater magnitude when comparing strains within serotype
for both the KPl and KP2 strains. Therefore, in general, all of
these organisms seem to reflect their ability to produce
extracellular capsular polysaccharide by the size of their
capsules. A notable exception is the KPl 2-70 strain which seems
to have a TD less than that of KPl-T but produces more ECPS per
cell than KPl-T.
Finally in the comparison between the three parameters of
polysaccharide production (TD, ECPS, and ELPS per cell at 48h
incubation) and the virulence studies in the standard mouse
virulence model, an inverse correlation was noted. Table 13
73
Table 12. Comparison of ECPS, ELPS, Capsule Size and Virulence of J<. pneumoniae Serotypes 1 and 2
Organism
KPl-0
KPl-T
KPl 2-70
KP2-0
KP2-T
KP2 2-70
ECPS
18.24
2.56
3.73
1.30
0.005
2.13
ELPS^
1.43
0.38
0.54
0.03
0.01
0.07
TD^
5.6
2.5
2.2
2.5
1.5
3.0
LD ^
4.9x10^
5.34x10^
7.30x10^
1.78x10^
>6.2xl0^
1.0x10°
^^50'
3.41x10^
1.53X10'
NP^
>7.3xlO'^
NP
4.7x10^
a - 1 - 6 ECPS yg ml" cell"(xl0 ) in dialyzed supernatants of 48h growth at 37 C in defined medium.
h - 1 - 1 fi
ELPS yg ml" cell" (xlO ) in dialyzed supernatants of 48h growth at 37 C in defined medium.
^transverse diameter.
LDrni 50% lethal dose, obtained from IP injections in mice. bU
^IDj-^; 50% infectious dose, obtained from transtracheal inoculations into the lungs of rats.
Not performed.
74
Table 13. Correlations Between Polysaccharide Production and Virulence in the Mouse Model
ECPS/cell^ ELPS/cell^ TD°
Serotype 1 ( L D ^ Q ) ^ -0.46 -0.37 -0.50
Serotype 2 ( L D ^ Q ) ^ -0.82 -0.72 -0.96
All strains (I-D^Q)^ -0.31 -0.15 -0.55
^Quantity of ECPS per cell in dialyzed supernatants at 48h growth in defined medium at 37 C.
Quantity of ELPS per cell as in footnote a.
^Transverse diameter of the capsule.
^The LDnn values obtained from 3 serotype 1 strains. bU
^The LDc^ values obtained from 3 serotype 2 strains. bU
^The LDj-f. values obtained from all 6 strains of type 1 and type 2 K_. pneumonVae.
75
represents the correlations obtained between the polysaccharide
parameters and the virulence data. The data from Table 13 show
that capsule size may be the best indicator of the ability of a
particular strain to be pathogenic, especially when the serotype is
unknown. However, the production of ECPS per cell correlates
nearly as well as does capsule size with these virulence
parameters. Finally the production of ELPS per cell gives the
least amount of information as to the virulence potential of a
given strain, though it still correlates with virulence. Again,
stronger correlations are found within serotype than in grouping
the two serotypes together, which is especially true for the KP2
strains where nearly perfect correlations were obtained.
Serum Sensitivities and Opsonophagocytic
Assays for K. pneumoniae
Of the six strains used in these studies, only one of the
strains (KP2-T) showed inhibition of growth in the presence of 90%
rabbit serum over a 60 min period. Data for all strains can be
seen in Table 14, which shows the change in the log-jQ CFU between
time 0 and 60 min of incubation in serum. The virulent KPl-0 and
KP2 2-70 strains seem to grow most favorably in 90% serum, but
their growth is not significantly different than those of the other
strains, with the exception of KP2-T.
The ability of these organisms to grow in the presence of
human leukocytes (WBC), type-specific antiserum (AB), or a
complement source (C) was then tested, as described in materials
76
Table 14. Serum Sens i t i v i t y of K_. pneumoniae
Strain^ ^^O^^Q C F U ^
KPl-0 + 0.97
KPl-T + 0.54
KPl 2-70 + 0.50
KP2-0 + 0.42
KP2-T - 0.08
KP2 2-70 + 0.64
^Log phase organisms were washed 3 times and resuspended in PBS in various concentrations and added to 9 parts normal rabbi t serum.
^The change in the number of colony-forming units (CFU) in log,Q uni ts a f te r 60 min incubation in 90% normal rabbi t serum at 3/ C.
77
and methods under Opsonophagocytic Assays (OPA). Table 15
summarizes the results after normalizing the data for comparative
purposes. Normalization of the data was as follows: 1) Colony
counts in log-jQ CFU that were obtained for each strain at 0 min
were subtracted from the log-jQ CFU at 60 min incubation in the OPA.
The net change in the log^^ CFU (Alog,Q CFU) was thus obtained. 2)
Secondly the Alog.Q CFU for the control assay, containing only
heat-inactivated serum (without AB, WBC or C), was subtracted from
the Alog-jQ CFU of all other assays within a given strain. The
final value then shows the Alog-.^ CFU from 0 to 60 min compared to
the serum controls and allows for comparisons among the strains
tested. The results in Table 15 show that the most important
variables which affect the Alog,Q CFU is the presence of WBC and
type-specific AB in the assay mixture. The effect of a complement
source did not seem to affect the net log-jQ CFU in these assays.
Also it seems that WBC do not significantly alter the net
log-,p. CFU of any of the strains in question in the absence of AB.
One notable exception to this conclusion involves the KPl-T strain
and its ability to be more readily phagocytosed in the presence of
complement (C-E) than in the absence of complement (D-E), while in
the absence of AB. But also for the KPl-T strain, type specific
antiserum enhances the ability of WBC to ingest KPl-T moreso than
does complement, though this was not a significant difference.
Table 16 shows the results obtained when KPl 2-70 or KP2-0 was
tested in the OPA in the presence of EPS from either KPl or KP2,
containing both ECPS and ELPS. When the EPS from a type 1 strain
78
Table 15. Opsonophagocytic Assay^
(AB+WBC+C) (ABC+WBC) (WBC+C) (WBC)
Strain Control Control^ Control^ Control^
KPl-0 -2.08^ -1.49 -0.10 -0.12
KPl-T -0.27 -0.27 -0.16 +0.16
KPl 2-70 -0.39 -0.42 -0.02 -0.03
KP2-0 -0.19 -0.30 -0.09 -0.13
KP2-T -0.40 -0.22 -0.01 -0.07
KP2 2-70 -0.81 -0.79 +0.37 +0.05
Log-phase organisms were washed in PBS and various concentrations were placed in 3 parts normal rabbit serum containing combinations of the following components: 1) type specific antiserum (AB)-and 2) Human peripheral white blood cells (WBC). The serum was heat-inactivated in some of the trials to test for the effect of a complement (C) source. The assay was performed at 37 C for 60 min and the net CFU in log-iQ units was determined. See appendix 4 through 9.
The net log-,p. CFU obtained from the assay in which AB, WBC and C were present, minus the net log-jQ CFU obtained from the control assay where none of these components were present (heat-inactivated normal rat serum only).
^The net log-.^ CFU obtained from the assay in which AB and WBC but no C source were present, minus the net log-jQ CFU from the control assay.
^The net log-jp, CFU obtained from the assay in which WBC and C but no AB were firesent, minus the net log-jQ CFU obtained from the control assay.
^The net log-.^ CFU obtained from the assay in which WBC but no AB or C were fDresent, minus the net log^Q CFU obtained from the control assay.
79
Table 16. Effect of the Addition of EPS on the OPA^
Strain EPS Alog^^ CFU^
KPl 2-70 PBS 0.56
KPl^ 1.19 p<0.0l3
KP2^ 0.67
KP2-0 PBS 0.41
KPl^ 0.35
KP2^ 0.68 p<0.005^
The OPA contained the following components in equal volumes: Human peripheral white blood cells at 1 x 10 gper ml in normal rabbit serum; J<. pneumoniae strains at 1 x 10 to 1 x 10 CFU/ml in normal rabbit serum; Rabbit antiserum against the homologous serotype, and either KPl or KP2 EPS in PBS or PBS alone. A total serum concentration of 75% was used.
Change in CFU from 0 to 60 min incubation at 37°C in log units.
^KPl EPS from dialyzed supernatants of KPl-0 at 339 yg ECPS/ml and 20 yg ELPS/ml.
^KP2 EPS from the neutral fraction of KP2-0 at 850 yg ECPS/ml and 25 yg ELPS/ml.
^KPl EPS from the LMW fraction of KPl-0 at 331 yg ECPS/ml and 23 yg ELPS/ml.
^KP2 EPS from the ethanol extracted fraction of KP2-0 at 489 yg ECPS/ml and 16 yg ELPS/ml.
^Statistical analysis comparing the Alog.« CFU after treatment with homologous EPS compared to treatment witn either PBS or heterologous EPS.
80
was added to the KPl 2-70 OPA mixture at 339 yg ECPS/ml and 20 yg
ELPS/ml there was a significant difference in the Alog,Q CFU from
the same mixture without ECPS present. However, when KP2-0 EPS,
containing 850 yg ECPS and 25 yg ELPS per ml was added to the same
OPA, no significant difference was seen compared to the non-ECPS
control. In the reverse experiment the KP2-0 strain with the
addition of KP2 EPS at 489 yg ECPS/ml and 16 y ELPS/ml grew
significantly better than both the antiserum control (PBS treated)
or the KPl ECPS treated trials (331 yg ECPS and 23 yg ELPS per ml).
Therefore EPS from a type-specific strain allowed for enhanced
growth of KPl 2-70 over controls in the presence AB, whereas EPS
from a heterologous serotype did not.
Purification of the EPS of K. pneumoniae
Organisms were grown in the defined medium at 37 C for 48h
while shaking at 200 rpm for these studies. The purification
protocol, as described in Materials and Methods, involved ethanol
fractionation, DEAE-Sephacel ion exchange and gel filtration
chromatography. Fractionation with ethanol produced a hygroscopic,
white and fluffy material which was not easily resuspended in
hydrophilic solutions and was precipi table in non-polar solvents
(i.e., methanol, ethanol, chloroform, hexanes). When suspended in
DHpO these materials produced highly viscous solutions, especially
at a concentration of 2 mg dry weight or more per ml. These
solutions were characteristically opalescent and, for certain
preparations, a white precipitate was noted. Removal of the
precipitate by centrifugation caused as much as a 25% loss of ECPS
81
from KP2 preparations but no detectable loss of ECPS from KPl
preparations. The KP2 preparations in general, dissolved less
easily in DH2O than the KPl preparations, but seemed to dissolve
much better in alkaline solutions above a pH of 11. A Tris buffer
was then used (0.01 M Tris, pH 12) in many of the subsequent
purification steps, especially to avoid the cessation of a column
run due to aggregates of KP2 EPS clogging the column filters.
Ethanol fractionated EPS was resuspended in 0.01 M Tris, pH
12, or in another low ionic strength buffer [i.e., 0.02 M
(NH4)2C02], and placed on a DEAE-Sephacel column equilibrated with
the same buffer. The column was eluted with approximately 200 ml
of buffer before the salt gradient was applied. It was found that,
for all of the ethanol fractionated samples from the strains used
in this study, yielded a fraction of uronic acid containing
material eluting from the column at this time. This fraction was
labeled the neutral (N) EPS fraction. When a salt gradient was
applied [up to IM concentrations of either NaCl or (NH^)2C02],
a second uronic acid and hexose containing fraction eluted. An
example of an elution profile on DEAE Sephacel can be seen in
Figure 6 for the KPl-T ECPS. Table 17 shows the ionic strength of
the buffer at which two KPl and two KP2 EPS samples were eluted.
The acidic (A) fraction of EPS for the various samples eluted
between 0.2 and 0.4 M (NH^)2C03.
The acidic and the neutral fractions, after dialysis and
lyophilization, were resuspended in the appropriate column buffer
(O.OIM Tris, pH 12, or 0.5M NaCl were commonly used) and placed on
82
Table 17. Elution Ionic Strength and Apparent Molecular Weights of the ECPS from Various Strains of KPl and KP2
Elution
ECPS
KPl-O(N)^
KPl-O(A)^
KPl-T(N)
KPl-T(A)
KP2-0(N)
KP2-0(A)
KP2-270(N)
KP2 2-70(A)
HMW^
>3xl0^
>3xl0^
>,3xlO^
>^3xlO^
>3xl0^
>^3xlO^
>3xl0^
>3xl0^
%total
15
10
52
53
60
70
100
100
LMW^
8.9x10^
5.0x10^
9.4x10^
1.1x10^
2.3x10^
2.0x10^
—
_ — —
%total
85
90
48
46
40
30
--
_—
Ionic strength
0.02
0.42
0.02
0.21
0.02
0.23
0.02
0.17
^HMW; the high molecular weight or void volume fraction of EPS eluted from S-2B gel filtration.
^LMW; the low molecular weight fraction of EPS found within the, inclusion capabilities of the gel filtration column.
^Elution Ionic Strength; the salt gradient molarity of (NH^)2C03 at which the various EPS fractions eluted from DEAE-Sephacel.
*^(N); neutral fraction.
^(A); acidic fraction.
83
84
20 30 40 50 60 70 80
Fraction Numbtr 90
85
gel filtration (S-2B or BGA-150m) columns. Examples of elution
profiles for KPl-0 (A), KPl-0 (N), KPl-T (A), KPl-T (N), KP2-0 (A)
and KP2 2-70(A) EPS on S-2B can be seen in Figures 7 through 13.
For all strains utilized in these studies, the acidic and the
neutral fraction of EPS produced a high molecular weight (HMW),
hexose and uronic acid containing fraction at the void volume of
the gel filtration profile. For the KP2 strains the vast majority
of the polysaccharide material eluted in this HMW fraction, whereas
only 10 to 15 percent of the acidic or neutral EPS from KPl-0 and
52 to 53 percent from KPl-T eluted at this HMW fraction. The
remainder of the hexose and uronic acid containing material for all
strains was within the inclusion capabilities of the gel filtration
column, and was typically contained in one fraction. This fraction
was labelled the low molecular weight (LMW) fraction. For the
KPl-0 and the KPl-T strains this fraction accounted for 85-90% and
46-48% of all hexose and uronic acid containing material,
respectively. In contrast, it was the lesser of the two fractions
for the KP2 strains, with the KP2-0 strains producing a LMW
fraction which accounted for 30 to 40% of the polysaccharide in the
sample, and the KP2 2-70 strain apparently producing no LMW
material. It was found later that the KP2 2-70 strain does produce
a LMW component which appeared after ethanol extracted material was
placed on a BGA 150 m column equilibrated with 0.01 M Tris, pH 12,
but did not appear when 0.5 M NaCl was used with the acidic or the
neutral EPS. Table 17 displays the percentages of EPS that were in
86
87
Uronic Add/ ig /ml(o) o o o o o o o o o o o o
-r T
.a E
u o
« ^ ""—'—'—'—r
(•)|UJ/B7y980X9H
88
89
Uronic Acid / ig /ml (o)
(•) IUJ/BT/ OSOXOH
90
91
Uronic Acid/ig/mKo)
w — o a> l O CSJ —
(•) I U I / C T / 9S0X9H
92
93
Uronic Acid/ig/mI(o)
o Q o o o ^ (O (\j _
o o o O 0> OD
P o o o f «o «
( • ) | U J / 6 T / 9 « O X 9 H
94
95
10 15 20 25 30
FRACTION NUMBER
35 40
96
97
T
LU CO
o X UJ X
15 20 25
FRACTION NUMBER
40
98
99
Uronic Acid /ig/ml(o)
o o O O o - <D iO -t <Si
- I — I t . i
o <0
- o 'J-
«>
E 3
O o
o CVJ ro
O O fO
O cn CM
( • ) I U J / B T / 980X8H
100
101
250
200-
3 ' 50 UJ
o X Ul X
100-
60
FRACTION NUMBER
102
the HMW and the LMW peaks as well as the apparent molecular weights
calculated from dextran calibration standards (Fig. 14 and Table
18). The HMW fractions from all strains were outside the range of
the column and were calculated to be at least 3 x 10^ daltons in
molecular weight. The LMW fractions were, however, retained by the
column and gave molecular weights as seen in Table 17. For the KPl r c
LMW EPS the molecular weight range is between 5 x 10 and 1 x 10
daltons, while for the KP2-0 LMW EPS a molecular weight of around 2
X 10 daltons was estimated.
As purification proceeded, fractions containing hexose and
uronic acid were pooled, dialyzed and lyophilized. Dried materials
were then- brought up in 1 mg/ml or 2 mg/ml solutions in DHpO and
tested for their content of ECPS, ELPS and protein. Table 19 shows
the percentage of these components to the total dry weight of the
samples. It can be seen that nearly 50% of the KPl ethanol
extracted [KPl (EtOH) EPS] material was in the form of ECPS, while
approximately 5 to 7% of the dry weight was accounted for by ELPS,
and about 4 to 7% was protein. For the KP2 strains, 78 to 84% of
the dry weight of the ethanol extracted [KP2 (EtOH) EPS] material
was ECPS, 2.4 to 2.5% was ELPS and 1 to 1.7% was protein. Between
37 and 45% of the dry weight of KPl (EtOH) EPS and between 12 and
18% of the dry weight of KP2 (EtOH) EPS was unaccounted for in
these preparations by the methods used.
A second study of this type was then performed on the uronic
acid and hexose containing materials that were purified by DEAE-
103
Table 18. Elution Volumes for Dextran Calibration Standards on
S-2B
Dextran^ V ° K ^ av
5-40 X 10^ 159.8 0.0
2.0 X 10^. 250.0 0.27
5.0 X 10^ 379.8 0.67
2.3 X 10^ 396.7 0.72
1.7 X 10^ 413.6 0.76
8.1 X 10^ 415.5 0.77
9.4 X 10^ 453.08 0.89
^Dextran standards in average molecular weight (daltons)
V ; Elution volume in ml at peak of fraction.
''K ; determined by the equation av
V^ - V^ V^=159.8 ml e 0 0
^av
V. - V„ V =490 ml t o t
104
Table 19. The Extracellular Products Found in the Ethanol Fractionated Supernatants of K_. pneumoniae
Strain
KPl-0
KPl-T
KP2-0
KP2 2-70
ECPS^
52.2
42.6
78.2
84.1
ELPS^
6.5
5.2
2.5
2.4
Protein^
3.9
•7.0
1.0
1.7
Other^
37.4
45.2
18.3
11.8
Values of ECPS, ELPS and protein are expressed as the percentage of the dry weight of the sample. The ECPS was calculated from the uronic acid determinations which were performed by the method of Blumencrantz et al. (7). The ELPS was determined by the method of Osborn et al. (57). Protein was determined by the procedure of Lowry et al. (51).
Other; the percentage of unidentified components contributing to the dry weight of ethanol extracted materials.
105
Sephacel ion exchange chromatography. Table 20 shows the amounts
of ECPS and ELPS contained in 2 mg/ml solutions and the percent of
the total dry weight of samples from two KPl and one KP2 strains.
The EPS from KPl-0 exhibited the least amount of ECPS per mg of dry
weight sample, with the KPl-0 (A) EPS being 12.8% ECPS by weight
and KPl-O(N) EPS 20.2% by weight. The figures for KPl-T (A) and
KPl-T (N) were somewhat greater, as seen in Table 20. The KP2 2-70
(N) and KP2 2-70(A) EPS were the most highly purified ECPS samples
at this stage of purification (70 to 76% of the total dry weight).
The amount of ELPS contained in these samples was seen to correlate
directly with the amounts of ECPS present for both the KPl EPS
samples, and it was determined that 1.6 yg of ELPS was present for
eyery 10 yg of ECPS. For the KP2 2-70 EPS, which was a much more
highly purified preparation, 2.5 yg of ELPS was present for ewery
100 yg of ECPS. From 64 to 86% of the total dry weight for the KPl
EPS samples and between 22 and 28% of the KP2 2-70 EPS was left
unaccounted for by these measures.
The extracellular products found in KPl-0 and KP2 2-70 EPS
after gel filtration and the percentages of the total dry weight
that these products comprise are listed in Table 21 . It can be
seen that in 1 mg of the HMW fraction from KPl-0, about 30% was
ECPS, 8% was ELPS and about 9% was protein, and for the LMW
fraction 53% was ECPS, about 4% was ELPS and 3% was protein. Thus
for KPl-0 EPS the fraction most enriched with ECPS and containing
the least amount of ELPS and protein was the LMW fraction (with
106
Table 20. Comparison of the ECPS and ELPS content in the Neutral (N) and Acidic (A) Fractions from DEAE-Sephacel
Sample ECPS (N=2)
KPl-O(N)^ 403.1+45.1
KPl-O(A)^ 255.4+24.7
KPl-T(N) 608.3^51.8
KPl-T(A) 284.2+13.0
KP2 2-70(N) 1400.1+87.3
KP2 2-70(A) 1515.3+66.3
Quantity of component in yg contained in 2 mg of dried sample.
Percentage of component contained in the dried sample.
^(N); The neutral fraction of hexose-containing material obtained from DEAE Sephacel.
(A); The acidic fraction as in footnote c.
%total^
20.2
12.8
30.4
14.2
70.0
75.8
ELPS^ (N=2)
79.5+7.5
29.3+3.7
105.3+18.2
69.3+10.3
36.1+0.8
38.1+1.3
%total^
4.0
1.4
5.3
3.5
1.8
1.9
107
Table 21. The Extracellular Products Foynd in KPl and KP2 EPS After Purification
Strain
KPl-0
KPl-0
KP2 2-70
KP2 2-70
Fraction
HMW^
LMW^
HMW
LMW
ECPS'^
29.6
53.0
80.7
83.9
ELPS'^
7.9
3.7
2.8
1.6
n ^ . b Protein
8.7
3.0
4.9
3.4
Other^
53.8
40.3
11.6
11.1
^Purification procedures include ethanol extraction, DEAE-Sephacel and gel filtration chromatography.
^Values of ECPS, ELPS, and protein are expressed as percentage of the weight of the samples.
^Other; the percentage of unidentified components contributing to the dry weight of the samples.
* HMW; the high molecular weight fraction.
^LMW; the low molecular weight fraction.
108
approximately 7 yg of ELPS and 6 yg of protein per 100 yg of ECPS).
The KPl-0 HMW EPS contained much more of these components
(approximately 27 yg ELPS and 29 yg protein per 100 yg ECPS). The
HMW and LMW fractions from KP2 2-70 contained far less quantities
of ELPS and protein, with the HMW fraction containing about 2 yg
ELPS and 6 yg of protein per 100 yg ECPS and the LMW fraction
containing 0.3 yg ELPS and 4 yg protein per 100 yg ECPS. The ECPS
accounted for the vast majority of the dry weight of KP2 2-70 ECPS
samples at this stage of purification (between 81 and 84%).
Finally, Table 22 shows the percent yield obtained for KPl-0
and KPl-T EPS at various stages of purification starting with a 200
ml culture of each strain grown in defined medium for 48h at 37 C.
The data reveal yields of between 24 and 80% throughout the
purification procedures. The final yield for KPl-0 ECPS was 22.5%
while the final yield for the KPl-T ECPS was 7.4%. Much of the
KPl-T ECPS was lost during purification on DEAE Sephacel, which was
also shown above not to further purify the ECPS from either the
KPl-0, KPl-T or the KP2 2-70 strain. Indeed, the eluted fractions
from ion exchange appear to be less pure than any of the fractions
before or after this purification step. These purification steps
apparently have not purified the KP2 ECPS beyond the level of
purification achieved by ethanol fractionation for the KPl ECPS.
Ethanol fractionation resulted in a product that is 43 to 52% ECPS
by weight, while no other subsequent fractions from the
purification schema, except for the LMW fraction from gel
109
Table 22. Percent Yield Obtained from ECPS Pur i f i ca t ion of KPl-0
Stage Preparation ygECPS/mg dry wt^ Total ECPS Yield(%)'
I 48h Dialyzed 79.3+10.2 108.2+2.0 100
Supernatants
II Ethanol 522.2+24.6 62.7+3.0 58
Fractionation
III DEAE 185.4^13.6 30.4+^2.2 28
Chromatography
IV BGA-150m
Chromatography HMW 287.0+38.3 2.4+0.3
IV BGA-150m 23
Chromatography LMW 471.8+96.4 21.9+4.5
The quantity of ECPS in the samples were determined from uronic acid measurements performed by the procedure of Blumencrantz et al. (8).
Yields were determined as the percent of ECPS at each stage of purification compared to the amount in the 48h dialyzed supernatant.
no
filtration, were of equal purity. The KPl LMW fraction retained
the highest purity of all the KPl fractions in that it contained
lower quantities of ELPS (7 yg per 100 yg ECPS) than the ethanol
extracted fraction (12 yg ELPS per 100 yg ECPS).
Due to the rather poor level of purity obtained for the ECPS
of the KPl strains by the procedures above, a different method was
utilized to purify the KPl ECPS from the KPl-0 strain. Ethanol
fractionated KPl-0 EPS was resuspended in DH^O and subjected to
electrodialysis (ED) at 2000 V. All of the DH^O in the cathode and
anode chambers were collected, lyophilized and referred to as
fraction I (Fr I). The electrodialyzed EPS was then fractionated
with cetavlon. The cetavlon precipitate, labelled Fraction II (Fr
II), and the cetavlon supernatant, labelled fraction III (Fr III)
were both washed with ethanol (3 times) and the product was tested
for its content of ECPS, ELPS and protein. The results of these
procedures are presented in Table 23. With 150 mg of starting
material, 130 mg total product were obtained gravimetrically after
these purification steps. Fr I was shown to contain no detectable
hexose or uronic acid and comprised at least 19 mg of material.
Therefore nearly 13% of the starting material was dialyzed free of
the EPS during ED. Fr II was found to comprise about 71% of the
weight of the starting material, and 77% of Fr II was found
chemically to be ECPS while 3% was ELPS, 3.5% was protein and 16%
was unaccounted for by these methods. Fr III, on the other hand,
comprised only 3% of the starting material. It was determined
Ill
Table 23. Pur i f i ca t ion of KPl-0 (EtOH) EPS by ED, Cetavlon and Gel F i l t r a t i o n
Sample
KPl-0 (EtOH)^
Fr 11^
Fr I I I ^
Fr I^
Fr I I , S-2B^
Fr I I , S-2B,ED
KPl-0 (EtOH)ED
mg dry wt
150.0
106.0
4.5
19.0
58.0
9 ^p
^ NP
ECPS
522.17+24.64
766.77+14.36
494.40+.56.15
ND" '
620.14j: 3.13
732.10+35.42
802.47^18.91
ELPS^
62.66+2.96
29.70+0.86^
212.73+2.96
ND
3 . 0 5 + 1 . 3 1
6 . 1 5 + 0 . 6 6
54.37+0.87
Protein
39.0+1.5
34.8+.7.6
NP^
ND
9 . 2 + 2 . 0
1 9 . 6 + 2 . 7
NP
Units are in yg per mg dry weight.
^Ethanol extracted EPS.
^Fr I I ; the cetavlon prec ip i ta te from KPl-0 (EtOH) EPS washed x3 wi th EtOH.
^Fr I I I ; the supernatant from the cetavlon step washed x 3 with EtOH.
^Fr I ; the dialysable material col lected during ED (2000 molecular weight c u t o f f ) .
^Fr I I , S-2B; Fr I placed on Sepharose - 2B and the LMW component co l lec ted .
^Fr I I , S-2B, ED; Material in footnote d re-electrodialyzed.
•^KPl-O (EtOH) ED; KPl-0 (EtOH) EPS subjected to ED (separate experiment).
^NP; not performed.
"^ND; none detected.
112
chemically that 49.5% of Fr III consisted of ECPS while 21.3% was
ELPS, which left 30% of Fr III unaccounted for by these methods. A
portion of Fr II was then placed on a S-2B gel filtration column
and an elution profile was obtained as seen in Figure 15. This
figure illustrates that now the vast majority of the hexose
containing material elutes at a well defined LMW peak. Tube
fractions 25-45 were collected, dialyzed and lyophilized and
retested for ECPS, ELPS and protein content as seen under the label
Fr II, S-2B in Table 23. Nearly 90% of the ELPS has been removed
from this fraction, when comparing it to the Fr II starting
material, as well as 75% of the protein. However, the ECPS per mg
dry weight was nearly 15% less than that of Fr II. The Fr II, S-2B
material was then electrodialyzed to remove the salts acquired from
gel filtration. This final preparation, labeled Fr II, S-2B, ED in
Table 23, is now comparable to Fr II in its ECPS content (73%
compared to 77% respectively) and still shows comparatively low
levels of ELPS and protein. Finally, in a separate experiment
KPl-0 (EtOH) EPS was subjected to ED and then tested for ECPS and
ELPS content as shown in Table 23 under the label KPl-0 (EtOH) ED.
The ED step shows a 28% increase in the quantity of ECPS per mg dry
wt with comparable levels of ELPS, when compared to the KPl-0
(EtOH) EPS. Table 24 shows the amounts of ELPS and ECPS that were
contained in 100 yg ECPS from all of these purification steps.
Greater than 90% of the ELPS and between 65 and 80% of the protein
were removed from the KPl-0 (EtOH) EPS by these methods as seen in
113
114
80 '
70
- 6 0 ^ E ^ 50 r =k
U 4 0 o ^ 30 X
2 0
10
—
-
-
A A
10 20 30 40 50 60
FRACTION NUMBER
115
Table 24 Purification of KPl-0 (EtOH) EPS bv Fn cetavlon and S-2B: Percent of^LPs! Protean'''
and Other Materials
^^m}± ELPS/ECPS^ Protein/Frpc;a n^u R ^^o^^^ri/tCPS Other material/ECPS^
KPl-0 (EtOH)^ 12.00 7.47
F^ n ^ 3.87 72.04
4-54 22.01
' ' ' ' ' " ^ 4 3 . 0 3 NPJ- .• d
^' I ' ND " Mnl
Fr I I , S-2B^ 0 . 1 6
Fr I I , S -2B , ED^ 0 . 8 4
NP
ND' i p J
T-48 5 9 . 2 8
2 - 6 8 3 3 . 0 8
a A^un t of ELPS, protein or other materials in pg per 100 .g ECPS.
'•^As In Table 23.
116
this table. Extracting the EPS with cetavlon alone without prior
ED gave a similar elution profile as was seen for the EtOH
extracted fractions and a similar level of purity (559.15 and
607.49 yg ECPS per mg dry weight and 20.89 and 52.05 yg ELPS per mg
dry weight for KPl-0 and KPl-T samples respectively).
Fr III (3.5 mg) was applied to a BioGel P-300 gel filtration
column (P-300), equilibrated with 0.1 M ammonium acetate, pH 8.1,
containing 0.1% SDS, after boiling for 5 min in the column buffer.
The elution profile in Figure 16 shows the hexose containing
fractions obtained. The Limulus Amoebocyte Lysate assay indicated
that the majority of the ELPS was located under the peak that
eluted at tube fractions 26-34. This latter pool was collected,
washed 3 times with EtOH and tested for its ECPS and ELPS content.
It was found that this pool was enriched with ELPS (45 yg of ELPS
per 100 yg of ECPS), more so than all other samples encountered.
The KP2-0 EPS was also subjected to the above purification
procedures (EtOH ED CET S-2B). No substantial difference was
seen in the quantitites of ECPS or ELPS between the KP2-0 (EtOH)
EPS and the EPS obtained by the above methods. However, the Fr III
material obtained from the supernatant after cetavlon extraction
was enriched with ELPS (38.1 yg ELPS per 100 y5 ECPS) compared to
the Fr II precipitate from cetavlon treatment (4.21 yg ELPS per 100
yg ECPS). Table 25 summarizes the data obtained from these studies
with the KP2-0 EPS. Figure 17 shows the elution profile of these
materials on S-2B. KP2 2-70 (A) EPS was also subjected to ED and
117
118
50
4 0 -
•g 3 0
C3>
3 U l CO
o X Ul X
20r
0 -
20 30 40 50 60
FRACTION NUMBER
119
120
FRACTION NUMBER
121
Table 25. Pur i f i ca t ion of KP2-0 EPS by ED and Cetavlon
Sample ELPS/ECPS^
KP2-0 (EtOH)^ 3.25+0.01
KP2-0 (EtOH)ED^ 3.20+0.27
KP2-0 (EtOH)ED, CET PPT^ 4.21+0.18
KP2-0 (EtOH)ED, CET SUPE^ 38.05+3.59
^Amourt of ELPS in yg found per 100 yg of ECPS (N=2).
^KP2-C (EtOH); Ethanol extracted KP2-0 supernatants from growth (48h) at 37 C in defined medium.
^KP2-0 (EtOH)ED; Electrodialysis of material in footnote b.
CET PPT; Ethanol washed precipitate from cetavlon extraction of material in footnote c.
^CET SUPE; Ethanol washed supernatant as in footnote d.
122
then fractionated with cetavlon, and both the cetavlon precipitate
(Fr II) and the cetavlon supernatant (Fr III) were ethanol
extracted. Table 26 shows the KP2 2-70 (A) EPS data utilizing
these purification methods. Figure 18 shows the elution profile
obtained for the Fr II material applied to the S-2B gel filtration
column. It can be seen in Figure 17 that these purification
procedures have produced a better separation of the two major
fractions of KP2-0 EPS (compare with Figures 11 and 22), but did
not dissociate the HMW form. Figure 18 shows that cetavlon
extraction did not further dissociate the HMW form of KP2 2-70 EPS
over that of ED alone, but, rather, tended to increase the
concentration of the HMW form with a decrease in the proportion of
lower molecular weight peaks. It can be seen in Figure 18 that
whenever ED was a part of the purification procedure, a more
prominent LMW fraction (tube fractions 22 to 44) can be seen,
regardless of cetavlon use in the purification protocol. Cetavlon
extraction alone does not reduce the proportion of HMW EPS to LMW
EPS, when compared to the profile for KP2 2-70(A) EPS, but the LMW
regions are markedly different from one another. Since cetavlon
extracts only acidic polysaccharides, the LMW peaks observed in
Figure 18 for the profile of KP2 2-70 (A) EPS, after tube fraction
36, are most likely composed of neutral polysaccharides that are
removed during cetavlon extraction. It is therefore likely that
only ED can effect a dissociation of HMW EPS, and that cetavlon
extraction serves to remove neutral polysaccharides, such as LPS,
from the EPS, which was found to be the case shown in Table 26.
123
Table 26. Purification of KP2 2-70 EPS by ED and Cetavlon
Sample^
KP2 2-70(A)
KP2 2-70(A), ED
KP2 2-70(A), ED CET PPT
KP2 2-70(A) CET PPT
KP2 2-70(A), ED, CET SUPE
KP2 2-70(A), CET SUPE
^KP2 2-70(A) EPS was subjected to ED (KP2 2-70(A), ED) at 2000 V and cetavlon extracted to give both the cetavlon precipitate (KP2 2-70(A), ED, CET PPT) and ethanol extracted material from the cetavlon supernatants (KP2 2-70 (A), ED, CET SUPE). KP2 2-70(A) EPS was also cetavlon extracted without ED (KP2 2-70, CET PPT) and ethanol extracted materials from the cetavlon supernatant were obtained (KP2 2-70(A), CET SUPE).
Amount of ELPS present in yg per 100 yg of ECPS.
^Amount of protein present in yg per 100 yg of ECPS.
^No ECPS detected.
^Not determined.
ELPS/ECPS^
3.67+0.19
3.26+0.23
3.42+0.29
2.89+0.12
46.15+0.11
d
Protein/ECPS^
2.51+0.09
0.04^0.03
0.07+0.04
ND^
2.38+1.50
d
124
125
6 0 ff
50
40
C7>
3 Ul 3 0 cn O X Ul X
20
10 -
• J — • -
FRACTION NUMBER
126
Effect of Purified Extracellular Products
from K. pneumoniae on Virulence
in a Mouse Model
The majority of the studies in this section were performed
with the moderately virulent KPl-T strain to test whether the
virulence of this strain could be enhanced by co-injection with
EPS at various stages of purity. These studies were performed
first by utilizing KPl EPS, as the material co-administered to mice
by IP injections, along with serial ten-fold dilutions of KPl-T.
At the dosages administered it was found that only the KPl-T (A)
and the LMW fractions of EPS from KPl-0 or KPl-T did not
significantly enhance the virulence of KPl-T over control values.
This was true for the LMW EPS fractions even at the high doses
(108-149 yg/mouse) administered. Table 27 displays the Alog-jQ LD^Q
per milligram of ECPS administered to these mice [A(log.|Q LDgQ)/mg
ECPS]. A wide range of differences are seen to exist among the
values presented in this table (from -1.00 to -40.00), and these
differences seem to correlate inversely with the degree of
purification. For example the A(log^Q LD^QJ/mg ECPS for the acid
or neutral ECPS from KPl-0 (-35.7 to -40.0) had at least three
times the virulence enhancing capability as did the same EPS
further purified by gel filtration (-3.89 to -11.78). These
differences reach significance at the levels shown in Table 28.
The KPl-0 HMW (N) EPS virulence enhancement values were also
significantly greater (p<0.01) than the KPl-0 LMW EPS values. Thus
127
Table 27. E f fec t of KPl EPS on KPl-T Vi ru lence in the Mouse Model
FCPS Adog^Q LD5Q)/mg ECPS a
KPl-0 (N)'^ -35.73 + 10.2
KPl-0 (A)^ -40.00 + 13.3
KPl-T (N)^ -9.12 + 0.99
KPl-T (A)^ -7.38 + 4.57
KPl-0 HMW (N)^ -11.78 + 0.52
KPl-0 LMW (N)^ -3.89 + 1.87
KPl-T HMW (N)^ -9.08 + 3.51
KPl-T LMW (N)^ -1.00 + 0.30
^A(log,Q LDr^)/mg ECPS; Change in the log.Q LD^Q compared to contrAV t r i a l s per mg ECPS co- in jected with tne KPl-T s t ra in ,
^Neutral EPS f rac t i on from DEAE-Sephacel.
^Acid EPS f rac t i on from DEAE-Sephacel.
*^High molecular weight f rac t ion from neutral EPS applied to Sepharose 2B gel f i l t r a t i o n .
^Low molecular weight f rac t ion as in footnote d.
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129
as purification proceeded for the KPl-0 EPS, virulence enhancement
significantly decreased.
There were no significant differences seen between the KPl-T
(A), KPl-T (N) and the KPl-T HMW EPS in their virulence enhancing
properties for the KPl-T strain. However, the KPl-T (N) and the
KPl-T HMW (N) ECPS were significantly greater virulence enhancers
for KPl-T than was the KPl-T LMW EPS (p values are shown in Table
28). The A(log^Q ^^^0^/^^ ^^P^ values for KPl-T (N), KPl-T (A) and
KPl-T HMW are all quite similar (-9.12, -7.38, and -9.08,
respectively) whereas the value for KPl-T LMW is -1.00. Thus for
both the KPl-0 and the KPl-T EPS, the LMW fraction showed
significantly less virulence enhancement for KPl-T in the mouse
model than the HMW fractions from either strain.
When comparing the KPl-0 EPS to the KPl-T EPS at various
stages of purity as to their virulence enhancing capabilities, it
was found that the KPl-0 (N) and the KPl-0 (A) EPS were
significantly more virulence enhancing than the same fractions of
KPl-T EPS, but the HMW (N) EPS fractions from either strain did not
differ significantly in this parameter (see Table 28). The LMW EPS
from KPl-0 (N) did, however, differ significantly (p<0.05) from the
KPl-T LMW EPS, even though both values were small compared to the
other values obtained for KPl EPS in Table 27. With the exception
of KPl-0 (N) and KPl-0 (A) EPS on one extreme, and KPl-0 LMW and
KPl-T LMW EPS on the other, the remaining four fractions of KPl EPS
show very similar A(log^Q LD^Q)/mg ECPS values, the mean of these
values being -10.58 ± 3.60.
130
Cetavlon extracted EPS from KPl-0 cultures [KPl-0 (CET) EPS]
was also tested in the mouse virulence assay, with both the KPl-T
and the KP2-0 s t r a i n , to see i f a d i f fe ren t pu r i f i ca t i on procedure
could a f fec t the virulence enhancement properties of KPl-0 EPS.
At 200 yg/mouse, the KPl-0 (CET) EPS was shown to enhance the
virulence of both the KPl-T and the KP2-0 s t ra in s i gn i f i can t l y over
control values. The A(log^Q ^^Q^^^ ECPS for KPl-0 (CET) EPS in
the virulence studies with the KPl-T s t ra in (-8.40 + 0.92) is not
s i g n i f i c a n t l y d i f f e ren t from the mean value (-10.58 ± 3.60)
obtained in the ea r l i e r studies in th is sect ion. Therefore the EPS
of KPl-0 retains i t s virulence enhancing properties whether an
ethanol-based or a cetavlon-based extract ion is u t i l i z e d .
The next series of studies were performed in order to
determine the a b i l i t y of the KP2 EPS to enhance the virulence of a
KPl organism. KP2 EPS, at various stages of p u r i f i c a t i o n , from
e i ther the v i ru len t KP2 2-70 or the moderately v i ru len t KP2-0
s t r a i n , were co-administered IP with the KPl-T s t ra in into mice,
and lOrr. values were obtained. Appendix 15 displays the dosages of
the various EPS f rac t ions along with the log-jg LDrg data and the
A(logiQ LDCQ) wi th respect to contro ls . S ta t i s t i ca l analysis of
these values showed that (1) administrat ion of KP2 2-70 ethanol
extracted [KP2 2-70 (EtOH)] EPS at 336.2 yg ECPS/mouse enhanced the
virulence of KPl-T s i gn i f i can t l y over control values (p<0.005); (2)
administ rat ion of KP2 2-70 (A) EPS into mice at a dosage of 454.1
yg ECPS/mouse, but not at 204.7 or 101.5 yg ECPS/mouse, enhanced
the virulence of KPl-T s i gn i f i can t l y over control values; and (3)
131
administration of KP2-0 (A) EPS at 428.6 yg ECPS/mouse did not
significantly enhance the virulence of KPl-T in the mouse model.
Table 29 shows the A(log^Q '-D5Q)/mg ECPS for each of the ECPS
fractions utilized in these studies. The KP2 2-70 (EtOH) fraction
is seen here to have produced the greatest increment of virulence
enhancement for the KPl-T strain in the mouse model (-5.38 +0.38),
followed by the KP2 2-70 (A) EPS at the higher doses (-3.56 + 0.48
and -4.21 + 1.18). The KP2-0 (A) EPS produced a relatively small
increment in virulence enhancement for KPl-T (-2.03 + 2.77) as well
as did the lowest dosage of KP2 2-70 EPS (-1.28 + 2.35). None of
these differences in virulence enhancement among the KP2 EPS
fractions were significantly different, however. Table 29 also
shows the A(log^Q '-' SO /' ^ ECPS obtained for the six KP2 2-70 (A)
EPS trials at three different doses. Four of the six values used
to calculate this mean also approximated the mean closely, whereas
two of the data points (one in the highest and one in the lowest
dosage) were unrelated to the mean obtained. Similarly, the two
data points used to obtain the mean A(logiQ LDrQ)/mg ECPS value for
KP2-0 (A) EPS were dissimilar. A high degree of variability was
thus seen in these experiments, as well as in the experiments
utilizing KPl EPS to enhance the KPl-T strain in the mouse model.
When comparing the ffect of KPl EPS to the effect of KP2 EPS
on the virulence of KPl-T in the mouse model, the following trends
were evident: (1) the HMW fractions or the fractions from
ion-exchange for the KPl EPS are between 2 and 10 times more
virulence enhancing for the KPl-T strain than are the KP2 EPS
132
Table 29. Effect of KP2 EPS on KPl-T Virulence in the Mouse Model
hPb
KP2 2-70 (EtOH)^
KP2 2-70 (A)^
KP2 2-70 (A)
KP2 2-70 (A)
KP2-0 (A)^
^As in Table 27.
yg ECPS/mouse
336.2
101.5
204.7
454.1
428.6
Adog^Q LD3Q)/mg ECPS
-5.38 ± 0.38
-1.28 +2 .35^
-3.56 + 0.48^
-4.21 + 1.18^
-2.03 + 2.77
Ethanol extracted EPS from 48h cu l tura l supernatants.
^Acid EPS f rac t ion from DEAE-Sephacel
^The mean and standard deviat ion of a l l the KP2 2-70 (A) EPS t r i a l s (N=6) was -3.02 ± 1.83.
133
fractions; and (2) the LMW fractions of KPl EPS are equal in
potency to the KP2 EPS in enhancing the virulence of KPl-T. Table
28 shows the p values for the comparison of the means of the
A(log^Q '- So'/' S ECPS for KPl and KP2 EPS. The KPl-0 (N), KPl-0
(A) and the KPl-0 HMW EPS are all significantly greater virulence
enhancers than any one of the KP2 EPS fractions, while the KPl-T
(N) EPS fraction is significantly more potent as a virulence
enhancer of KPl-T than two of the three KP2 EPS fractions [KP2 2-70
(A) and KP2-0 (A), but not KP2 2-70 (EtOH) EPS]. The KPl-T HMW
EPS was a significantly greater virulence enhancer than the KP2
2-70 (A) EPS (p<0.02) but did not differ significantly from the
KP2-0 (A) or the KP2 2-70 (EtOH) EPS. There were no significant
differences seen between the KPl-T (A) or the KPl-0 LMW EPS when
compared to any of the KP2 EPS fractions in virulence enhancement
of KPl-T. Finally, the A(log^Q ^^SO^^^^ ^^^^ ^ ° ^ ^^^ ^^^''^ "" ^
EPS was significantly lower (p<0.005) than the value obtained for
the KP2 2-70 (EtOH) EPS, but not different significantly from the
other KP2 EPS fractions.
The reverse experiment was then performed, wherein a KP2
strain was utilized in standard mouse virulence assays, with the
additional co-administration of EPS from KPl or KP2, to see if EPS
from either serotype could enhance the virulence of the KP2 strain.
The moderately virulent KP2-0 strain was chosen for these trials,
and either KPl-0 (N) EPS at 40.3 or 200.0 yg ECPS per mouse were
co-administered. The A(log^Q ldc^Q)/mg ECPS are given in Table 30.
These data reveal that the virulence of the KP2-0 strain was
134
Table 30. E f fec t of KPl or KP2 EPS on the V i ru lence o f KP2-0 in the Mouse Model
EPS Adog^Q LDgQJ/mg ECPS^
KP2 2-70 (EtOH)^ -1.70 + 0 . 4 6
KPl-0 (N)^ - 3 . 9 7 + 0 . 0 0 (p<0.005)^
KPl-0 (CET)^ -5.08 + 0.88
^As i n Table 27.
^KP2 2-70 (EtOH); Ethanol ex t rac ted EPS from 48h supernatants of KP2 2-70 grown in def ined medium a t 37 C.
^KPl-0 (N) ; Neutral EPS f r a c t i o n from DEAE sephacel.
*^KPl-0 (CET); Cetavlon ex t rac ted EPS
^ S t a t i s t i c a l ana lys is o f the means comparing the A(log•,f^ LDt-p,)/mg ECPS of KP2 2-70 (EtOH) EPS to t ha t o f KPl-0 (N) EPS.""
135
enhanced in the mouse model by co-injection of EPS from either the
KPl-0 or the KP2 2-70 strain. When cetavlon extracted KPl-0 EPS
was co-injected with the KP2-0 strain, a A(logiQ LDgQ)/mg ECPS
value of -5.08 j 0.88 (N=2) was obtained, which was not
significantly different than the value obtained for KPl-0 (N) EPS.
Both values, however, were significantly greater than that obtained
for the KP2 2-70 EPS with the KP2-0 strain. In general, however,
the extent of virulence enhancement was relatively small compared
to that manifested by the KPl-T strain with these same EPS
fractions. The KP2 2-70 (EtOH) EPS enhanced the virulence of KPl-T
with approximately three times the magnitude of that which it
enhanced the KP2-0 strain (-5.38 compared to -1.70, respectively)
per mg of ECPS. This difference is significant at the p<0.001
level. The same phenomenon was observed for the effect of KPl (N)
EPS on KP2-0 and KPl-T virulence, namely that the KPl-0 (N) EPS was
significantly more virulence enhancing (p<0.01) for KPl-T than for
KP2-0 (-35.7 compared to -3.97, respectively) per mg of ECPS.
Finally, when comparing the effect of KPl EPS versus the KP2 EPS in
the virulence enhancement of KP2-0, it was seen that the KPl-0 (N)
EPS was significantly more enhancing (p<0.005) than the KP2 2-70
(EtOH) EPS. It has thus been determined that the KPl EPS was
significantly more virulence enhancing for both serotype 1 and
serotype 2 K.. pneumoniae than the KP2 EPS in the mouse model.
Furthermore, the virulence of the KPl-T strain was affected
significantly more so by the co-administration of either KPl or KP2
EPS than was the KP2-0 strain.
136
The next set of experiments were performed to determine
whether the virulence enhancement properties of EPS could be
influenced by electrodialysis (ED) of the EPS as a purification
step before utilization in the mouse model. Both the KP2 2-70
(EtOH) and the KP2-0 (EtOH) EPS were electrodialyzed at lOOOV until
no further marked increases in mA were observed over a 30 minute
period. Both of these types of KP2 EPS were then injected
separately at various dosages with the KPl-T strain into mice.
Table 31 summarizes the results for KP2 2-70 (EtOH) EPS in terms of
the A(logiQ LDcgj/mg ECPS. It can be seen from this table that the
EPS before ED enhanced the virulence of KPl-T to a significantly
greater extent (p<0.005) than did the EPS after ED (-5.38 compared
to -3.01, respectively). Table 32 summarizes these results in
terms of the A(logiQ LDcQ)/mg ECPS. In contrast to the effect of
KP2 2-70 (EtOH) EPS, the KP2-0 (EtOH) EPS enhanced KPl-T virulence
significantly less (p<0.005) before ED. However, after subjecting
KP2-0 (EtOH) EPS to ED, its virulence enhancement capabilities
significantly increased (p<0.005) over that of the same EPS before
ED. There was no significant difference between the effect of
electrodialyzed KP2-0 (EtOH) EPS and the effect of electrodialyzed
KP2 2-70 (EtOH) EPS on KPl-T in the mouse model. There still
remained a significant difference (p<0.02) between the
nonelectrodialyzed KP2 2-70 (EtOH) and the electrodialyzed KP2-0
(EtOH) EPS as to their virulence enhancement capabilties. These
experiments then showed a difference in the effects of ED upon the
ECPS from one strain (KP2-0) having manifested an increase in
137
Table 31 . Effect of E lect rodia lys is (ED) on the Virulence Enhancement of KPl-T by KP2 2-70
EPS in the Mouse Model
KP2 2-70 EPS^ Adog^Q LD5Q)/mg ECPS*
Before ED -5.38 ± 0.66
Af ter ED^ -3.01 +0 .56 (p<0.005)
^KP2 2-70 EPS; obtained by ethanol extract ion of 48h supernatants of 37°C cul tures grown in defined medium.
^ As in Table 27.
^Electrodialysis proceeded at lOOOV until no marked increase in mA occurred over a 30 min period.
^Statistical comparison of the means of the A(log.jQ LDgQ)/mg ECPS obtained before and after electrodialysis.
138
Table 32. Effect of ED on the Virulence Enhancement of KPl-T by KP2-0 EPS in the Mouse Model
KP2-0 EPS^ ^loSlO '-^50'^^^ ^^^^- (^^^'^^^
Before ED -1.15 + 0.36 (N=2)^
Af ter ED^ -3.81 +0 .14 (N=3) (p<0.005)^
^KP2-0 EPS; obtained by ethanol extract ion of 48h supernatants of 37 C cultures grown in defined medium.
^As in Table 27.
^N equals the number of LD^Q t r i a l s performed.
^S ta t i s t i ca l analysis (Student's t tes t ) of the Alog^Q ' -^SQ/^^ ECPS for KP2-0 EPS before and a f te r e lec t rod ia lys is .
139
virulence enhancement, while in the other strain (KP2 2-70),
virulence enhancement of the ethanol extracted EPS decreased as the
result of ED. Moreover, the EPS from the less virulent KP2-0
strain was seen to be significantly less virulence enhancing for
KPl-T than was the EPS from the highly virulent KP2 2-70.
The next set of experiments were performed to determine the
effect of saponification, as a means to remove covalently linked
fatty acid esters from EPS, on the virulence enhancing properties
of EPS in the mouse model. In these trials the neutral EPS from
KPl-0 [KPl-0 (N)] was saponified before utilization in mouse
virulence tests. The A(logiQ LDrgj/mg ECPS is displayed in Table
33. Both the KPl-T and the KP2-0 strain were tested. It was found
that saponification of the KPl-0 (N) EPS.significantly decreased
its virulence enhancing properties both for the KPl-T and the KP2-0
strain in the mouse model without affecting the type specificity of
the ECPS. The A(log^Q LD5Q)/mg ECPS dropped from -35.7 +_ 10.20 to
-3.4 +_ 0.92 for KPl-T after saponification while, in tests
involving KP2-0, it dropped from -3.97 ± 0.00 to -0.70 J: 0.01.
These data suggest that certain ester linkages (presumably fatty
acids esterified to the ECPS or ELPS of these preparations, as
addressed in Results, Section H) were contributing to the virulence
enhancing capabilities of these materials. Tables 34 and 35 show
the A(log.Q L D ^ Q ) per yg of ELPS contained in a variety of the
polysaccharide samples of KPl and KPl EPS, respectively, utilized
in these virulence enhancement studies on the KPl-T strain. These
tables show that, when comparing the ELPS content of these
140
Table 33. Effect of Saponif ication^ on the Virulence Enhancement of KPl-T and KP2-0 by KPl-0 (N)
EPS in the Mouse Model
Stra in in jected EPS A(log^Q L^5o)/"^9 ^^^^^
KPl-T KPl-0 (N)^ -35 .70 + 10.2
KPl-0 (N) - 3 .40 + 0.92 (p<0 .05 )^
(saponified)
KP2-0 KPl-0 (N) -3.97 + 0.01
KPl-0 (N) -0 .70+ 0.01 (p<0.001)^
(saponified)
^KPl-0 (N) EPS (10 mg) was placed in 0.5 M NaOH overnight at room temperature and dialyzed 3 times against 8L DHpO while stirring.
^As in Table 27.
^KPl-0 (N); the neutral fraction of ethanol fractionated EPS placed on DEAE-Sephacel at 200 yg/mouse.
^Statistical analysis (Student's t test) of the A(log.^ LDc.^)/mg ECPS for saponified and untreated EPS in the KPl-T stady.^^
^Statistical analysis as for the KP2-0 study as in footnote d.
141
Table 34. Virulence Enhancement of KPl-T in the Mouse Model: Comparison to the Dosage of ELPS in
KPl EPS Samples
Sample^
KPl-0 (N)
KPl-T (N)
KPl-0 (N)
KPl-0 (N)
HMW
LMW
yg ELPS/mouse
8.0
12.2
16.8
6.8
Adog^Q LD5Q)/yg ELPS^
-0.18 + 0.08 (N=3)
-0.09 + 0.01 (N=3)
-0.06 + 0.002 (N=2)
-0.07 + 0.04 (N=3)
Samples were resuspended in PBS at 2 mg dry weight per ml and 0.1 ml was co- in jected with the KPl-T s t ra in IP into mice.
b A(log,Q LDcQ)/yg ELPS; the change in the log^Q LD^Q per yg injected.
^N equals the number of LDrQ trials performed.
142
Table 35. Virulence Enhancement of KPl-T in the Mouse Model: Comparison to the Dosage of ELPS in KP2 EPS Samples
Sample^ ng ELPS/mouse A(log^Q L^5o'/^9 ^ ^ b
KP2 2-70 (EtOH) 9.44 -0.19 + 0.13
KP2 2-70 (A)
KP2 2-70 (A)
KP2 2-70 (A)
11.40
5.70
2.85
-0.17 + 0.07^
-0.13 +0 .06^
-0.04 + 0.17^
KP2-0 (A) 9.97 -0.09 +0 .12
Samples were resuspended in PBS at various concentrations and 0.1 ml was CO-injected wi th the KPl-T s t ra in IP into mice.
^As in Table 33.
^The mean and standard deviation of all the KP2 2-70 (A) EPS trials (N=6) is -0.11 + 0.10.
143
preparations, no significant differences between different EPS
samples were seen as to their virulence enhancement capabilities
per yg of ELPS. The average A(log^Q "-^so'/^S ELPS for all of the
KPl EPS and KP2 EPS samples was calculated to be -0.10 ± 0.05 and
-0.12 +_ 0.06, respectively. These results argue strongly for a
central role of ELPS in the virulence enhancing properties of these
preparations.
To further clarify the roles of both ELPS and ECPS in
virulence enhancement, the KPl-0 (EtOH) EPS was subjected to an
alternative purification procedure involving electrodialysis (ED),
followed by cetavlon fractionation (CET) and gel filtration on
Sepharose -2B. This alternative procedure allowed for separation
of the ECPS from the ELPS in this sample, as seen in Table 24. The
Fr III sample was then re-extracted with cetavlon after ED at 2000V
to give a sample of ELPS, found in the cetavlon supernatant, that
was free of ECPS as demonstrated by the absence of uronic acid
activity in the preparation. This ELPS fraction was also shown to
precipitate with anti-type 1 antiserum in double diffusion studies
but did not exhibit identity with the ECPS from Fr II. It was
found that, at a dosage of 400 yg of ECPS (and 15.5 yg ELPS) per
mouse, the Fr II material enhanced the virulence of KPl-T
significantly over controls (p<0.01). However, after Fr I was
passed over S-2B and the majority of the ELPS removed, and then
subjected to ED to remove salts obtained from the column [KPl-0 Fr
II, S-2B (ED) EPS], the resultant material at 400 yg/mouse did not
enhance the virulence of KPl-T over controls. The Fr III material
144
at 9.4 yg ELPS/mouse and with no detectable ECPS decreased the LDrg
of KPl-T by 0.45 log,Q units but this was not significantly
different than control LD^Q values (p<0.10). Table 36 shows the
A(log.jQ LD^Q)/mg ECPS and the A(log^Q LD^gj/yg ELPS which resulted
from these studies using the alternative purification methods. The
points to be made here are that, 1) with further purification the
A(log-.Q LDrQ)/mg ECPS values decreased, 2) regardless of the extent
of purification the A(logiQ LD^QJ/yg ELPS remained essentially the
same and 3) a dosage of somewhere between 10 and 15 yg of ELPS per
mouse was sufficient to significantly enhance the virulence of
KPl-T over control values. In comparing the A(log-jQ LDcQ)/yg ELPS
values in this study with those from Table 34, it can be seen that
there were no significant differences in these values for all of
the polysaccharide samples tested in the mouse model. The mean
value for the A(logiQ LDrQ)/yg ELPS for KPl samples turns out to be
-0.086 j: 0.044 (N=7). Therefore, approximately 11.6 yg of ELPS
from KPl-0 or KPl-T should decrease the LDCQ of KPl-T in the mouse
model by one log-jQ unit. For the KP2 EPS the mean value for the
A(log,Q LD5Q)/yg ELPS was determined to be -0.12 + 0.06 (N=5).
Therefore 8.3 yg of KP2 ELPS were apparently needed to decrease the
LD -f value of KPl-T by one log-jQ unit. Due to the high variability
in these assays, no significant difference could be seen between
the effects of KPl and KP2 samples on KPl-T virulence enhancement,
with respect to the dose of ELPS.
145
Table 36. Effect of an Al ternat ive Pur i f i ca t ion of KPl-0 EPS on the Virulence Enhancement of KPl-T in the Mouse Model
Sample^ ^(^OS^Q LDggJ/mg ECPS ^(^09lO ^^50 ' /^^ ^^^^^
KPl-0 Fr I I - 2 . 6 8 + 0 . 4 4 - 0 . 0 7 + 0 . 0 1
KPl-0 Fr I I - 0 . 7 4 + 1 . 1 4 - 0 . 0 8 + 0 . 1 3
S-2B (ED)
KPl-0 Fr I I I - ^ -0.05 + 0.00
^Samples were prepared from ED of the ethanol f ract ionated prec ip i ta ted from 48h supernatant f l u i d of KPl-0 cultures followed by cetavlon f rac t i ona t ion . The Fr I I sample is the cetavlon p rec ip i t a te . The Fr I I S-2B (ED) sample is material obtained from placing the Fr I I sample on S-2B, co l lec t ing the LMW f rac t ion and e lect rod ia lyz ing at 2000V. The Fr I I I sample is the ethanol extracted material obtained from the supernatants a f te r cetavlon ex t rac t ion .
As in Table 27: footnote a.
^As in Table 34: footnote b.
No ECPS was detected in th is f rac t ion as determined by the uronic acid assay of Blumencrantz et a l . (7 ) .
146 Structural Studies on the EPS Produced
by K. pneumoniae
The primary focus of these studies was on the effects of ED on
the EPS from the KP2 strains. Ethanol extracted KP2 2-70 [KP2 2-70
(EtOH)] or KP2-0 [KP2-0 (EtOH)] EPS from 48h supernatants were
resuspended in column buffer (0.01 M Tris, pH 12) and applied to
either BGA-150 m or S-2B gel filtration columns equilibrated with
the same buffer. A portion of these samples were subjected to ED
for various time periods before application to the column. Figure
19 shows the effect of ED on the elution profile from BGA-150 m for
the KP2 2-70 (EtOH) EPS at 4 mg dry weight per ml. Before ED there
were two prominent hexose containing peaks, one eluting at the void
volume of the column (HMW) and one retained by the column (LMW)
with a peak of hexose activity at fraction 33-36. After subjecting
the KP2 2-70 (EtOH) EPS to ED at 400V (intermediate ED), an elution
profile on BGA-150m was obtained, denoted by the profile seen in
Figure 19. In this profile at least two thirds of the HMW fraction
from the profile before ED is now absent, and the LMW component has
increased in magnitude. The 400V ED product was then subjected to
ED at lOOOV and the resulting material was placed on BGA 150m. The
elution profile for this final ED product of KP2 2-70 (EtOH) EPS
can be seen in Figure 19. The HMW component in this final ED
profile is now virtually absent and the LMW peak is apparently more
refined (higher concentrations of hexose containing material in a
fewer number of fractions). It was found that approximately 10% of
the hexose activity was lost during ED and the majority of this
hexose activity was recovered in the fluids collected outside of
147
148
I I 1 1 1 1 — O «rt O lO O uo r o CM CM — —
( I O J / D T / ) 9S0X9H
149
the ED dialysis bag (in the cathode and anode fluids). Subsequent
studies were performed utilizing dialysis tubing with a 1000 or
2000 molecular weight cutoff rather than a 10,000 molecular weight
cutoff, so as to retain all of the hexose activity within the
dialysis tubing.
The KP2 2-70 (EtOH) EPS at 4 mg/ml was again subjected to ED,
this time at 400V, lOOOV and then at 2000V and subjected to RID in
agarose impregnated with antiserum prepared in rabbits against the
KP2 2-70 organism. Figure 20 shows the precipitin zones formed from
these antigen-antibody interactions in agarose at various stages of
ED. Well a in Figure 20 shows the KP2 2-70 (EtOH) EPS before ED.
In this figure a number of diffuse precipitin zones can be seen
within a rather broad and hazy background. When this material was
subjected to ED at 400V the RID profile seen in well b in Figure 20
was obtained. At this stage one predominant precipitin zone was
observed, with a diameter far less than that in well a. Apparently
a small portion of the EPS was also precipitating with antibody at
the periphery of the sample well. It was not certain, however,
whether this precipitate was due to antibody- antigen interactions.
Figure 20, well c, shows the RID profile for KP2 2-70 (EtOH) EPS
after ED at lOOOV, and is essentially the same profile as that seen
in well b. To be sure that ED was complete, the EPS was subjected
to 2000V, and the RID profile seen in Figure 20, well d, was
obtained. In this figure it can be seen that the zone diameter of
the precipitin ring has apparently increased over that of well c,
even though the concentration of ECPS placed in this well was
150
151
i^isissssas^
152
approximately the same as that in the other RID profiles.
From the data obtained from gel filtration and RID studies on
electrodialyzed products of KP2 2-70 (EtOH) EPS, it can be
concluded that ED affects the EPS in two different fashions: 1) HMW
components have apparently dissociated and have given rise to LMW
components and, 2) The LMW components have become more homogeneous
in molecular size as ED proceeded. To substantiate these
conclusions the KP2 2-70 LMW EPS from the BGA-150m profile of KP2
2-70 (EtOH) EPS electrodialyzed at lOOOV (Figure 19) was pooled,
collected, dialyzed and lyophilized to dryness. A comparatively
small sample (2mg dry weight) of the LMW EPS was reapplied to
BGA-150m before and after another round of ED at 1000 V. The
elution profile for the KP2 2-70 (EtOH) LMW EPS before and after ED
can be seen in Figure 21 . Before ED it can be seen that the
elution profile contains a predominant HMW peak that eluted at the
void volume (fractions 16-18), even though no HMW material was
carried over from the initial column run. Also at this low
concentration of EPS one can now see that the LMW fraction
apparently consisted of a number of distinct lower molecular weight
species that, with larger sample volumes, seemed to coalesce into
one broad fraction. These individual LMW fractions were somewhat
symnetrical and occured in the profile at a rather distinct
periodicity (peaks occured for every 60 ml of eluant on the
average). After ED an elution profile was obtained as seen in
Figure 21. The HMW component was again seen to diminish, while the
LMW components seem to have become somewhat less broad and more
refined toward the center of the LMW region. Moreover a new peak
153
154
8
o» 6 U l CO
o X Ul X i
30 40
FRACTION NUMBER
155
was seen near fraction 70.
The ethanol extracted 48h supernatant from the KP2-0 strains
[KP2-0 (EtOH) EPS] was then subjected to ED at up to 2000V to
compare the elution profiles of this ECPS on gel filtration before
and after ED. Figure 22 depicts the elution profile of the KP2-0
(EtOH) EPS before and after ED at 2000V on BGA-150m (6 mg dry
weight applied). Before ED the hexose and uronic acid activity was
limited largely to the void volume of the column, with some
evidence of a second fraction manifested as a shoulder of hexose or
uronic acid activity to the right of the void volume peak at around
fraction 24, and a third small fraction that peaked at fraction 42.
After ED a slight shift to the right is noted, with more hexose
activity found in later fractions (fractions 27-60) and a small,
though not well-defined peak at fraction 57. To demonstrate the
existence of two juxtaposed peaks of activity at or near the void
volume, a much smaller sample of KP2-0 (EtOH) EPS was applied to
the same column and the elution profile can be seen in Figure 23.
In Figure 23' the peak to the right of the void volume fraction was
somewhat more evident in the profiles before and after ED but in
contrast to the EPS from KP2 2-70, the HMW fraction was not
drastically diminished in magnitude after extensive ED.
The ability of ED to dissociate higher molecular weight KP2
2-70 EPS to that of lower molecular weight components argues for
the presence of electrophilic interactions between polysaccharide
strands. During ED it was found that as the mA increased across
the terminals, there was a concommitant increase in pH at the
cathode and a decrease in pH at the anode. The surge in mA during
156
157
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cr Ul
m
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Li-
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o CM
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o o
1
o GO
1
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1
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(|ai/Brr)3S0X3H
158
159
CP
3 U l CO
o X Ul X
FRACTION NUMBER
160
ED tended to produce high temperatures, so it was necessary to
change the DH^O in all chambers when a current of approximately 25
mA was reached. The column labeled "Wash" in Table 37 refers to
these DH^O changes as ED proceeded, the washes being numbered
progressively. The pH of the DH2O was measured, as well as was the
pH in the cathode and the anode, once this 25 mA current was
achieved, for each wash. The change in the pH in the cathode or
the anode was calculated by subtracting the pH of DHpO from the
resulting pH in either chamber. The ratio of the change in the pH
in the cathode to the change in pH in the anode (ApH cathode/ApH
anode) was then determined, and these results are listed in Table
37. From this table it can be seen that the highest ratio between
the pH changes at the cathode and the anode occured within the
first three washes, where over 2 pH unit changes have occurred in
the cathode for ewery one pH unit change in the anode. Washes 4 to
10 produced a ratio of 1.76, and washes 11-19 produced a 1.60 pH
change ratio, which were both significantly less in magnitude than
the ratio obtained in the first three washes, but were not
significantly different from each other. Therefore the very early
stages of KP2 2-70 (EtOH) EPS electrodialysis showed a greater
surge of cations to the cathode than the latter stages of ED in
relation to the surge of anions to the anode. In a total of 19
washes the overall ApH cathode/ApH anode value was determined to be
1.75.
During ED of KP2 2-70 (EtOH) EPS, the DH2O from both the anode
and the cathode chambers were pooled separately, lyophilized and
resuspended in concentrated volumes to test for certain divalent
161
Table 37. The pH Differential of the Anode and Cathode Chambers During Electrodialysis
ApH Cathode/ApH Anode^
2.16 + 0.15
1.76 + 0.06
1.60 +. 0.13
1.75 +0.22 (N=19)
The number of changes of DHpO in the electrodialysis chambers as electrodialysis proceeded.
ApH Cathode/ApH Anode; the ratio of the pH changes in the cathode chamber and the anode chamber with respect to the pH of dH^O (pH = 6.10). ^
Wash^
1 - 3
4 - 10
11 - 19
Total
162
cations [calcium (Ca"*" ) and magnesium (Mg"^^)] in the cathode wash
and phosphate (P0^~^), which is the major anion in the defined
medium, in the anode wash. Table 38 shows the results of the
quantitation of these ions in the first three washes during ED. It
was found that the major divalent cation dissociated from the KP2
2-70 EPS during the first 3 washes of ED was Mg" ^ (1648.8 yM),
+2 while 829.0 yM of Ca was determined from the same cathode sample. _3
No PO. was found in the cathode sample but 779.3 yM was found in
the corresponding anode sample after 3 ED washes. Comparatively
+2 +2 small amounts of Mg and Ca were found in the anode also (22 and
51 yM, respectively). These data indicate that approximately 3.2
moles of these divalent cations are retrieved at the cathode for _3
ewery 1 mole of PO^ retrieved at the anode.
+2 +2 Table 39 shows the quantities of Mg and Ca found in yg per
mg of ECPS for both the KP2 2-70 (EtOH) and the KP2-0 (EtOH) EPS
before and after ED at 400V. In this table it can be seen that the
vast majority of Mg is lost from either EPS fraction after ED at
+2 400V, while approximately two-thirds of the Ca is lost during the
_3 same interval. Table 40 shows the effect of ED on the PO^
concentration for both KP2 2-70 (EtOH) and KP2-0 (EtOH) EPS.
Nearly 50% of the PO." was found to be extractable from both KP2
EPS fractions after ED at 400V. Further ED at lOOOV removed 18%
more PO.'^ from the KP2 2-70 (EtOH) EPS and 32% more PO^'^ from the
KP2-0 (EtOH) EPS. Figure 24 shows a histogram of the effect of ED
on the concentration of PO "' [P04"^] in mM, both in the sample
(open bars) and in the anode (stippled bars) for KP2-70 (EtOH) EPS
at various intervals of ED. The cross-hatched bars reflect the
163
"°'^^ 22.0+0.3 51.0+18.4 779.3+2.80
Cathode 1648.8+14.7 829.0+7.1 ND"
ND; none detected.
164
Table 39. Effect of ED on the Quantity of Divalent Cations Found in KP2 EPS
EPS
KP2-70 (EtOH) Before ED
After ED^
KP2-0 (EtOH) Before ED
After ED^
yg Mg'*" /mg ECPS
8.44+1.07 (N=4)^
0.10+0.05 (N=3)
8.50+2.76 (N=4)
0.54+0.37 (N=4)
yg Ca'^^/mq ECPS
1.70+0.15 (N=2)
0.62+0.09 (N=3)
1.14+0.05 (N=2)
0.38+0.00 (N=2)
ED proceeded at 400V until no marked surge in mA was noted over a 30 min period.
'N equals the number of determinations made from two separate preparations.
165
Table 40. Effect of ED on the Quantity of Phosphate Found in the KP2 EPS
EPi yg P04"^/mq ECPS (N=2)^
KP2 2-70 (EtOH) Before ED 36.81 + 1.31
400V ED^ 17.83 + 2.41
lOOOV ED^ 11.05 + 1.64
KP2-0 (EtOH) Before ED 2 9 . 2 8 ^ 2 . 8 2
400V ED 15.69 +_ 0.58
lOOOV ED 6.24 + 0.13
N equals the number of phosphate determinations made on the same sample.
400V ED; Electrodia lys is at 400V un t i l no marked surge in mA was noted over a 30 min period.
lOOOV ED; Electrodia lys is at lOOOV as in footnote b.
166
167
( V ) (l"^/^iJLi)Sd03 i n ^ fO CVJ —
I I i I I
O
GO
1 r I "1—r ro OJ
(TV)(i^^)*'0<d
168
concentration of ECPS in mg/ml. The [PO^"^] in the sample before
ED (position A) was calculated to be 55.5 ymoles, but after ED at
400V (position B) the [PO^"^] in the sample diminished to 30.9
ymoles, while the [PO^"^] recovered in the anode equalled 19.9
ymoles (92% recovery of [PO^"^] from position A to position B).
After ED at lOOOV (position C), 15.4 ymoles of [PO^"^] remained in
the sample, while 9.1 ymoles were recovered in the anode (a
recovery of 79% from position B to position C). Finally, the ECPS
concentration in the sample remained essentially the same
throughout ED as seen in this figure. The results from this study
and the above data show that the EPS from two strains of KP2
contained appreciable amounts of both cations and anions even after
extensive dialysis, and the majority of the cations and anions
measured were extractable by the ED procedures, as shown both in
the concentrations of ions retained by the sample and the ions
recovered at the ED terminals.
The major difference between the two KP2 EPS was that the KP2
2-70 EPS was seen to readily dissociate into a LMW component after
ED while the majority of the KP2-0 EPS stayed in the HMW form even
after extensive ED. A study was then undertaken to see whether the
KP2-0 EPS could be dissociated into a lower molecular weight form
by the addition of sodium dodecyl sulfate (SDS). The KP2 (EtOH)
EPS was first electrodialyzed at 2000V to rid of all possible
hydrophilic associations of EPS due to inorganic cations and
anions. The electrodialyzed EPS was then subjected to boiling in
2% SDS for 5 min to disrupt noncovalent, hydrophobic interactions.
169
and applied to a Sepharose 2B column equilibrated with 0.01 M Tris,
pH 12, and 0.1% SDS. The elution profile obtained is displayed in
Figure 25, and fractions were monitored for both hexose and uronic
acid activity. Both the hexose and uronic acid profiles exhibited
two major fractions on S-2B; the HMW void volume fraction and a
second fraction immediately to the right of the HMW fraction, as
was seen in the profiles for KP2-0 EPS after ED alone (Figure 22).
A third and a fourth minor fraction can also be seen in this
profile and correspond proportionally to the same minor fractions
seen after ED alone (Figure 22). Although the two major fractions
produced on gel filtration after ED in the presence of SDS seem to
have been more clearly separated from one another than in the
profile obtained from ED alone, the differences are minor at best.
Thus, with the methods used in these studies to (1) remove all
dialysable ions that may contribute to the aggregation of the KP2-0
EPS and (2) to disrupt any noncovalent, hydrophobic interactions
between KP2-0 EPS polymers, it was found that no major shift to a
LMW form of ECPS could be achieved, although a slight shift to a
still rather high molecular weight fraction was evident.
The EPS from KPl-0 48h ethanol extracted supernatants was also
subjected to ED at 2000V to determine the effect of ED on the S-2B
elution profile of this material, which is shown in Figure 26.
Again, a relatively small quantity (1 mg total ECPS) was placed on
the column to avoid the coalescence of adjacent peaks (see Figure 7
for comparison). In both the profile before ED and after ED, a
number of symmetrical and rather evenly spaced peaks can be seen.
170
171
9 0^
7 0 -
E 50 C7»
3 Ul 8 30H X UJ X
20 30 40
FRACTION NUMBER
172
173
cr UJ 00
Z
< cr.
(J> 00 r^ c^ in
|iJU/&^3S0X3H
CM —
174
which do not necessarily correspond to the fractions seen in
earlier profiles (see Figure 7). Both profiles in Figure 26 have
retained a similar quantity of the HMW component of KPl-0 EPS,
though there appears to be a shift to the right of 4 fractions for
the ED profile. Most of the other fractions coincide as to
fraction number when comparing the two profiles in the LMW region,
except for the materials eluting at the far right of the profile
(between tube fractions 55 and 72). There were, however,
quantitative differences between many of the coincidental fractions
in the two profiles. For example, the fractions which eluted at
tube fractions 23, 27 and 34 for the ED profile were of greater
magnitude with regard to hexose than were the corresponding
fractions seen in the profile before ED, whereas the peaks of
hexose at tube fractions 38 and 43 were much larger in the profile
before ED than in the corresponding fractions after ED. There was
then an apparent shift of hexose from later to earlier fractions in
the LMW region as a result of ED, as well as an apparent shift of
the HMW fraction to a higher elution volume. There were also two
prominent peaks of hexose in the material after ED at tube
fractions 53 and 65 that were in contrast to the material before
ED.
Two different methods were utilized to ascertain the
contribution of hydrophobic interactions in the KPl-0 EPS, both of
which were based on the assumption that lipid groups are covalently
linked to either the ECPS or to the contaminating ELPS and that
these hydrophobic groups may tend toward micellar formation in
175
aqueous solutions (see Results, Section G). The first of these
methods was hydrolysis of KPl-0 EPS with 60% hydrofluoric acid (HF)
at 0 C, which has been shown to liberate acylglycerols and
diacylglycerols from the capsular polysaccharides of the Group B
meningococci, but leave the glycosidic linkages intact (37).
Figure 27 shows the elution profile on S-2B of KPl-0 EPS obtained
before and after HF .treatment, utilizing the EPS obtained from
dialyzed 48h supernatants. The profile after HF treatment is seen
here to be a more homogenous preparation of LMW EPS, with much of
the higher molecular weight hexose activity absent and a more
refined singular LMW fraction. The second method used to define
these putative hydrophobic interactions was that of treatment of
the KPl-0 EPS with 0.5 M NaOH (saponification). Figure 28 shows
the effect of saponification of KPl-0 (N) ECPS on the elution
profile obtained on S-2B. The profile obtained before
saponification contained a number of prominent peaks. After
saponification the vast majority of the hexose activity was found
to elute between tube fractions 32 and 50. Thus with HF treatment,
or with NaOH treatment of KPl-0 EPS, the fractions in the HMW
region were not as prominent and the LMW fraction apparently became
more homogeneous. It was also found that ED of the KPl-0 EPS,
followed by cetavlon fractionation, produces essentially the same
profile, with the LMW peak predominant (Figure 15). However, there
is a difference in the elution volume between the LMW fractions
from HF and NaOH treatment on the one hand and the LMW fraction in
Figure 15.
176
177
130
FRACTION NUMBER
178
179
FRACTION NUMBER
180
Further studies on the saponif icat ion of KPl-0 EPS, as well as
on various f ract ions of KPl and KP2 EPS from other sources,
revealed that f a t t y acids (FA) were being released from a l l of
these f rac t i ons , even a f te r the EPS was pre-extracted with organic
solvents to remove any noncovalently attached l i p i d s . The presence
of covalently l inked f a t t y acids was demonstrated by the i r release
from the EPS treated with 0.5 M NaOH, and the i r conversion to
v o l a t i l e methyl esters to be detected in gas l i qu id chromatography
(GLC). A known concentration of bacterial methyl ester standard
was run along with j<. pneumoniae EPS to quanti tate the to ta l amount
of f a t t y acid methyl ester (FAME) released by a known quantity of
ECPS (yg FAME/100 yg ECPS). Table 41 shows the results obtained
fo r the ethanol extracted ECPS from 5 strains of K pneumoniae. I t
can be seen from th is table that the KPl-0 variant produced ethanol
extractable material that contained s ign i f i can t l y more FA than did
i t s covar iant, the KPl-T s t r a i n . Among the KP2 (EtOH) EPS
f ract ions tested, the KP2 2-70 EPS and the KP2-0 EPS did not d i f f e r
s i gn i f i can t l y in the i r content of FA. However KP2 8052 EPS had
s i gn i f i can t l y less FA than did the EPS from KP2 2-70.
A s imi lar study was then performed on the HMW and LMW
fract ions from gel f i l t r a t i o n of both the KPl-0 and KPl-T EPS.
Various amounts of these f ract ions were saponi f ied, methylated and
assayed fo r the i r to ta l quanti ty of FAME on gas- l iqu id
chromatography. Table 42 shows the ra t ios of FAME to ECPS and to
ELPS in yg per lOOyg. The HMW samples from ei ther the KPl-0 or
181
Table 41. Quantitation of Fatty Acid Methyl Ester (FAME) Released from EPS after Saponification
FAME/ECPS^
6.13 + 1.45 (N=3)^
2.39 ± 1.61 (N=3) (p < 0.05)^
5.05 t 0.16 (N=3)
4.33 + 1.51 (N=2)
1.35 ± 0.54 (N=3) (p < 0.001)^
EPS; All fractions of EPS from the 5 strains tested were ethanol extracted from 48h supernatants.
FAME/ECPS: The amount of fatty acid methyl ester in yg found in 100 yg of ECPS as quantitated by the procedure of Blumencrantz et al. (7).
^N equal the number of trials performed, each from a different preparation of EPS.
Statistical analysis (Student's t test) between the mean values obtained for KPl-0 and KPl-T EPS.
^Statistical analysis (Student's t test) between the mean values obtained for KP2 2-70 and KP2 8052 EPS.
EPS^
KPl-
KPl-
KP2
KP2-
KP2
•0
•1
2-
-0
70
8052
182
Table 42. Quantitat ion of FAME Released from the EPS of KPl-0 and KPl-T Obtained from Gel F i l t r a t i o n on S-2B
Sample^ yg FAME/100 yg ECPS yg FAME/100 yg ELPS^
KPl-0 HMW 5.49 + 2.91 (N=4)^ 13.11 + 6.95 (N=4)
KPl-0 LMW 0 . 2 7 + 0 . 0 8 (N=4 ) 4.92 + 1.40 (N=4)
KPl-T HMW 3 . 7 7 + 1 . 3 3 (N=3) 8.91 +3.14 (N=3)
KPl-T LMW 0.18 + 0.02 (N=3) 6.25 + 0.54 (N=3)
^The HMW and LMW fract ions from KPl-0 and KPl-T obtained from gel f i l t r a t i o n on S-2B.
The number of yg of f a t t y acid methyl ester obtained per 100 yg of ECPS.
The number of yg of f a t t y acid methyl ester obtained per 100 yg of ELPS.
The number of determinations of FAME performed on two separate preparations.
183
KPl-T strain was seen to contain significantly more yg FAME than
the LMW samples per lOOyg ECPS (p < 0.02 and p < 0.005,
respectively). Essentially 20 to 25 times the amount of FAME per
lOOyg ECPS in the LMW fractions was found in the HMW fractions. In
contrast, the differences in the amount of FAME determined per
lOOyg of ELPS in the HMW versus the LMW fractions of these two
organisms was not nearly so marked. There were no significant
differences seen in the yg FAME/1 OOyg ELPS between any of the
samples, though there was a trend toward higher values being found
in the HMW component. These data suggest that the FAME content of
these samples was primarily related to the amount of ELPS present
in the fractions, rather than to the amount of ECPS.
The last structural study in this section involved growing the
KP2 2-70 strain in the same defined medium, with the exception that _o
the PO. buffer was eliminated and replaced by a 10 mM Hepes 3
buffer. The final molarity of PO. in the new medium (DMH) was
one-thousandth that of the old medium. Figure 29 shows the elution
profile of KP2 2-70 (EtOH) EPS obtained from growth in DMH on
BGA-150m. It can be seen in this profile that a HMW, void volume
fraction was present (tube fraction 18) even under conditions of
low [PO.''^] in the medium. The LMW fractions differed somewhat
from that seen for this EPS grown in the phosphate-buffered defined
medium (see Figure 19) in that there were two prominent fractions
(at tube fraction 24 and 33) rather than one LMW fraction. Thus, _3
even with the [PO. ] at 3 orders of magnitude less in
concentration in the defined medium, the HMW component was
produced.
184
185
FRACTION NUMBER
186
Gel Immunodiffusion Studies for Identification
and Quantitation of ECPS Produced
by K. pneumoniae
Ouchterlony double diffusion studies were performed in gels
for the various EPS fractions obtained from ion exchange for a
number of KPl and KP2 strains. 1 mg of lyophilized samples in one
ml DH2O were prepared for these studies. One precipitin line
appeared for each of the EPS fractions and each was shown to be
immunologically identical to one another. Therefore, the acid and
the neutral fractions were identical serologically within a given
strain and identical between strains of the same serotype. The
same patterns were seen for the acid and neutral EPS isolated from
three different strains of KP2.
When various fractions of KPl EPS were tested by RIE using
type-specific antiserum (AB), all of the fractions tested, except
for the HMW fractions, appeared to contain a mobile component that
reacted with AB with a peak as far as 2 to 3 or more cm above the
well, whereas the fractions, containing only HMW EPS at the same
ECPS concentration show some difficulties in entering the gel, and
show distortion of the materials that did enter. This was
especially true for the KPl HMW fraction, where it was seen that
only a small portion of EPS had entered the gel. Table 43 shows
the calibration of the standard curve obtained in RIE for 1:2
dilutions of both KPl HMW and KPl-O LMW EPS. The standard curve
for the LMW EPS produced a straight line with a correlation
coefficient(r) of 0.94, while the curve for the HMW fractions did
187
Table 43. Standard Curve for the RIE^ of KPl-0 HMW and KPl-0 LMW EPS
Sample
KPl-0 HMW
KPl-0 LMW
ECPS(yg/ml)
628.
314.
157,
78.
1081.
540.
270,
135,
.5
,3
.1
.6
,3
.6
.3
.2
Rocket
1.
1.
1.
0,
4.
3.
3.
2.
Hei
,10
.05
.12
.74
,25
,80
,00
.45
ght (cm)
^Electrophoresis took place at 4-6V/cm for 3h in 0.2% agarose impregnated with anti-KPl antiserum. Ten yl of sample were added to each wel l .
188
not assume a straight line function (r=0.57). Moreover the
majority of the HMW EPS precipitated by AB at about 2mm above the
well in all four dilutions, and this EPS was believed not to have
entered the gel, but was rather a distortion of the sample well.
It was found that the ECPS present in the serum of rats
infected- with KPl-0 was in the HMW form, or of even a higher
molecular weight form, since an immunological precipitate was seen
around the upper periphery of the sample well after RIE, but no
ECPS appeared to enter the gel. After a number of unsuccessful
manipulations, the quantitation of KPl EPS in rat sera was
abandoned. However, RIE was used successfully to quantitate the
ECPS in rat sera infected with the KP2 2-70 strain. Out of the six
sera studied, only two were found to contain measurable quantities
of ECPS, and one of these sera were selected to perform
quantitative RIE. This serum sample was isolated from a rat which
was found to have nearly 1 x 10 bacteria per total lung
homogenate; the highest level of organisms found for all the rats
in this particular study. The other rat serum that had measurable 9
ECPS came from a rat that had approximately 5 x 10 bacteria per
total lung homogenate. The rats with the next 3 highest
concentrations of bacteria in their lungs (between 1.2 and 2.4 x
10 CFU per total lung homogenate) were found not to have
measurable ECPS in their sera by RIE.
Table 44 shows the RIE rocket heights obtained on the serum
from the rat that had the highest CFU titer of the KP2 2-70 strain
189
a Table 44. RIE of Standard Concentrations of KP2 2-70
ECPS and Serum from an Infected Rat
Sample
KP2 2-70 (A)^
KP2 2-70 HMW
ECPS (yq/ml)
756.9
378.5
189.2
1614.8
807.4
403.7
201.9
Rocket Height
2.25
1.55
0.45
3.30
2.27
2.00
0.70
Rat Serum 820" 2.50
^Electrophoresis took place at 4-6V/cm for 3 h in 0.2% agarose impregnated with anti-KPl antiserum. Ten yl samples were used,
The acid f rac t ion of KP2 2-70 EPS from ion exchange chromatography.
^The HMW f rac t i on of KP2 2-70 EPS from gel f i l t r a t i o n chromatography.
Calculated from the standard curve.
190
in its lungs and the rocket heights produced from 2-fold dilutions
of KP2 2-70 (A) EPS, as well and the rocket heights obtained from
2-fold dilutions of KP2 2-70 HMW EPS. In Figure 30 a graph was
drawn to show the standard curve produced by the rocket heights in
cm against the quantity of ECPS that produced these rockets. Five
of the seven data points obtained conformed to a straight line,
which was then used to calculate the amount of ECPS in the rat
serum. The dotted lines in the graph depict the rocket height (2.5
cm) of the unknown sample and the calculated yg of ECPS. Thus this
rat serum was found to contain approximately 820 yg ECPS per ml.
Radial immunodiffusion was also performed to quantitate the
ECPS found in the sera of these rats infected with KP2 2-70. Table
45 shows the zone diameters of the serological precipitin reaction
obtained both for the known KP2 2-70 ECPS standards and for the
sera from the same rat as used above. By these methods the
concentration of ECPS in this rat serum was calculated to be 496.8
yg/ml. So by the two methods used in these studies, the level of
KP2 2-70 ECPS in the serum of a rat infected with high levels of
KP2 2-70 in its lungs, was found to be between 500 and 800 yg/ml.
However, in four of the five remaining sera obtained from animals
less moribund from the infection, no ECPS was measurable. There
was however, an apparent ring of precipitation around the periphery
of the wells and within the gel well itself for most of these sera.
Whether or not these precipitation reactions were immunological was
not readily demonstrable and, therefore, not determined.
191
192
0.5 1.0 1.5
Peak Height (cm)
2.0 2.5
193
Table 45. Quantitation of KP2 2-70 ECPS in the Serum of Infected Rats by Radial Immunodiffusion
Sample^
KP2 2-70 (A)"
yg ECPS
1914.6
957.3
478.65
239.33
Zone diameter
9.15 + 0.07 (r
8.15 + 0.49
7.00 + 0.14
6.30 + 0.42
Rat Serum 496.8 6.95 j 0.76 (N=4)^
^10 yl of sample were placed in the gel wells and incubated overnight for 24h.
Measurement of the immunologic precipitin zone around the wells at 24h in mm (N=2).
^The acid fraction of KP2 2-70 EPS.
Correlation coefficient for the standard curve.
^N equals the number of determinations made.
194
Survey of the Outer Membrane Proteins
of K. pneumoniae
The Triton insoluble outer membrane proteins (OMP) from
strains of both serotypes 1 and 2 were prepared and examined by SDS
polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing
8.0 M urea. Figure 31 shows the Coomassie blue-stained OMP from
the KPl strains. Lanes A and B in Figure 35 are the OMP profiles
for KPl-0 and KPl-T, respectively. Lanes C and D are OMP profiles
from revertants in the KPl-O and KPl-T population, respectively
[Lane C is from a KPl-T like strain isolated from the KPl-0
population (KPl-Or), and Lane D is from a KPl-0 like strain
isolated from the KPl-T population (KPl-Tr)]. Lanes E and F are
OMP profiles from a large and a small capsular variant from the KPl
2-70 population, respectively. No significant differences could be
detected among the profiles of the six strains examined. Lane G
shows, for reference, the OMP profile of i- coli strain CS138,
which had been induced for the LamB protein (3). There are two
major protein bands from the profiles of the six KPl strains that
are apparently analogous to two major OMP of E_. coli namely the
OmpC and the OmpA proteins.
Figure 32 illustrates a similar study on the OMP from three
KP2 strains. Lanes A and D are profiles from KP2-0, with lane D
containing twice the volume of sample as Lane A. Lanes B and E are
OMP profiles from the KP2-T strain, and Lanes C and E are OMP
profiles from the KP2 2-70 strain, again with double the volume of
sample in the latter profiles. The OMP composition was
195
196
A B C D E F G
Lam B a & OmpC
OmpF ^ ^ H ^ O m p A
197
B rm •»•-•
D
i^,v •• MiM
198
4i# •-.^if'
j^sii-m-
.f-<^'^
Sft?".
199
fundamentally the same for these three strains also, and these
strains appear to possess the £. coli OmpC and OmpA protein
analogues, as did the KPl strains. A minor difference in the
profiles was observed in the high molecular weight region of the
KP2-T strain compared to that of the KP2-0 and KP2 2-70 strain,
namely that a distortion of the p.rotein bands was observed. This
distortion is best visualized in Lane E in Figure 32. It is known
that this high molecular weight region of the gel typically
contains the largest mass of LPS and these types of distortions are
usually attributed to contamination with LPS. It is then likely
that the KP2-T sample contained more LPS than did the other
samples.
CHAPTER IV
DISCUSSION
Classically the histologic and pathologic features of
Klebsiella pneumonia in humans include a massive, confluent lobar
consolidation consisting primarily of PMN, a voluminous edema, and
abscess formation with massive cavitation. Unfortunately, at least
for the development of animal models, there exists a wide
diversity of clinical manifestations of this disease process.
Several classifications have been proposed which distinguish
between an acute and chronic pneumonia pattern (29,34,43,70), a
primary versus secondary (suprainfecting) pneumonia (34,43,70), and
endogenous or epidemic sources of the organism (56,65). The
progression of the disease and its prognosis are primarily related
to age and predisposing variables (77). Since most Klebsiella
lobar pneumonias are seen in debilitated, middle-aged males (43,
65,77), it is difficult to establish a model that closely
approximates the human condition, especially when the large numbers
of predisposing factors are considered. The rats used in the
present study were healthy, young males more likely to effectively
combat the experimental infection than the aged and debilitated
human patient. This may explain the relatively low mortality and
the greater chronicity of infection seen in this rat model.
Nevertheless, in the present study, we were able to establish a rat
model which displayed the classical symptoms for J<. pneumoniae
pneumonia found in humans.
200
201
Berendt et al. (4) produced a bronchopneumonia in rats using
intranasal inoculations of 5 x 10^ l<. pneumoniae (strain A-D). The
authors eventually abandoned their rat model, concluding that the
squirrel monkey provided a more satisfactory experimental model for
lobar pneumonia. This non-human primate model allows measurements
of clinical signs that a rodent model does not afford, such as
fever, respiratory rate, and throat cultures (5). However, the
squirrel monkey pneumonia pattern mimics only the acute form of the
disease. The rapidity with which death occurred and the low
frequency of abscess formation severely restricted the utility of
this.model. Moreover, in their discussion (4), the authors noted
the economic, practical and statistical advantages of using a
rodent model to study this type of infectious process.
Sale and Wood (71) reported the production of a lobar
pneumonia in rats. They described a highly acute infection, with
the majority of their rats succumbing to their pneumonia by day 3
post-exposure. Again, their model simulated, at best, the acute
pattern of Klebsiella pneumonia in humans. The high mortality
encountered by these researchers was most likely due to the
administration of mucin into the lungs as an adjuvant, a procedure
that other authors felt could have "profound effects" on the
experimental animal (4).
Establishing the threshold of infection in the rat model at a
4 TBC of 5 X 10 was based on several meaningful observations.
First, it was at this approximate titer when morphological changes
in the lungs were seen. Berendt et al. (4) reported that lysozyme
202
levels did not become elevated until the number of bacteria in the
lungs reached four to five logs. Serum lysozyme is a convenient
assay for determining the extent of infection because it reflects
the appearance, frequency and severity of pyogranulomatous lesions
(11). Second, with only a few exceptions, the present study showed
an all or none response to the j<. pneumoniae challenge, with rat
lungs containing either well over this 5 x 10^ CFU threshold or
well below it. Third, changes in lung weight also supported a TBC 4
of 5 X 10 CFU as the threshold of infection. None of the rats
with a lung content of under 5 x 10 bacteria had any marked
elevation in lung weight.
In the present study we were able to produce a chronic lobar
pneumonia in rats without the aid of adjuvants. This model allows
for the colonization and infection of the rat lung by j<. pneumoniae
for at least 28 days. Previous attempts at establishing an
experimental paradigm for Klebsiella respiratory infections have
disregarded the infectivity or virulence of the bacterium. In this
report, two serotypes and variants within these serotypes were
examined for their ability to produce a chronic lobar pneumonia in
rats, and a comparision was made to their virulence in mouse
lethality tests. The results show how important it is to know the
pathogenic nature of the organism before model construction is
possible.
A relationship of both practical and economical significance
developed from the comparison of the ability of a given strain to
infect the lungs of rats and to be virulent in the mouse model. A
203
positive correlation of 0.95 was obtained by comparing the four
strains in which both a quantifiable LD^Q and ID^Q were determined.
In essence it is possible to gain insight into the ability of a
strain of l<. pneumoniae to produce an infection in the lungs of
rats by performing the simpler and cheaper mouse virulence assay.
If this relationship holds true for other strains of K,. pneumoniae,
performing a mouse virulence test, obtaining and LD^Q and then
adding 1.7 log-jQ units to this LD^Q calculation should approximate
the IDrQ for that particular strain.
It was seen that in 3 of the 4 strains tested, a large and a
small encapsulated variant could be isolated by various means. Two
of these strains revealed prominent variants without manipulation
while the third strain required passage through mice to-enrich for
a large encapsulated variant. These variations in capsule size
within a defined population have been described elsewhere for J<.
pneumoniae (26, 46), though little is known about the reasons for
the existence of these variations. The smaller capsule size has
been suggested (46) as the more stable structure, and it may be
that the large capsule (or what could be an unregulated synthesis
of CPS) is a mutation occurring at low frequency. Passage through
mice, or infection in general, gives a selective advantage to the
large encapsulated form and thereby enriches its presence in the
population. Since most of the strains in this study were isolated
from human disease, it is not surprising that the large
encapsulated variant was seen to be predominant in 2 of the 3
strains in question, both of which were lung isolates. The KPl
204
ATCC 8047 strain manifested two variants with respect to capsule
size, one which had a TD of 5.6 ym and the other a TD of 2.5 ym.
The KP2 ATCC 29011 strain exhibited one variant with a TD of 2.5 ym
and the other with a TD of 1.5 ym. The KP2 strain then produced a
large encapsulated variant which had the same TD as the small
variant in the KPl strain. The KPl CDC 2-70 strain, which was the
remaining strain shown to produce two capsular variants, had a TD
of 2.2 ym. It was seen, however, that a large encapsulated variant
appeared under India ink preparations at a frequency of
-4 approximately 10 , which had a TD as large as the KPl-0 strain
(5.6 ym on the average). After mouse passage, many of these large
encapsulated variants were isolated from the KPl 2-70 population.
It was seen then that the KPl strains in these studies generally
were capable of producing much larger capsules than the KP2
strains.
These variations in capsulze size within a given bacterial
population were considered to be isogenic variations for a number
of reasons. First of all, these variations were seen to arise
commonly from a single colony. Secondly, the biotypes of these
variants, determined on API-20E strips, were found to be identical,
as well as was their OMP profiles as seen in SDS PAGE gels. If
these variants in capsule size were really a mixture of
contaminating j<. pneumoniae, one would expect to see several
serotypes represented, since there are 72 known serotypes of this
organism. On the contrary, all variants possessed the same
serotype within a given population, and the ECPS isolated from
205
these variants exhibited immunological identity in gel
immunodiffusion. Therefore, it is likely these variants arose from
a single strain and are isogenic with respect to capsule
production.
The KP2 2-70 strain was not seen to produce two separate and
distinct variants in capsule size even after extensive
manipulation, which included both mouse passage and low speed
differential centrifugation. Both manipulations originally were
able to separate large and small encapsulated variants, but this
distinction was again lost on subsequent plating. An increase in
capsule size for this strain was also seen during culture,
exhibiting a TD of 2.5 ym at 18h growth and a TD of 3.3 ym at 48h
growth in defined medium. It was also noted that the KP2 2-70
strain produced rather small (1 mm), nonmucoid colonies at 18h on
TSA, and contained organisms possessing a rather homogenous TD of
2.5 ym. On the other hand, plating these same organisms on
nutrient agar produced a relatively large (3-4 mm) and mucoid
colony type at 18h, with organisms possessing an assortment of TD
ranging from 2.5 to 5.0 ym. This phenomenon was not seen for any
of the other strains in these studies. The KP2 2-70 strain then
has the peculiar ability to regulate its capsule diameter with
changing environmental influences. This is further substantiated
by the changing kinetics regarding the production of ECPS by this
organism in defined medium. At up to 18h of culture, comparatively
small quantities of ECPS were produced, even though the stationary
phase of growth had begun approximately 10 hours earlier. After
206
18h the rate of production of ECPS increased tremendously. It is
possible that there were certain components in the medium that
regulated the production of capsule and, that by 18h of culture,
these components were at low enough concentrations to permit
deregulation of CPS production. This is also supported by the fact
that nutrient agar is of a much lower ionic strength than TSA and
seems to allow for this deregulation of CPS production much
earlier in culture. By 48h the colonies of KP2 2-70 on TSA became
mucoid and large, and exhibited the large assortment of capsular
types seen on nutrient agar at 18h.
Although the identity of these regulating components for CPS
production by KP2 2-70 have not been determined, it is believed
that metal ions, such as magnesium and calcium, may play a role.
It has been demonstrated that these ions are important for the
stability and organization of the outer membranes of gram negative
bacteria (16). Divalent cations are thought to play a role both in
reduction of change repulsion between highly anionic polysaccharide
molecules and are thought to bridge adjacent LPS molecules and to
link LPS with membrane proteins (16). Chelating agents have been
shown to effect the release of up to 50% of the LPS from the whole
cells (49). The CPS of J<. pneumoniae has been shown in this study
to be complexed with divalent cations. When these metal ions
attain a low concentration in the medium, much of the CPS (and LPS)
may begin to loosen from the outer membrane and exude into the
growth medium. The loss of these polymers from the cell may
trigger a constitutive response of CPS and LPS production to
207
replace these lost surface polymers. The end result manifests as a
larger complex of CPS surrounding the bacterium, coupled with a
higher rate of escape of CPS into the medium.
Although the KP2 2-70 strain seems to be regulated and,
therefore, conservative in CPS production, other strains seem to
regulate CPS production by producing low levels of CPS and are not
inducible during the same conditions that affect the KP2 2-70
strain. Alternatively these other strains may undergo low level
mutations for high capsule production, which are then selected for
under the right conditions, such as during infection. Both the
variations in phenotypic expression of the KP2 2-70 strain and the
genotypic mutational variations of other K_. pneumoniae for capsule
production may then serve the same requirement for these cells,
that is the need to produce extraordinary quantities of both
capsule and slime (ECPS) under certain conditions.
It is plausible that this high rate of CPS production plays
some functional role for K_. pneumoniae, otherwise the metabolic
expense would seem too costly. In light of the evidence that much
of this CPS is found in the extracellular milieu, it is suggested
that the ECPS also functions for the benefit of the organism in
some manner. In a more static environment, as opposed to cultures
shaking at high speeds, the ECPS may exist as a large extension of
the bacterium and may surround huge numbers of bacteria in the form
of microcolonies. These static conditions are probably found in
the lungs of infected animals. If the capsule is envisioned as a
large chelating complex for divalent cations, these large
208
extensions of the capsule (microcolonies) may not only provide
better protection against phagocytosis, but may also serve as an
ion escalator for certain cations needed for growth. Since it has
been shown by Fukutome et al. (32) and others (31, 45), that the
primary host defense against I<. pneumoniae involves the production
of opsonic AB specific to the capsular type, it is reasonable to
view these putative microcolonies of J<. pneumoniae as being more
resistant to phagocytosis than an individual bacterium, even in the
presence of AB to the capsule. These arguments then consider
freely soluble ECPS to be, in part, an artifact of rotating
cultures, and call for a distinction in capsular organization
between the in vivo and in vitro situation. It has been shown by
Pollack (66) and in the present study, however, that circulating
cell free CPS can be demonstrated in the sera of infected animals,
especially in cases of severe infection.
In the OPA studies performed, we were able to support the
hypothesis that PMN will not phagocytose l<. pneumoniae in the
absence of AB. In view of the theories posited in the Introduction
as to the possible functional roles of the ECPS in virulence, the
OPA studies have apparently shed some light as to which of these
functions may apply. It was shown .that the KPl 2-70 strain was
able to thrive significantly better in the OPA in the presence of
AB to the type 1 capsule, when homologous KPl EPS was present
rather than when twice the quantity of heterologous KP2 EPS was
present. Although the KP2 EPS at these dosages imparted much more
viscosity to the OPA, it did not help the KPl 2-70 organism survive
209
any better than control trials, which contained no EPS. In the
reverse experiment KP2 EPS allowed the KP2-0 organism to grow
significantly above that of either KPl EPS treated or control
assays. Therefore, the notion that EPS influences the ability of
PMN to phagocytose K_. pneumoniae due to its viscosity is not
supported in these studies. There also appears not to be any
immediate biological effect of the EPS on the WBC. However, the
type-specific, homologous EPS allowed these organisms to survive
the effects of 90% serum, WBC and AB to the capsule significantly
better than both controls and heterologous EPS treated trials.
Therefore, it is most likely an AB neutralization phenomenon taking
place. It was also found in the OPA that the large encapsulated
KPl-0 variant is not better protected from phagocytosis under these
conditions than is the small-capsuled KPl-T variant. Whether the
same level of protection holds true in vivo is an entirely
different question.
It was determined in these studies that the production of
ECPS, ELPS and the size of the capsule were all positively related
to the ability of an organism to infect the lungs of rats or to
kill mice. This relationship was greatest for the cell associated
capsule size parameter, but was also seen to correlate highly with
ECPS and ELPS production. These correlations were, however, much
greater when examined within, rather than among serotypes. It was
also apparent that even within a given serotype, a strict
correlation between total polysaccharide production and virulence
does not hold true, nor does the positive relationship always hold
210
between the capsule size and ECPS production. A primary example of
deviations from the rule involves the KPl 2-70 strain, which
produces more ECPS per cell in defined medium than does the KPl-T
strain, but harbors a smaller capsule and is at least two orders of
magnitude less virulent in the mouse model. Although KPl 2-70 was
not seen to be sensitive to 90% serum and not subject to in vitro
phagocytosis by PMN in the absence of AB, it may be that the
antiphagocytic properties of the relatively small capsule of this
strain are more readily overcome by host defenses in vivo. Yet
this organism was seen to produce more ECPS than the large
encapsulated KPl-T strain. It may be that it is not possible to
compare unrelated strains, regardless of a common serotype, as to
their capsule to ECPS ratio. A strict relationship between these
two parameters of CPS production may only apply within isogenic
pairs, and may be reflective of other surface components (LPS, OMP)
that are intimately involved in the stabilizing forces of the outer
membrane of K_. pneumoniae. It was seen, however, that there were
no remarkable differences in the OMP profiles of these organisms.
This strict capsule to ECPS ratio was observed, however, for the 4
variants obtained from one KP2 strain in a study by Ehrenworth and
Baer (28). Duguid and Wilkinson (21) also demonstrated that, with
varying culture conditions, the capsular and slime (EPS)
polysaccharide generally increased or decreased together, though
not in strict proportion. Thus a general relationship does exist
within serotype as to capsule and ECPS production but only in the
isogenic situation does it appear to strictly apply.
211
The capsule size and the quantity of ECPS produced by a given
strain are considered to be manifestations of the same phenomenon.
It appears to be the actual rate of total CPS production which
distinguishes a small from a large capsular variant, and a high
from a low ECPS producer. • The capsule is viewed as a dynamic
intermediate between CPS production and release into the growth
medium. A high CPS producer may harbor a large cap-sule due to the
rate of CPS production being faster than the rate of release into
the medium.
If the association between the capsule size and ECPS
production holds true, then the question still remains as to
whether the capsule or the ECPS or both are responsible for the
virulence of j<. pneumoniae. It is well established that the
presence of a capsule is essential for this organism to be
pathogenic (28, 32, 45). A relationship between the rate of
production of CPS and the degree of pathogencity has been shown
with J<. pneumoniae (28) as well as with Streptococcus pneumoniae
(55, 74). These studies suggest that the antiphagocytic properties
of the capsule increase as the cell-associated capsular material
increases in volume. There also exist acapsular variants of l<.
pneumoniae that produce copious amounts of ECPS but are avirulent
(28) as was also seen with the KP2 8052 strain utilized in this
study (L-D-Q > 7.3 x 10 ). Although it is generally true that when
a large capsule is being produced, large quantities of ECPS are
being released, the reverse phenomenon is not always true, and high
ECPS producers exist which are acapsular. These latter variants
212
These lines of evidence support the role of the capsule as a
necessary structure for virulence, but do not rule out the ECPS as
a virulence factor when a capsule is present. It was found in the
present study that as low as 25.5 yg of KPl-0 (A) ECPS could
enhance the virulence of KPl-T significantly over control values as
could approximately 400 yg of KP2 2-70 ECPS. It was also seen that
in one of the rats infected with the KP2 2-70 strain, between 500
and 800 yg of ECPS were detected per ml of serum. These values are
also in line with the amount of KP2 EPS used by Batshon et al. (2)
to suppress the immune response to this antigen. KPl EPS was shown
also to be present, but not quantifiable, in the serum of rats
infected with KPl-0 or KPl-T, since the EPS was presumably in a
high molecular weight form and was not mobile in RIE or RID assays
even in 0.2% agarose. The KPl EPS was also shown to suppress AB
production to the capsule at a dosage of between 10 and 100 yg per
mouse (58). These values are also in line with the virulence
enhancement data obtained in the present study. It is therefore
conceivable that the capsular and slime polysaccharide work
together to enhance the pathogenicity of j<. pneumoniae; the capsule
providing protection against nonspecific immune defense postures of
the host, and the ECPS possibly enhancing the antiphagocytic
potential of the organism by neutralizing AB, by suppressing the
antibody response via a tolerance phenomenon, and by affecting the
proper functioning of the macrophage. The effect shown by Yokochi
et al. (85, 86) on the macrophage could also explain the tolerance
phenomenon in that the poorly functioning macrophage may not be
213
processing the ECPS to present it to B-lymphocytes for AB formation
to occur. Unfortunately due to the contaminating LPS in the above
preparations, the notion that the LPS may be responsible for these
effects can not be ruled out. Macrophages have been shown to be
particularly sensitive in vivo to LPS administration (81). In
fact, from the results on virulence enhancement described in the
present work, the LPS may be the virulence component in the studies
from these other investigators.
In regard to the associations that hold the CPS to the cell
wall, it is believed that the LPS is intimately involved for a
number of reasons. First it was seen that both the ECPS and ELPS
are released together in a consistent ratio among all strains
within a given serotype. This ratio of ELPS to ECPS was constant
within serotype and the KPl strains were found to release nearly
twice the quantity of ELPS to ECPS as did the KP2 strains. These
stable proportions of ELPS to ECPS within serotype may then be a
reflection of the actual surface composition of these polymers.
Electron micrographs of these bacteria support this idea, since the
extracellular capsular materials were seen to slough off of these
bacteria in large masses (Fig. 5). If the KPl strains actually
have twice the quantity of LPS on their surface as the KP2 strains
this may explain why they were seen to produce much larger
capsules, since it is possible that the CPS is anchored to the
outer membrane in association with LPS. An alternative explanation
for the larger capsules seen among the KPl isolates has to do with
the extra negative charge per repeat unit of the CPS polymer
imparted by the pyruvyl linkage (30). This extra anionic component
214
may increase the adhesiveness of the CPS to itself, thereby
allowing for greater aggregation.
Secondly, the ECPS and ELPS were seen to retain their
association and to co-purify together, and were shown in gel
filtration and in gel immunoelectrophoresis studies to exist in a
highly aggregated form. In gel filtration both the KPl and the KP2
EPS aggregates eluted at the void volume of the column, so no
molecular weight differences could be discerned by these methods.
However, in the RIE and RID studies, the HMW aggregates from the
KPl strains were noticeably larger than the HMW EPS from the KP2
strains. The HMW aggregates from the KPl strains also contained at
least twice the quantity of ELPS as did the HMW KP2 EPS. It is
believed that the ELPS functions to hold together these aggregates
and is responsible for the larger size of the KPl HMW EPS.
Finally, it was shown that the EPS from the acapsular KP2 8052
strain contained significantly less fatty acids than the KP2 2-70
EPS. As the quantity of FA was considered to be related to the
ELPS content of the samples tested, it is apparent then that the
KP2 8052 EPS contains significantly less ELPS than the KP2 2-70 EPS
or, alternatively, that the KP2 8052 ELPS contains significantly
less FA per polymer than does the KP2 2-70 ELPS. The latter
conclusion is supported by the fact that the KDO content in yg per
mg of ECPS was quite similar. In any case the KP2 8052 strain
apparently has one fourth to one-fifth the FA content in its
extracellular polysaccharides as do the polysaccharides of the
encapsulated KP2 strains. If the extracellular polysaccharides are
215
truly reflective of the surface composition on the cell wall, it is
suggestive that acapsular, mucoid strains are the result of
anchored LPS polymers on their surfaces. In summary, LPS may be
responsible for holding the apsule together on the cell surface due
to the intimate association seen between CPS and LPS polymers, due
to the constant ratios seen within serotype in the extracellular
material, and in light of the data obtained from an acapsular
variant.
The ECPS:ELPS complex is felt to be held together not only by
ionic interactions in the form of salt bridges between anionic
components of the polymers, but also by hydrophobic interactions
imparted by the lipid A of the LPS and possibly by a lipid terminus
on the CPS polymer, as was shown for £. coli and meningococcal ECPS
(37). These aggregates were seen to dissociate for the KP2 2-70
EPS after ED only, which argues that the predominant association
between these polymers is that of salt bridging. However, for the
KPl-0 EPS the aggregates did not dissociate after ED alone and were
found to require an additional cetavlon extraction step or
treatment with either 0.5 M NaOH or 60% HF at 0°C, both of which
are known to remove covalently linked fatty acid esters. Cetavlon
is a cationic detergent and is known to separate LPS from the CPS
of K_. pneumoniae (72). Cetavlon alone, however, was not able to
disrupt the KPl-0 EPS aggregates as seen on gel filtration.
Therefore it is believed that both hydrophilic and hydrophobic
interactions are dominant in the HMW EPS of KPl-O. The KP2-0
strain, on the other hand, produced a HMW aggregate that was not
216
seen to dissociate to any appreciable extent after both ED and
cetavlon extraction or after both ED and boiling in SDS. The
possibility of covalent linkages in the KP2-0 EPS is suggested. It
has been demonstrated, for at least the KPl-0 and the KP2 2-70
EPS, that ED serves as a powerful tool for the purification of
these polymers.
The LMW forms of EPS from either the KPl or KP2 strains were
shown to contain 50% or less ELPS than their respective HMW forms.
We do not believe that the ECPS and ELPS in the LMW regions of gel
filtration are linked together in any regular fashion, since these
LMW samples exhibit a great deal of variability in their ELPS
content. It was possible to remove virtually all ELPS from the LMW
EPS of KPl-0 by ED and cetavlon extraction. The LMW ECPS and ELPS
are probably not efficiently separable on the gel filtration
resins that were used in the earlier studies (S-2B and BGA-150m).
The utilization of a P-300 column was shown to produce better
separation of ECPS and ELPS (Fig. 16).
The OMP profiles of K.. pneumoniae were unremarkable as to
their differences between high and low CPS producers. This is in
contrast to was seen in the OMP profiles of different isolates of
£. coli, where marked variation from strain to strain is the rule
(44). Also, in E_. col i, there was demonstrated a new OMP that was
associated only with encapsulated organisms (63). The OMP profiles
of K_. pneumoniae did not show these differences, but did reveal an
interesting phenomenon that distinguished high from low ECPS
producers, especially in the KP2 group. A greater distortion of
217
OMP bands in the HMW region of the gels were seen in the OMP
profiles of low ECPS producers as compared to high producers. This
distortion is usually attributable to high LPS levels in the
Triton-insoluble membrane preparations. If, for instance, KP2-T
actually has much more LPS on its surface, it may be that LPS
replaces the CPS in low producers or nonproducers of CPS.
Therefore CPS and LPS may be freely exchanged for one another on
the cell surface in order to fill any gaps. If the CPS contains a
lipid terminus, it may utilize the same space in the outer membrane
as does LPS for anchorage. Yet if the lipid terminus is absent in
J<. pneumoniae, it may be anchored through electrophilic (and
possibly covalent) interactions with LPS. Electrodialysis of whole
organisms at 2000V did not reveal any differences in the capsule
size of the KPl-0 strain and allowed for the release of less than
1% of the ECPS that was obtained in the cultural supernatant.
Therefore a hydrophobic linkage is more than likely present.
In light of the evidence obtained on the quality and quantity
of undialyzable inorganic ions attached to the KP2 2-70 EPS, a
hypothetical structure was drawn to explain these associations, as
can be seen in Figure 33. Two ECPS strands are here held together
by magnesium phosphate bridges, thus forming a highly stable
complex through structural complementarity. The negatively charged
uronic acid moiety in the repeating tetrasaccharide of the ECPS
polymer may orient at precise angles from the next uronic acid in
the same chain, thereby allowing for three dimensional
electrophilic interactions. Uronic acids lining up in the same
218
219
0
I-MG-O-P-O-MG
0 6'
0
•MG-0-P-O-MG l l
OUTER
ME.MBRANE
220
plane may form a ladder-like structure with another juxtaposed ECPS
strand, due to these putative divalent cation phosphate bridges.
In such an arrangement, a free negative charge resonates between
the two unoccupied oxygens in the phosphate ion. This structure
may then function as an ion escalator to transport positive ions
from the outer periphery of the capsule to the outer membrane,
where a convenient ion acceptor or porin may be located. The
capsule may then serve at least two important functions for j<.
pneumoniae; one being as an antiphagocytic structure, and the other
for the provision of metal ions from the external milieu.
The major problem that existed in the present study and in
many of the studies cited on ECPS as a virulence factor involved
the extent of purification of the ECPS and, in particular, the
amount of LPS contained in the EPS fractions. Batshon (2) worked
with 3 different preparations of KP2 EPS and showed that two of
these preparations were lethal for a significant proportion of mice
injected with a dose of 250 yg. The third preparation, which had
similar values of hexuronic acid as the two others, appeared to be
completely nontoxic even in doses of 2500 yg. The authors
concluded that there was a possibility of endotoxin contamination
in two of the 3 preparations. It has been shown in the present
study that doses of KP2 2-70 (A) up to 800 yg per mouse were not
lethal, although the mice became quite ill and listless for a 24 to
48 h period at these doses. Much of the early work performed by
Kato's group (58) spoke of the powerful adjuvanticity of the CPS of
K. pneumoniae. These publications were then followed by a focus on
221
the strong adjuvanticity of the LPS of l<. pneumoniae (87) without
providing the reader many clues as to what role CPS actually
played. It was most likely that it was the LPS contamination in
the ECPS preparations that produced these biological effects. It
has long been known that LPS is a virulence factor in gram-negative
bacteria (6), and has been shown to cause a transient leukopenia,
(6, 14, 81) in the host. The macrophage is particularly sensitive
to LPS, which was seen to both inhibit macrophage migration and to
cause severe morphologic damage to these cells (40, 81). A
significant reduction in the number of mature macrophages in the
peritoneum of mice was demonstrated after a dosage intravenously of
0.1 to 20 yg of Salmonella LPS (81). The response to LPS is also
known to be biphasic, with a transient leukopenia in the first 3h
after administration followed by an augmentation of phagocytic
activity by 48h. Furthermore, approximately 10 yg of E . coli LPS
was shown to increase the infectivity of pathogenic staphylococci
in rabbit skin, with an absence of leukocytic infiltration into the
focal area of infection (14). Therefore, there is evidence in the
literature to support the correlation of ELPS with virulence
enhancement seen in the present study.
It has been seen here that without appropriate precautions,
the ELPS will co-purify with the ECPS. Gotschlich et al. (37) were
able to separate meningococcal LPS and CPS by the use of
zwitterionic detergents. The method used in the present study to
remove ELPS was that of electrodialysing the ECPS in order to
222
remove the large quantities of small molecular weight ions which
contaminate these preparations. The ECPS is in many ways a magnet
for both positive and negative ions in that it produces salt
bridges presumably between adjacent uronic acid (or pyruvyl)
groups. Not only is the ED step removing these ions, but it is
also allowing for the dissociation of ECPS and ELPS with the
subsequent cetavlon extraction. ELPS is most likely associated
with the ECPS hydrophilically through the phosphate groups on LPS
covalently linked to lipid A. A divalent cation could provide a «3
salt bridge between the PO- on the LPS and a negatively charged
group on the ECPS. There is still the possibility of the bond
between ELPS and ECPS being covalent, and labile under acidic
conditions with a rise in temperature above 60°C, as can happen in
ED without a proper cooling apparatus to keep the temperature down.
Whether the bond is covalent or noncovalent, this procedure has
allowed for separation of the two polysaccharide components to a
far greater degree than without ED, at least for the KPl-0 ECPS.
Having finally separated the KPl-0 ECPS from the ELPS and
protein, virulence studies were then performed using these
preparations. It was found that our purest ECPS preparation
(containing <1% ELPS) in doses of up to 400 yg per mouse, did not
significantly enhance the virulence of KPl-T over control values.
Again, with further purification of the ECPS, the A(logiQ LD^Q)/mg
ECPS decreased accordingly. Both protein and ELPS, as well as
other contaminating materials (i.e., salts), were removed from the
ECPS during these purification steps. However, it was found that
223
the ability of these materials to decrease the LD^Q of KPl-T was
directly correlated to the amounts of ELPS present in the samples,
with approximately 11.6 yg of KPl ELPS or 8.3 yg of KP2 ELPS needed
to decrease the LD^Q of KPl-T by one log^Q unit, regardless of the
quantity of protein or ECPS present. Therefore, the ECPS of K,.
pneumoniae is believed not to have significant virulence
enhancement properties by itself, but the ELPS appears to be a
powerful virulence enhancer at low dosages. In light of the fact
that LPS from j<. pneumoniae has been shown to possess much more
powerful adjuvant activity than E_. coli LPS (58, 87), there is good
reason to believe that the ELPS of j<. pneumoniae acts as an
important virulence factor during infection. This is also
supported by the apparent loss of virulence enhancement seen after
mild saponification of the polysaccharide samples, which has been
shown to decrease the biologic activity of endotoxin (69).
In conclusion, the polysaccharides of K_. pneumoniae have shown
to be highly complex structures that aid the organism in a number
of ways during pathogenesis. The capsule plays an antiphagocytic
role, as does the ECPS, at least in AB neutralization, while the
ELPS seems to be responsible for certain biological effects that
decrease the host's resistance to infection. The ECPS was seen
also to act as a magnet for a variety of inorganic ions. This avid
attraction for ions may serve to provide necessary nutrients in the
highly competitive host environment and may also affect complement
activation and phagocytosis, both of which need divalent cations
for proper functioning. It was also seen that KPl-0 ELPS alone did
224
not significantly enhance the virulence of KPl-T at 9.4 yg/mouse
(Appendix 21) whereas the KPl-0 (N) ECPS significantly enhanced
KPl-T virulence while containing only 8.0 yg ELPS per mouse dosage.
It is suggested that the ECPS may augment the biologic activity of
ELPS in these virulence studies. This may in fact be why the 1<.
pneumoniae LPS is considered to be a more potent biologic effector
molecule than the E. coli CPS.
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81. Wiener, E., A. Beck, and M. Shilo. 1965. Effect of bacterial
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236
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APPENDICES
Page
1. CPS Structural Repeat Unit for K_. pneumoniae
Serotypes 1 and 2 239
2. ECPS Production 240
3. Serum Sensitivity 241
4. OPA for KPl-0 242
5. OPA for KPl-T 243
6. OPA for KPl CDC 2-70 244
7. OPA for KP2-0 245
8. OPA for KP2-T 246
9. OPA for KP2 2-70 247
10. Effect of Addition of EPS to the OPA 248
11. The Extracellular Products Found in the Ethanol
Fractionated Supernatants of K_. pneumoniae 249
12. The Extracellular Products Found in KPl and KP2 EPS
After Purification 250
13. Effect of KPl EPS on KPl-T Virulence in the Mouse Model.. 251
14. Effect of Cetavalon Extracted EPS on KPl-T and KP2-0
Vi rul ence in the Mouse Model 252
15. Effect of KP2 EPS on KPl-T Virulence in the Mouse Model.. 253
16. Effect of KPl or KP2 EPS on the Virulence of KP2-0
in the Mouse Model 254
17. Effect of Electrodialysis (ED) on the Virulence
Enhancement of KPl-T by KP2 2-70 EPS
in the Mouse Model 255
237
238
Page
18. Effect of Electrodialysis (ED) on the Virulence
Enhancement of KPl-T by KP2-0 EPS
i n the Mouse Model 256
19. Effect of Saponification on the Virulence Enhancement
of KPl-T by KPl-0 (N) EPS in the Mouse Model 257
20. Effect of Saponification on the Virulence Enhancement
of KP2-0 by KPl-0 (N) EPS in the Mouse Model 258
21. Effect of an Alternative Purification of KPl-0 EPS
on the Virulence Enhancement of KPl-T
in the Mouse Model 259
22. Quantitation of FAME Released from the EPS of KPl-0
and KPl-T Obtained from Gel Filtration 260
239
1. CPS Structural Repeat Unit for K. pneumoniae
Serotypes 1 and 2
Serotype 1^
-4)-e-D-GlcAp-(l^)-a-L-Fuc (1.3)-6-D-Glc ( U / \ H p 2 3 V / c
/ \ H3C C02H
Serotype 2
a-D-GlcA P
j^
^3)-a-D-Glc -(l->4)-3-D-Man (l->4)-e-D-Glc (1-
a Erbing et al., 1976 (30).
^Park et al., 1967 (64).
240
2. ECPS Production
yg/ml^ yg/ml yg/ml X + S.D.
KPl-0 18h 82.00 96.58 15.78 64.79+43.06 24h 110.94 135.67 51.32 99 .31^43 .36 36h 126.84 168.43 81.96 125.74+43.25 48h 204.78 231.18 204.16 213.37^15.42
KPl-T 18h 23.95 25.63 24.79+ 1.19 24h 37.38 43.25 40.32 + 4.15 36h 59.52 61.23 60.38 + 1.21 48h 92.12 97.00 94.56 + 3.45
KPl 2-70 18h 10.65 22.34 29.24 20.74 + 9.40 24h 153.71 33.44 66.79 84 .65+62.09 36h - - - - 379.60 379.60 48h 293.87 489.96 416.91 400.25+99.10
KP2-0 18h 309.50 576.60 443.05 +188.87 24h 571.59 720.85 646.22+105.54 36h 906.24 907.78 907.01 + 1.09 48h 1035.68 1205.49 1120.59 +120.07
KP2-T 18h 1.89 24h 2.35 4.81 36h 48h 4.46 4.69
KP2 2-70 18h 31.14 16.04 24h 164.66 154.71 36h 602.19 691.11 48h 861.28 796.37
^yg/ml of ECPS calculated from uronic acid data on dialyzed supernatants.
1.89 3.58 +
4.58 +_
23.59 + 159.69 + 646.65 + 828.83 +
1.74
0.16
10.68 7.04
62.88 45.90
241
3. Serum Sensitivity
Strain
KPl-0
KPl-T
KPl 2-70
KP2-0
KP2-T
KP2 2-70
log^QCFU^
0 min
5.49 + 0.15
8.30 + 0.04
8.21 + 0.01
8.62 + 0.07
8.43 + 0.06
8.67 + 0.03
log^QCFU^
60 min
6.46 + 0.15
8.84 + 0.09
8.71 + 0.01
9.05 1 0.08
8.35 + 0.18
9.31 + 0.05
^Log phage organisms were washed 3 times and resuspended in PBS in
various concentrations, and added to 9 parts normal rabbit serum.
" The number of colony forming units (CFU) in log.|Q units at 0 time
(N=3).
^The CFU after 60 min incubation in 90% normal rabbit serum (N=2).
242
4. OPA for KPl-0
Time 0 Log^Q CFU = 5.28 + 0 . 1 4 (N=4)
60 min
A)
B)
c)
D)
E)
AB^
+
+
-
-
-
c^
+
-
+
-
-
WBC^
+
+
+
+
-
log^Q CFU (N=2)
4.15 1 0.16
4.74 + 0.06
6.13 + 0.02
6.11 + 0.39
6.13 + 0.12
Alog^QCFU^
-1.13
-0.54
+0.85
+0.83
+0.95
^AB, Type specific antibody.
C, complement source (normal rabbit serum).
^WBC, white blood cells (human peripheral leukocytes).
Net growth from 0 to 60 min in log-jQ units.
243
5. OPA for KPl-T
Time 0 Log^Q CFU = 6.16 10.16 (N=2)
As in Appendix 4.
As in Appendix 4.
60 min
AB^ C^
A) + +
B) +
c) - +
D) -
E) -
^As in Appendix 4.
As in Appendix 4.
WBC^
+
+
+
+
-
log^QCFU (N=4)
6.79 + 0.18
6.79 + 0.05
6.90 1 0.07
7.22 + 0.28
7.06 + 0.24
Alog^Q CFU^
+0.63
+0.63
+0.74
. +1.06
+0.90
244
6. OPA for KPl CDC 2-70
Time 0 Log^Q CFU = 6.75 1 0 . 0 9 (N=4)
Time 60 min
AB*
A) +
B) +
0 -D) -
E) -
c"
+
-
+
-
WBC
+
+
+
+
^
60 min
log^QCFU (N=2)
7.34 + 0.15
7.31 + 0.01
7.71 + 0.08
7-70 ± 0 . 0 1
7.73 + 0.18
Alog^QCFU^
+ 0.59
+ 0.56
+ 0.96
+ 0.95
+ 0.98
^As in Appendix 4.
As in Appendix 4.
^As in Appendix 4.
^As in Appendix 4.
245
OPA for KP2-0
Time 0 Log^QCFU = 7.15 + 0.21 (N=2)
60 min
A)
B)
0 D)
E)
*As
"AS
=As
''AS
^As
AB* c"
+ +
+
+
-
-
in Appendix 4.
in Appendix 4.
in Appendix 4.
in Appendix 4.
in Appendix 4.
WBC^
+
+
+
+
-
log^QCFU (N=2)
7.99 1 0.02
7.87 + 0.12
8.08 1 0.12
8.04 1 0,06
8.17 + 0.04
Alog^QCFU
+0.83
+0.72
+0.93
+0.89
+1.02
246
8. OPA for KP2-T
0 min 60 min
A)
B)
C)
D)
E)
AB^
+
+
-
-
-
c"
+
-
+
-
-
WBC^
+
+
+
+
-
log^QCFU (N=2)
7.66 + 0.07
7.26 + 0.09
6.96 + 0.24
7.19 + 0.08
7.00 + 0.24
log^QCFU (N=2)
7.50 1 0.25
7.28 1 0.19
7.19 1 0.27
7.36 + 0.18
7.24 + 0.18
Alog^QCFU
-0.16
+0.02
+0.23
+0.17
+0.24
^As in Appendix 4.
As in Appendix 4.
^As in Appendix 4.
As in Appendix 4.
247
9. OPA for KP2 2-70
Time 0 Lo9in
AB*
A) +
B) +
0 -D) -
El -
CFU =
c"
+
-
+
-
.
6.57 1 0.02
WBC^
+
+
+
+
^
(N=2)
60 min
log^QCFU (N=2)
6.70 + 0.07
6.72 + 0.04
7.88 + 0.02
7.56 + 0.14
7.51 + 0.21
Alog^QCFU^
+ 0.13
+ 0.15
+ 1.31
+ 0.99
+ 0.94
^As in Appendix 4.
As in Appendix 4.
^As in Appendix 4.
As in Appendix 4.
248
10. Effect of Addition of EPS to the OPA
Time 0 Log^QCFU = 6.75 +0.09 (N=4) for KPl 2-70
Time 0 Log^QCFU = 6.33 +0.15 (N=4) for KP2-0
Time 60 min
60 min
Strain AB^ C* WBC^ EPS log^^CFU (N=2) Alog^QCFU^
KPl 2-70 + - + - 7.31 + 0.01 0.56
+ - + KPl^ 7.94 + 0.07 1.19
+ - + KP2^ 7.42 + 0.11 0.67
KP2-0 + - + - 6.74 + 0.12 0.412
+ - + KPl^ 6.68 1 0 . 1 6 0.350
+ - + KP2' 7.01 + 0.12 0.675
a As i
b
n Appendix 4.
n Appendix 4.
n Appendix 4.
n Appendix 4.
n Table 16: footnote c.
n Table 16: footnote d.
^As in Table 16: footnote e.
' As in Table 16: footnote f.
As i
^As i
^As i
^As
^As
249
11. The Extracellular Products Found in the Ethanol
Fractionated Supernatants of J<. pneumoniae
Strain
KPl-0
KPl-T
KP2-0
KP2 2- 70
ECPS'' (N=3)
522.2124.6
425.6115.8
782.4110.0
840.6+11.5
ELPS'' (N=2)
64.514:5
52.111.2
25.412.1
23.6+0.12
Protein' ' (N=2)
39.211.5
70.115.3
9.610.6
16.9+0.2
^Values for ECPS, ELPS and protein expressed in yg per mg dry
weight.
^N equals the number of determinations performed on separate samples.
250
12. The Extracellular Products Found in KPl and KP2 EPS
After Purification^
Strain Fraction ECPS^ ELPS^ Protein^
KPl-0 HMW 296.2131.1(N=3) 79.4ll6.3(N=3) 86.612.1(N=2)
KPl-0 LMW 529.9118.1(N=3) 37.41 5.8(N=3) 30.0l0.2(N=2)
KP2 2-70 HMW 807.4ll6.7(N=3) 28.ll 0.0(N=2) 48.7l0.7(N=2)
KP2 2-70 LMW 838.7ll0.4(N=3) 15.7l 0.9(N=2) 33.7l0.2(N=2)
^Purification procedures included ethanol extraction, DEAE-Sephacel
and gel filtration chromatography on S-2B.
^Values of ECPS, ELPS and protein are expressed in yg per mg dry
weight.
251
13. E f f e c t o f KPl EPS on KPl-T V i r u l ence
i n the Mouse Model
EPS yg ECPS/mouse log^Q LD^Q ^ ^ ° 9 I O ^^50^
KPl-0 (N)^ 40.3 3.3410.64(N=3)^ -1 .4410.41(p<00.001)^
KPl-0 (A )^ 25.5 3.7510.56(N=3) - 1 . 02 l 0 .34 (p<0 .01 )
KP l -T (N)^ 121.7 3 .67 l0 .12(N=3) - 1 . l l l 0 . 1 2 ( p < 0 . 0 0 5 )
KPl -O(A)^ 56.9 4.3610.26(N=3) -0 .4210.26(p<0.02)
KPl-0 HMW(N)^ 62.8 4.0410.41(N=3) - 0 . 74 l 0 .03 (p<0 .05 )
KPl -0 LMW(N)^ 108.0 4.3610.20(N=4) -0 .4210.20(p<0.10)
KPl-T HMW(N)^ 96.9 3.90l0.52N=2) -0 .8810.34(p<0.05)
KPl-T LMW(N)^ 149.0 4.63l0.37(N=3) -0.15l0.04(p<0.80)
Control PBS 4.78l0.44(N-10)
^Alog.Q LD^Q; Change in the log^Q LD^Q over PBS control values.
^Neutral EPS fraction from DEAE-Sephacel.
^Acid EPS fraction from DEAE-Sephacel.
^High molecular weight fraction from neutral EPS applied to
Sepharose 2B gel filtration.
^Low molecular weight fraction as in footnote d.
'N equals the number of LDnn trials performed.
^Statistical analysis (Student's t test) between experimental (EPS
treated) and control (PBS treated) trials.
252
14. Effect of Cetavlon Extracted EPS on KPl-T
and KP2-0 Virulence in the Mouse Model
Strain
Injected
KPl-T
KP2-0
EPS
KPl-O(CET)^
Control
KPl-O(CET)^
Control
yg ECPS/mouse log-jQ LD^Q ^"'oQio ^^50
200.0
200.0
PBS
200.0
200.0
PBS
3.23 -1.55
2.97 -1.81(p<0.0001)^
4.7810.44(N=10)
4.87 -1.14
5.12 -0.89(p<0.05)^
6.01+0.21(N=2)^
^Alog-|Q LDCQ; Change in the log^Q LD^Q compared to control values.
^KPl-0 (CET); cetavlon fractionated supernatant of cultures of
KPl-0 grown in defined medium for 48h at 37 C
^Statistical analysis (Student's t test) of the comparison of the
means of the log^Q LD^Q values for experimental (ECPS) and control
(PBS) trials.
^N equals the number of LD^Q trials performed.
15. Effect of KP2 EPS on KPl-T
Virulence in the Mouse Model
253
EPS
KP2 2-70 EtOH^
KP2 2-70 (A)^
KP2 2-70 (A)
KP2 2-70 (A)
KP2-0 (A)^
ug ECPS/mouse
336.2
101.5
204.7
454.1
428.6
PBS
log^o LDgQ (N=2)^ Alog^Q LD 50
2.97 1 1.27
4.67 1 0.49
4.07 1 0.35
2.87 1 0.77
3.91 + 1.19
-1.81(p<0.005)'
-0.11(p<0.80)
-0.71(p<0.01)
-1.91(p<0.001)
-0.87(p<0.10)
4.78 + 0.44 (N=10)
^Alog,Q LDCQ; change in the log-jQ LD^Q over PBS control values.
^Ethanol extracted EPS from 48h cultural supernatants.
^Acid EPS fraction from DEAE-Sephacel.
^Statistical analysis (Student's t test) between experimental (EPS
treated) and control (PBS treated) trials.
^N equals the number of determinations performed from two separate
trials.
254
16. Effect of KPl or KP2 EPS on the Virulence
of KP2-0 in the Mouse Model
EPS yg ECPS/mouse log.|Q LD^Q ^^°9IO ^^50
KP2 2-70 (EtOH)^
KPl-0 {Hf
432
467
467
536
40.
40.
3
3
5.42
5.16
5.42
4.80
5.86
5.86
-0.60
-0.86
-0.60
-1.22
-0.16
-0.16
Control PBS 6.0210.22 (N=2)
^Alog,Q LDCQ; change in the log^Q LD^Q over PBS control values.
' KP2 2-70 (EtOH); ethanol extracted EPS from 48h supernatants of
KP2 2-70 grown in defined medium at 37 C
^KPl-0 (N); neutral EPS fraction from DEAE-Sephacel.
255
17. Effect of Electrodialysis (ED) on the Virulence Enhancement
of KPl-T by KP2 2-70 EPS in the Mouse Model
KP2 2-70 EPS^ yg ECPS/mouse LD^Q log^Q LD^Q ^^^9^Q LD^Q"^
Before ED
After ED^
470
470
470
380
380
409
432
1.17x10^
1.17x10^
4.07x10^
4.11x10^
2.56x10"^
7.30x10'^
2.75x10^
2.07
2.07
2.61
3.61
3.41
3.86
3.44
-2.71
-2.71
-2.17
-1.17
-1.37
-0.92
-1.34
Control PBS 6.03x10^ 4.78l0.44(N-10)^
^KP2 2-70 EPS obtained by ethanol extraction of 48h supernatants of
cultures grown in defined medium at 37 C
^Alog^Q LD^Q; the change in the log^Q LD^Q from the PBS control
data.
^Electrodialysis proceeded at lOOOV until no marked increase in mA
occurred over a 30 min period.
^As in Table 31.
256
18. Effect of Electrodialysis (ED) on the Virulence
Enhancement of KPl-T by KP2-0 EPS in the Mouse Model
KP2-0 EPS^ yg ECPS/mouse LD^Q log^Q LD^Q ^^^9-^Q LD^Q^
Before ED 441 1.45x10^ 4.16 -0.62
470 2.31x10^ 4.36 -0.42
After ED^ 392 2.08x10^ 3.32 -1.46
392 2.08x10^ 3.32 -1.46
421 1.30x10^ 3.11 -1.67
Control PBS 6.03x10^ 4.78 (N=10)^
^KP2-0 EPS; obtained by ethanol extraction of 48h supernatants of
cultures grown in defined medium at 37 C
^Alog^Q LD^Q; the change in the log^Q LD^Q from the PBS control
data.
^Electrodialysis proceeded at lOOOV until no marked increase in mA
occurred over a 30 min period.
^N refers to the number of LD^Q trials performed.
257
19. Effect of Saponification on the Virulence Enhancement
of KPl-T by KPl-0 (N) EPS in the Mouse Model
Strain
KPl-T
EPS
KPl-O(N)^
KPl-O(N)^
(Saponified)
KPl-O(N)^
(Saponified)
yg ECPS/mouse
40.3
200.0
200.0
Control PBS
log^Q LD^Q Alog^Q LD^Q
4.23
3.97
3.34 + 0.64 -1.28 + 0.41
-0.55
-0.81
4.78 + 0.44 (N=10)
^Alog-|Q LDCQ; the change in the log.|Q LD^Q from PBS controls.
^KPl-O(N); the neutral fraction of ethanol fractionated EPS placed
on DEAE-Sephacel (N=3).
^KPl-O(N) (Saponified); neutral EPS as in footnote b placed in 0.5
M NaOH overnight at room temperature and dialyzed 3 times against
8LDH2O in the cold.
258
20. Effect of Saponification on the Virulence Enhancement
of KP2-0 by KPl-0 (N) EPS in the Mouse Model
Strain EPS yg ECPS/mouse log^Q LD^Q ^^^^-[Q LD^Q^
KP2-0 KPl-O(N)'^
KPl-O(N)
KPl-O(N)^
(Saponified)
KPl-O(N)^
(Saponified)
Control
40.3
40.3
200.0
200.0
PBS
5.86 -0.16
5.86 -0.16
5.87 -0.15
5.87 -0.15
6.02+0.22(N=2) d
^Alog,Q LDCQ; the change in the log^Q LD^Q from PBS controls.
^ KPl-O(N); the neutral fraction of ethanol fractionated EPS placed
on DEAE-Sephacel.
^KPl-O(N) (Saponified); neutral EPS as in footnote b placed in 0.5
M NaOH overnight at room temperature and dialyzed 3 times against
BLDH^O in the cold.
^N equals the number of LD^Q trials performed.
259
21. Effect of an Al ternat ive Pur i f i ca t ion of
KPl-0 EPS on the Virulence Enhancement
of KPl-T in The Mouse Model
Sample^
KPl-0 Fr II
KPl-0 Fr II
KPl-0 Fr II
S-2B (ED)
KPl-0 Fr II
S-2B (ED)
KPl-0 Fr II
S-2B (ED)
KPl-0 Fr III
KPl-0 Fr III
ECPS/mouse°
400.6
400.6
402.7
201.3
100.7
ND^
ND
ELPS/mouse^
15.5
15.5
3.4
1.7
0.9
9.4
9.4
^^910^^50
3.58
3.83
4.58
4.83
4.58
4.33
4.33
Alog^Q LD5Q
-1.20
-0.95
-0.20
+0.05
-0.20
-0.45
-0.45
Control PBS PBS 4.7810.44 (N=10)^
^As i n Table 23.
^The quantity of ECPS in yg co-administered with serial dilutions
of the KPl-T strain IP into mice.
^The quantity of ELPS in yg as in footnote b.
^The change in the log^Q LD^Q with respect to controls.
^ND; none detected.
^N equals the number of separate trials performed.
260
22. Quantitation of FAME Released from the EPS of
KPl-0 and KPl-T Obtained from Gel F i l t r a t i o n
Sample FAME ECPS ELPS^
KPl-0 HMW 24.22112.84 (N=4)^ 440.76137.97 184.7312.36
KPl-0 LMW 34.0419.72 (N=4) 12769.951596.59 692.56114.18
KPl-T HMW 59.84121.11 (N=3) 1587.14155.22 671.6813.55
KPl-T LMW 20.1211.73 (N=3) 11516.921867.26 321.71114.18
^The HMW and LMW fract ions from both KPl-0 and KPl-T EPS obtained
from gel f i l t r a t i o n .
The quanti ty of f a t t y acid metyl esters in yg obtained from
saponi f icat ion of EPS.
^The quant i ty of ECPS in yg (N=3).
* The quant i ty of ELPS in yg (N=2).
^N equals the number of determinations performed on two separate
preparations.