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A 200 kDa protein is associated with haemagglutinating isolates of
Moraxella (Branhamella) catarrhalis
Margaret Fitzgeralda, Riona Mulcahy
b, Susan Murphy
b, Conor Keane
c,
Davis Coakleyb, Thomas Scott
a;
*
aDepartment of Biological Sciences, Dublin Institute of Technology, Kevin St., Dublin 8, Ireland
bMercer's Institute for Research on Ageing, St. James's Hospital, Dublin 8, Ireland
cDepartment of Clinical Microbiology, St. James's Hospital, Dublin 8, Ireland
Received 2 May 1997; accepted 5 May 1997
Abstract
Moraxella catarrhalis adheres to human erythrocytes by means of a proteinaceous, trypsin sensitive, heat modifiable
haemagglutinin. A 200 kDa protein was found to be associated with haemagglutinating isolates of M. catarrhalis. This protein
was present on all haemagglutinating isolates (n=17), but was absent on the non-haemagglutinating isolates (n=23) examined.
This protein demonstrated heat-modifiable properties in sodium dodecyl sulfate and was degraded by trypsin. Immunoblot
assays with polyclonal antiserum indicated that the 200 kDa protein was associated exclusively with haemagglutinating isolates
and antibodies to this protein did not recognise epitopes on non-haemagglutinating isolates. This protein, which appears to be
a surface expressed protein may be a haemagglutinin of M. catarrhalis.
Keywords: Moraxella (Branhamella) catarrhalis ; Haemagglutination; Surface protein; Heat modi¢ability; Immunoblotting
1. Introduction
In recent years Moraxella (Branhamella) catarrha-
lis has been increasingly recognised as an important
human pathogen [1,2]. Previously dismissed as a
commensal of the oro-nasopharyngeal tract, it is
now reported as the third most common isolate after
Haemophilus in£uenzae and Streptococcus pneumo-
niae as the causative agent in lower respiratory tract
infections such as bronchitis and pneumonia in the
elderly, especially in those with underlying pulmo-
nary disease [3,4]. M. catarrhalis has been sporadi-
cally implicated in invasive infections including sep-
ticaemia in both the immunocompetent [5] and the
immunocompromised patient [6].
The recent recognition of M. catarrhalis as an im-
portant upper and lower respiratory tract pathogen
has prompted researchers to investigate the possible
virulence mechanisms employed by the organism in
order to understand the pathogenesis of M. catarrha-
lis infection. Bacterial adherence is believed to be the
initial event in most infectious processes [7]. There
have been a number of studies on the adherence of
M. catarrhalis to human oropharyngeal cells and
human erythrocytes [8^11]. The adhesive processes
0928-8244 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 9 2 8 - 8 2 4 4 ( 9 7 ) 0 0 0 4 1 - 2
FEMSIM 766 27-8-97
* Corresponding author. Tel. : +353 (1) 4024747;
Fax: +353 (1) 4024995.
Abbreviations: PBS A, Dulbecco A phosphate buffered saline;
NOG, N-octyl-L-D-glucopyranoside; TBS, Tris buffered saline
FEMS Immunology and Medical Microbiology 18 (1997) 209^216
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involved remain unresolved. It has been established
that the haemagglutinin is a trypsin sensitive protein
[11,12] and is associated with tracheal cell adherence
[11]. Two phenotypes of the haemagglutinin have
been identi¢ed, phenotype I agglutinates human er-
ythrocytes only, while phenotype II agglutinates hu-
man and rabbit erythrocytes [12]. A recent study has
reported that a signi¢cantly higher percentage of M.
catarrhalis isolates recovered from infected elderly
patients, with respiratory tract infection, haemagglu-
tinated human erythrocytes when compared with the
haemagglutinating activity of isolates recovered from
colonised healthy elderly people, suggesting that the
haemagglutinin may play a role in the infectious
process in respiratory tract infection in the elderly
population [13].
In this present study 40 isolates of M. catarrhalis
were examined by SDS-PAGE and a 200 kDa pro-
tein, which was found to be associated with haemag-
glutinating isolates was investigated.
2. Materials and methods
2.1. Bacterial isolates and growth conditions
Forty isolates of M. catarrhalis were examined.
These isolates were from a collection acquired during
a study of the epidemiological aspects of M. catar-
rhalis infection and colonisation [13]. Isolates were
identi¢ed according to the criteria previously out-
lined [14] and were stored at 370³C using the Protect
Bead System (Technical Service Consultants). Prior
to all tests isolates were grown on Columbia blood
agar at 37³C for 18 h, unless otherwise stated.
2.2. Haemagglutination
The ability of the 40 isolates to agglutinate human
group O red blood cells and rabbit erythrocytes was
determined by a micotitre method [12].
2.3. SDS-PAGE of crude whole-cell protein
preparations
Whole cell preparations of 17 haemagglutinating
and the 23 non-haemagglutinating isolates of M. ca-
tarrhalis were analysed by SDS-PAGE.
Bacterial suspensions were centrifuged in a micro-
fuge at 10 000Ug for 5 min. The pellet was boiled in
200 Wl sample bu¡er containing 0.065 M Tris, 2% (w/
v) SDS, 5% (v/v) L-mercaptoethanol, 10% (w/v) glyc-
erol and 0.001% (w/v) bromophenol blue. The result-
ing protein extracts were resolved by SDS-PAGE on
10% polyacrylamide gels by the technique of
Laemmli [15]. The resolved proteins were visualised
by staining with Coomassie brilliant blue.
Isolate K48 showed variability on repeated hae-
magglutination testing. Separate colonies were sub-
cultured and three phenotypes were identi¢ed. These
were titled W, X and Y and gave titres of 4, 2 and 0
against human erythrocytes and 32, 16 and 1 against
rabbit erythrocytes. These phenotypes were tested by
SDS-PAGE, using standardised suspensions.
Bacterial suspensions of phenotype I and pheno-
type II haemagglutinating isolates (B4, K29, S109,
S407 and S580) were treated with an equal volume
of trypsin solution (2 mg ml
31) in PBS A at 37³C for
2 h. Following washing in PBS A the bacterial pellets
were prepared for SDS-PAGE analysis as described
above. Control preparations were treated identically,
using PBS A instead of trypsin solution.
2.4. Heat modi¢ability of the 200 kDa protein
The heat modi¢ability of the 200 kDa protein was
examined by incubating crude whole-cell protein ex-
tracts of isolate S407 in SDS-PAGE sample bu¡er at
various temperatures ranging from 20³C to 100³C
for 5 min, before resolving the preparations by
SDS-PAGE on 5% polyacrylamide gels.
2.5. Isolation of outer membranes
The outer membranes of two haemagglutinating
isolates (S407 and S580) and two non-haemaggluti-
nating isolates (K16 and S89) were obtained using
the broth culture supernatant technique previously
described [16], with some slight modi¢cations. Brain
Heart Infusion broth (100 ml) was inoculated with
several colonies of bacteria from a Columbia blood
agar plate and incubated at 37³C for 24 h, with
shaking. The cells were centrifuged at 10 000Ug for
15 min at 4³C. The supernatant fraction was col-
lected and centrifuged again at 10 000Ug for 15
min at 4³C. The resulting supernatant fraction was
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M. Fitzgerald et al. / FEMS Immunology and Medical Microbiology 18 (1997) 209^216210
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then centrifuged at 100 000Ug for 90 min at 4³C.
The pellet was suspended in sample bu¡er and the
proteins present in these outer membrane vesicles
were resolved by SDS-PAGE using 10% polyacryl-
amide gels.
These blebs consist of outer membrane vesicles
that can be observed by electron microscopy; they
contain OMPs and lipooligosaccharide and are free
of cytoplasmic membrane contamination [17].
2.6. Preparation of anti-serum
Polyclonal antiserum against whole cells was ob-
tained by immunisation of a rabbit with a formalin
treated suspension of isolate S407. In addition, poly-
clonal antiserum was prepared against a protein ex-
tract of isolate S407, obtained by ion-exchange chro-
matography. Following elution of the proteins from
the ion-exchange column, fractions containing pro-
tein were analysed by SDS-PAGE. The fractions
containing the 200 kDa protein were pooled, dia-
lysed with several changes of sterile water at 4³C
for 48 h and used in the production of antisera. In
the preparation of each anti-serum a New Zealand
white rabbit was given three injections, subcutane-
ously, of the whole cell preparation or the protein
extract (800 Wg protein per dose) in Freund's com-
plete adjuvant on day 0, and in Freund's incomplete
adjuvant on days 14 and 35.
Following immunization, antisera were obtained
on day 49 and the immunoglobulin fraction of the
antiserum was separated by the method of Harboe
and Ingild [18]. Antisera were adsorbed by using (i) a
cocktail of non-haemagglutinating isolates (21, 25,
K2, K16, S22, S68, S75, S76, S89) and (ii) a cocktail
of haemagglutinating isolates (B4, K38, K44, K51,
S109, S274, S407, S540, S580).
2.7. Immunoblot analysis
Both adsorbed and unadsorbed antisera were
studied using immunoblot analysis. Western immu-
noblots were performed, with polyclonal anti-serum
to whole cell preparation of isolate S407, by electro-
phoretic transfer of crude protein extracts of hae-
magglutinating isolates (B4 and S407) and a non-
haemagglutinating isolate (isolate 21) of M. catarrha-
lis resolved by SDS-PAGE to nitrocellulose, employ-
ing the technique described by Bollag and Edelstein
[19]. The membranes were blocked in 2% (w/v) skim
milk (Difco) prepared in Tris bu¡ered saline (TBS)
for 1 h. Following washing, the membrane was
probed with the polyclonal antiserum as the primary
antibody. The membranes were washed in TBS, in-
cubated in 1:1000 dilution of swine anti-rabbit,
horseradish peroxidase-conjugated antibody for 2 h,
washed again and developed in a solution of 4-
chloro-1-naphthol for 10 min.
Antiserum to the protein extract of isolate S407
which had been adsorbed with the cocktail of non-
haemagglutinating isolates (described above) was ex-
amined in more detail using immunoblot analysis by
probing against a selection of haemagglutinating iso-
lates (B4, K29, K38, K48, S64, S109, S407, S540 and
S580) and non-haemagglutinating isolates (21, K1,
K3, K5, K10, S68 and S89) of M. catarrhalis.
3. Results
3.1. Haemagglutination and SDS-PAGE analysis
Seventeen of the 40 isolates examined agglutinated
human erythrocytes, while the remaining 23 isolates
were non-haemagglutinating. Of the 17 haemaggluti-
nating isolates, 10 isolates haemagglutinated human
erythrocytes (phenotype I), while 7 isolates haemag-
glutinated both human and rabbit erythrocytes (phe-
notype II). The protein pro¢les obtained following
the resolution of crude protein extracts of the 17
haemagglutinating and the 23 non-haemagglutinat-
ing isolates of M. catarrhalis by SDS-PAGE demon-
strated the presence of a 200 kDa protein in all prep-
arations of haemagglutinating isolates which was
absent in all preparations of non-haemagglutinating
isolates examined (Fig. 1). The three phenotypes of
K48 showed di¡erences in the amount of the 200
kDa protein that was present. The strength of the
200 kDa band was related to the haemagglutinating
activity of the phenotypes (Fig. 2). The trypsin-
treated haemagglutinating strains showed no 200
kDa protein. The apparent molecular mass (Mr) of
the 200 kDa protein varied slightly between some
isolates from 185^205 kDa. However, the Mr of
the `200 kDa' of a particular isolate was repeatedly
the same when di¡erent preparations of the same
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M. Fitzgerald et al. / FEMS Immunology and Medical Microbiology 18 (1997) 209^216 211
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isolate were compared. In addition, no apparent dif-
ference in protein pro¢le was noted when phenotype
I isolates were compared with phenotype II isolates,
thus inter-strain variability in Mr was unrelated to
haemagglutination phenotype.
The 200 kDa protein demonstrated heat-modi¢-
ability, it migrated as a protein of higher Mr when
solubilised at temperatures less than 80³C. Determi-
nation of the Mr of this protein proved di¤cult, but
it appeared to be greater than 400 kDa.
OMP preparations were obtained using the broth
culture supernatant technique. The puri¢ed outer
membranes of two haemagglutinating (S407 and
S580) and of two non-haemagglutinating (K16 and
FEMSIM 766 27-8-97
Fig. 2. SDS-PAGE analysis of phenotypes W, X and Y of isolate K48. Lane 3, phenotype W (titre against rabbit erythrocytes 32 and hu-
man erythrocytes 4); lane 2, phenotype X (titre against rabbit erythrocytes 16 and human erythrocytes 2) and lane 4, phenotype Y (titre
against rabbit erythrocytes 1 and human erythrocytes 0). The 200 kDa protein is marked `s '. Proteins were stained with Coomassie bril-
liant blue on a 10% polyacrylamide gel. Mr markers are shown in lane 1.
Fig. 1. SDS-PAGE analysis of crude whole-cell protein extracts of M. catarrhalis. Lanes 2^6, haemagglutinating isolates and lanes 7^11,
non-haemagglutinating isolates. The presence of a 200 kDa protein in the extracts of haemagglutinating isolates is indicated by `s '. The
10% polyacrylamide gel was stained with Coomassie brilliant blue. Mr markers are shown in lane 1.
M. Fitzgerald et al. / FEMS Immunology and Medical Microbiology 18 (1997) 209^216212
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S89) M. catarrhalis isolates were resolved by SDS-
PAGE. The protein band patterns were similar to
those obtained by Bartos and Murphy [16] and Mur-
phy and Loeb [17] with the eight major outer mem-
brane proteins (A^H) being present in all prepara-
tions. However, in the haemagglutinating isolates
FEMSIM 766 27-8-97
Fig. 4. Western blot analysis of crude whole-cell protein preparations of M. catarrhalis. Haemagglutinating isolate S407 (lane 1) and iso-
late B4 (lane 2) and non-haemagglutinating isolate 21 (lane 3) probed with unadsorbed polyclonal antiserum (left), polyclonal antiserum
adsorbed with a cocktail of non-haemagglutinating isolates (middle) and polyclonal antiserum adsorbed with a cocktail of haemagglutinat-
ing isolates (right). The 200 kDa protein is indicated by `s '.
Fig. 3. SDS-PAGE analysis of outer membrane preparation of isolate S407 (bottom panel) and isolate S 580 (top panel). In (bottom)
upper lane, outer membrane bleb preparation; middle lane, whole cell preparation. In (top) middle lane, outer membrane bleb prepara-
tion; bottom lane, whole cell preparation. The 200 kDa protein is marked `s '. Proteins are stained with 1% Coomassie blue on a 10%
polyacrylamide gel. Mr markers are shown in the bottom lane in the bottom panel and in the top lane in the top panel.
M. Fitzgerald et al. / FEMS Immunology and Medical Microbiology 18 (1997) 209^216 213
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examined (S407 and S580), the 200 kDa protein ob-
served in the crude protein whole cell extracts was
also evident, albeit weakly in the outer membrane
preparations. (Fig. 3). This band was not found in
the outer membrane preparations of the non-hae-
magglutinating isolates (K16 and S89).
3.2. Western immunoblotting
The polyclonal antiserum to a whole cell prepara-
tion of M. catarrhalis (isolate S407) was analysed in
immunoblot assays (Fig. 4a). When this antiserum
was adsorbed with a cocktail of non-haemagglutinat-
ing isolates and examined by immunoblotting analy-
sis, the adsorbed antiserum recognised epitopes on
the 200 kDa protein on the two haemagglutinating
isolates studied (S407 and B4), while antibodies rec-
ognising epitopes on other proteins had been signi¢-
cantly adsorbed out (Fig. 4b). Adsorption of the
antiserum with a cocktail of haemagglutinating iso-
lates resulted in the signi¢cant removal of antibodies
recognising the 200 kDa protein (Fig. 4c).
To determine if antiserum adsorbed with a cock-
tail of non-haemagglutinating isolates, which con-
tained antibodies predominantly to the 200 kDa pro-
tein, demonstrated reactivity with a larger selection
of M. catarrhalis isolates, a total of 16 isolates were
examined by immunoblot analysis, using antiserum
to the protein extract of M. catarrhalis (isolate 407).
This adsorbed antiserum recognised epitopes on the
200 kDa protein in all nine haemagglutinating iso-
lates examined (B4, K29, K38, K48, S64, S109, S407,
S540 and S580), but failed to detect a 200 kDa pro-
tein in all seven non-haemagglutinating isolates (21,
K1, K3, K5, K10, S68 and S89) of M. catarrhalis
studied (Fig. 5).
4. Discussion
M. catarrhalis is now a well recognised cause of
upper and lower respiratory tract infections in chil-
dren and in the elderly, respectively. Due to the in-
creased awareness of the pathogenic role played by
M. catarrhalis in infection, much work has evolved
around establishing the virulence factors employed
by the organism. Adherence of bacteria to host cells
is generally considered the initial step in the patho-
FEMSIM 766 27-8-97
Fig. 5. Western blot analysis of crude whole-cell protein preparations of haemagglutinating and non-haemagglutinating isolates of M. ca-
tarrhalis probed with polyclonal antiserum adsorbed with non-haemagglutinating isolates of M. catarrhalis. The haemagglutinating isolates
were S407 (lane 1), B4 (lane 2), S109 (lane 3), S580 (lane 4), K38 (lane 5), S64 (lane 6); the non-haemagglutinating isolates were S68
(lane 7), K1 (lane 8), K3 (lane 9), K5 (lane 10) and K10 (lane 11). The 200 kDa protein is indicated by `s '.
M. Fitzgerald et al. / FEMS Immunology and Medical Microbiology 18 (1997) 209^216214
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genesis of many infections [7]. It has been proposed
that the adherence of M. catarrhalis to oropharyn-
geal cells and human erythrocytes may be mediated
by ¢mbriae [8]. While Ahmed et al. [9] report an
association between nasopharyngeal adherence of
strains and haemagglutination, this association is
tenuous as a number of strains, which were highly
¢mbriated, were non adherent to nasopharyngeal
cells and were non-haemagglutinating. Similarly, Ri-
kitomi et al. [10] have observed no signi¢cant di¡er-
ence in the adhesive capacity to oropharyngeal cells
or in haemagglutination between ¢mbriated and
non-¢mbriated strains of M. catarrhalis. In the latter
study, it was also noted that no correlation between
haemagglutination and oropharyngeal cell adherence
was evident. In an ongoing study no correlation be-
tween haemagglutination and adherence to HEp-2
cells by M. catarrhalis has also been observed (M.
Fitzgerald, unpublished observations). Thus it would
appear that haemagglutination and epithelial cell ad-
herence are mediated by separate adhesins and that
non-¢mbrial adhesins, may be involved in haemag-
glutination.
Recently, a correlation was found between the
haemagglutination titre of isolates of M. catarrhalis
and their ability to adhere to tracheal epithelial cells
[11]. This ¢nding may be of importance as the or-
ganism is commonly associated with tracheobronchi-
tis in the elderly [20]. Furthermore, the fact that 75%
of strains isolated from the sputum of the elderly
with respiratory tract infections were haemaggluti-
nating in comparison to 15% of the strains isolated
from healthy elderly individuals would suggest a role
for the haemagglutinin in the aetiology of M. catar-
rhalis infection in the elderly [13].
A recent study has established that M. catarrhalis
has at least two haemagglutination phenotypes [12].
Phenotype I isolates haemagglutinate human eryth-
rocytes, while phenotype II isolates haemagglutinate
human and rabbit erythrocytes. Haemagglutination
by both phenotypes was mediated by trypsin-sensi-
tive proteins that were heat-labile at 70³C. The ob-
servation that haemagglutination is mediated by pro-
teinaceous structures has also been reported by
others [11]. To date no study characterising these
haemagglutinins has been documented.
In this present study it was established that a 200
kDa protein is exclusively associated with haemag-
glutinating isolates of M. catarrhalis. There was
some variability of the Mr of this protein from one
isolate to another, but the Mr was consistent for
each isolate. This protein was found on both hae-
magglutinating phenotypes and there was no corre-
lation between Mr and phenotypes. In addition, it
was established that in the SDS-PAGE of the three
phenotypes of K48, the density of the 200 kDa band
correlated closely with the haemagglutinating titre of
the phenotype. SDS-PAGE analysis of the bleb prep-
arations of haemagglutinating isolates demonstrated
the presence of the 200 kDa protein, indicating that
it may be an outer membrane protein. However,
while it was identi¢ed in the outer membrane
vesicles, the relative amount was not greater than
the lysate, thus the possibility exists that this protein
may not be an outer membrane protein, but may be
a surface protein bound to the membrane. Evidence
that the 200 kDa protein was surface expressed was
provided by the fact that: (a) antibodies recognising
epitopes on this protein were removed following ad-
sorption of the antiserum with haemagglutinating
isolates, as demonstrated by immunoblot analysis
and (b) ongoing studies have shown that antibodies
to the 200 kDa protein are present in the serum of
children and adults when analysed by immunoblot-
ting, indicating that the protein is expressed in vivo
(R. Mulcahy, unpublished observations).
Electron microscopy studies have shown that hae-
magglutinating strains have an outer ¢brillar coat
which is removed by trypsin treatment and adher-
ence to red blood cells appears to be mediated by
this outer ¢brillar coat [12]. Haemagglutination ap-
pears to be mediated by a trypsin sensitive, heat
sensitive, protein haemagglutinin. The 200 kDa pro-
tein is heat sensitive and trypsin sensitive and be-
cause of the association between the 200 kDa protein
and haemagglutinating strains it is possible that this
protein may be a haemagglutinin. Further work is
currently underway to investigate this.
Acknowledgments
We thank Professor Cyril Smyth for his useful
discussion and his critical comments on this manu-
script. This work was supported by a grant to M.F.
from the Health Research Board of Ireland.
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