INFECTION AND IMMUNITY, Oct. 1979, p. 143-1490019-9567/79/10-0143/07$02.00/0

Vol. 26, No. 1

Pathogenic Species of the Genus Haemophilus andStreptococcus pneumoniae Produce Immunoglobulin Al

ProteaseMOGENS KILIAN,t* JIRI MESTECKY, AND RALPH E. SCHROHENLOHER

Department ofMicrobiology, Institute ofDental Research and Division of Clinical Immunology andRheumatology, University ofAlabama in Birmingham, Birmingham, Alabama 35294

Received for publication 4 May 1979

Thirty-seven strains ofthe genus Haemophilus and five strains ofStreptococcuspneumoniae were examined for their ability to produce extracellular enzyme thatcleaves immunoglobulin molecules. All strains of H. influenza, H. aegyptius, andS. pneumoniae elaborated enzyme that selectively cleaved human immunoglob-ulin Al (IgAl) myeloma proteins but was inactive against a variety of otherproteins including human IgA2, IgG, and IgM, porcine and bovine secretary IgA,human and bovine serum albumins, and ovalbumin. Although susceptible, humansecretary IgA remained largely undigested. Two strains of H. pleuropneumoniaeisolated from fatally infected pigs cleaved porcine secretary IgA, but had no effecton human IgA proteins. None of 16 strains that belonged to nonpathogenicHaemophilus species produced IgA protease. Analyses of the cleavage productsof human IgAl and secretary IgA proteins by immunochemical methods, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, and analytical ultracentrifu-gation revealed that Fab and Fc fragments were produced. Since the productionof IgAl protease by Neisseria meningitidis has been reported previously, ourfinding that H. influenzae and S. pneumoniae produce an IgAl protease indicatesthat this is a property of all three major etiological agents of bacterial meningitis.This suggests that IgAl protease production may be an important factor in thepathogenesis of this disease.

Mucosal surfaces of the upper respiratorytract are inhabited by a broad spectrum of bac-teria some of which may, under conditions thatare poorly understood, invade mucosal tissuesand produce systemic infection. Certain bacteriawhich colonize mucosal surfaces, such as Neis-seria meningitidis, N. gonorrhoeae, and Strep-tococcus sanguis, were shown to produce anextracellular enzyme that cleaves serum immu-noglobulins belonging to immunoglobulin Al(IgAl) but not IgA2, IgG, or IgM classes (11-15).Secretory IgA (S-IgA), the principal carrier ofspecific humoral defense on mucous surfaces, isonly partly susceptible to digestion by bacterialIgA proteases. This relative insusceptibility maybe explained by the presence of a higher propor-tion of IgA2 in S-IgA than in serum IgA (12), bythe association of IgA with secretary componentwhich renders S-IgA resistant to various pro-teases, and by the presence of S-IgA-associatedantibodies that may neutralize IgA protease ac-tivity (A. G. Plaut et al., Fed. Proc. 38:5275,1979). Since cleavage of IgAl by such enzymes

t Present address: Department of Microbiology, RoyalDental College, DK-8000 Aarhus, Denmark.

resulted in a considerable loss of antibody activ-ity (14), it appears probable that these IgA pro-teases represent a significant virulence factor.A classic example of a systemic infection

which is initiated by bacteria that gain accessinto the circulation through the mucous mem-branes of the respiratory tract is bacterial men-ingitis. N. nningitidis, Haemophilus influ-enzae, and S. pneumoniae represent the princi-pal bacterial species responsible for this disease.It was previously reported that pathogenicstrains ofN. meningitidis produce IgAl protease(11), and we report in this communication thatthe other two leading causative agents of bac-terial meningitis, H. influenzae and S. pneumo-niae, also produce an enzyme which cleavesIgAl and to a limited extent S-IgA, but has noeffect on IgA2, IgM, and IgG.

MATERIALS AND METHODSBacterial strains. Thirty-seven human and por-

cine isolates of the genus Haemophilus and five strainsof S. pneumoniae isolated from human blood, cerebro-spinal fluid, and sputum were included in the study.The Haemophilus strains that represent each of fivebiotypes ofH. influenza and the species H. aegyptius,

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144 KILIAN, MESTECKY, AND SCHROHENLOHER

H. parainfluenzae, H. aphrophilus, H. paraphrophi-lus, H. segnis, and H. pleuropneumoniae have beenpreviously described in detail (5). The Haemophilusand S. pneumoniae strains are listed in Table 1, whichprovides information on serotype and site of isolation.S. sanguis strain ATCC 10556 obtained from theAmerican Type Culture Collection, Rockville, Md.,and a clinical isolate of N. meningitidis (strain VK 4)served as positive controls for IgAl protease activity.Haemophilus, S. pneumoniae, and Neisseria strainswere cultivated on chocolate agar (blood agar base[Difco Laboratories, Detroit, Mich.] with 10% [vol/vol] heated, defibrinated horse blood). Agar plateswere incubated at 37°C in air with an additional 5%CO2 in accordance with the growth requirements ofthe individual strains. S. sanguis was cultivated onbrain heart infusion agar (Difco) incubated under mi-croaerophilic conditions.Immunoglobulin preparations. IgA, IgM, and

IgG paraproteins were isolated from plasma of patientswith multiple myeloma or Waldenstrom's macroglob-ulinemia. Details of the purification procedures, whichincluded ammonium sulfate precipitation, gel filtrationon Sephadex G-200 and Sepharose 6B, and ion ex-change chromatography on diethylaminoethyl-Seph-adex, have been described (9, 10). The properties ofthe IgA myeloma proteins such as sedimentation con-

stant, carbohydrate composition, L-chain type, pres-ence of J chain, and IgA subclass (determined bymonospecific antisera and carbohydrate compositions)have been reported (9). Polyclonal IgG was isolatedfrom normal human serum. S-IgA was isolated frompooled colostrum and processed as described previ-ously (10). Immunochemical and ultracentrifugationanalyses revealed that colostral S-IgA consisted of 15Sand llS forms, with a preponderance of the latter type(23). Purified S-IgA isolated from pig milk was a giftfrom P. Porter, Unilever Research, Bedford, U.K.Bovine S-IgA, partly purified by the above-describedmethods (10), was a gift from A. Bhown, University ofAlabama in Birmingham.

Preparation of IgA protease. IgA protease wasprepared as described by Higerd et al. (4). Suspensionsof the respective bacterial strains in sterile saline wereinoculated onto the surface of presterilized dialysismembranes placed on chocolate or brain heart infusionagar plates. After incubation for 2 days at 37°C themembranes were removed from the agar surface andwashed in a minimal volume of 0.05 M potassiumphosphate buffer, pH 7.5, with 0.85% (wt/vol) NaCl(PBS). The wash was clarified by centrifugation at30,000 x g for 15 min at 4°C and was concentrated 20times by positive-pressure ultrafiltration using an

Amicon PM 10 membrane.Detection of IgA protease activity. Concen-

trated protease preparations were incubated for 3 h at37°C with equal volumes of the respective immuno-globulins dissolved in PB3S at a concentration of 5 mg/ml. Controls contained buffer instead of enzyme prep-

aration. For the initial screening of strains for IgAprotease activity, individual colonies from 1- to 2-daychocolate agar cultures were suspended in 50 p1 of a 5

mg/ml immunoglobulin solution which was then in-cubated for 15 to 18 h at 37°C. Proteolysis was de-tected by three methods: (i) immunoelectrophoresis,

(ii) sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis, and (iii) analytical ultracentrifugation.

Immunoelectrophoresis. Immunoelectrophoresisof digested proteins and undigested controls was per-formed in 2% agar in Veronal buffer, pH 8.6. Antiseraused for development of immunoelectrophoresis wereunabsorbed rabbit anti-human S-IgA (10), and com-mercial, monospecific antisera against K- or A-lightchains (Meloy, Springfield, Va.) and a-chain of IgA(Hyland Laboratories, Costa Mesa, Calif., and BehringDiagnostics, Somerville, N.J.).SDS-polyacrylamide gel electrophoresis. Sam-

ples were electrophoresed in sodium phosphate buffer,pH 7.2, in the presence of 0.1% SDS and 7 M urea bythe method of Weber and Osborn (21). To cleavedisulfide bridges, immunoglobulins were reduced bymixing samples with an equal volume of a solution of8 M urea, 2% SDS, and 0.2 M 2-mercaptoethanol for30 to 60 min prior to gel electrophoresis. Apparentmolecular weights offragments observed in SDS-poly-acrylamide gels were calculated as described by Weberand Osborn (21), with the following proteins withknown molecular weights used as standards: humanIgG paraprotein (150,000), ovalbumin (45,000), chy-motrypsinogen (25,000), and chicken egg white lyso-zyme (14,400). The molecular weights of fragments inreduced samples were determined with reference toreduced and purified L- and H-chains of IgG.

Analytical ultracentrifugation. Digested andundigested proteins were centrifuged concurrently ina Spinco model E analytical ultracentrifuge (BeckmanInstruments, Inc.) at 56,000 rpm and 20'C. Samples,which contained 10 mg of protein per ml, were dialyzedagainst PBS prior to analysis. A 600 phase-plate anglewas used for photographs taken at 8-min intervals.Sedimentation rates were calculated by the method ofSchachman (16) on the basis of measurements carriedout with a Nikon microcomparator (Nippon Kogaku,Tokyo, Japan).

Gel filtration. A 20-mg amount of polymeric IgAlparaprotein (Car) was digested with an IgAl proteasepreparation from H. influenzae strain HK 393 for 16h at 37°C. The digested protein was chromatographedon a Sephadex G-200 column (1.6 by 90 cm) in 1%ammonium bicarbonate buffer. Pooled fractions (seebelow) were lyophilized, redissolved in Veronal buffer,pH 8.6, and used for antigenic and molecular-weightanalyses.

RESULTSIgAl protease-producing bacteria. Ex-

amination ofthe bacterial strains (listed in Table1) for IgA protease activity revealed that allstrains of H. influenzae, H. aegyptius, and S.pneumoniae cleaved IgAl proteins. The IgAlprotease-producing strains ofH. influenzae rep-resented all five biotypes of this species andincluded encapsulated strains isolated from pa-tients with meningitis and other infections aswell as nonencapsulated strains from upper res-piratory tracts of healthy individuals. None ofthe 16 strains of other Haemophilus speciesproduced an enzyme with the ability to cleave

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VOL. 26, 1979

TABLE 1. Bacterial stroSpecies/biotype and strain

H. influenzae IHK 66HK 193, 194, 195HK 393 - NCTC 8467HK 395 = CIP 5423

H. influenza IIHK 21, 24, 25, 30HK 208HK 402 = NCTC 7918

H. influenza IIIHK 387 = NCTC 4560HK 57

H. influenza IVHK 368 = CIP 5424HK 397 = NCTC 8470

H. influenza VHK 223, 238, 240

H. aegyptiusHK 368 = ATCC 11116HK 270

H. parainfluenzaeHK 47HK 82, 90, 133HK 409

H. aphrophilusHK 308, 315HK 371 = NCTC 5886

H. paraphrophilusHK 83HK 414 = NCTC 10556HK 415 = NCTC 10557

H. segnisHK 84HK 307HK 316 = NCTC 10977

H. pleuropneumoniaeHK 405 = ATCC 27088HK 407

S. pneumoniaeVK 3VK 5VK 6VK 7,8

IMMUNOGLOBULIN Al PROTEASE 145

ains used in the study, their origin and ability to cleave human IgAl and S-IgAaSerotype Origin IgA 1 protease activity

bb

bf

ThroatMeningitis

ThroatMeningitisPus, mastoid

Throat

d Throat

Otitis media

ConjunctivitisConjunctivitis

ThroatSalivaSeptic finger

Dental plaqueEndocarditis

SalivaAbscessParonychia

SalivaDental plaqueDental plaque

31ND

Pleuropneumonia, pigPleuropneumonia, pig

SputumBloodMeningitisBlood

+

+

+

+

+

+

Cleave porcine s-IgAbut not human IgA

a Abbreviations: HK, strain collection studied by Kilian (5); NCTC, National Collection of Type Cultures,London, U.K.; CIP, Collection de l'Institut Pasteur, Paris, France; ATCC, American Type Culture Collection,Rockville, Md.; ND, not done.

IgAl. As a control, the IgAl proteins used in theassay were cleaved by S. sanguis ATCC 10556and N. meningitidis VK 4, both of which areknown producers of IgAl protease (12). IgAlprotein (Car) digested by the two latter strainswas compared, by immunoelectrophoresis, withthe same protein incubated with H. influenzaeHK 393 and S. pneumoniae VK 7. Apparentresemblance was noted in the immunoelectro-phoresis pattern of IgAl protein digested by S.sanguis and S. pneumoniae. IgAl digested byH. influenza and N. meningitidis resulted in amutually similar immunoelectrophoresis pattern

which, however, differed from that observedwith the two streptococcal species (Fig. 1).Substrate specificity. Enzyme preparations

from strains of the species H. influenza, H.aegyptius, and S. pneumoniae cleaved each ofthree IgAl paraproteins including polymeric andmonomeric IgA ofboth kappa and lambda types.SDS-polyacrylamide gel electrophoresis and an-alytical ultracentrifugation (see below) indicatedthat fully active enzyme preparations causedvirtually complete cleavage of IgAl within 3 hof incubation. The same enzyme preparationswere also capable of cleaving S-IgA, but only a

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146 KILIAN, MESTECKY, AND SCHROHENLOHER

molecular weight of 84,000. This protein waspreviously identified as secretary component(10). After incubation with IgA protease, mono-meric IgAl was cleaved into two fragments withapparent molecular weights of 65,000 (majorcomponent) and 54,000 (minor component). Di-gested polymeric IgAl protein showed twobands with molecular weights of 65,000 (majorband) and 127,000. In contrast, the major portionof S-IgA remained undigested. However, twobands with molecular weights of 65,000 and193,000 were also observed. Pig S-IgA incubatedwith strains of H. pleuropneumoniae wascleaved into several components (Fig. 3C andD). Because of the limited amount of pig S-IgAavailable, further analyses were not performed.To purify the fragments of cleaved IgAl pro-

tein, we applied 20 mg of a polymeric IgAl-Kparaprotein (Car), digested by protease derived

FIG. 1. Immunoelectrophoresis of IgAl myelomaprotein (Car) incubated with: (A) PBS, (B) H. influ-enzae HK 393, (C) S. pneumoniae VK 7, (D) N. men-ingitidis VK 4, and (E) S. sanguis ATCC 10556.Antiserum: rabbit anti-S-IgA, not adsorbed for L-chain reactivity. Anode to the left.

part of the available protein was digested afterincubation for up to 18 h. Proteolysis was notobserved with IgA2, IgG, and IgM paraproteins,or with normal serum IgG and porcine or bovineS-IgA. Human serum albumin, bovine serumalbumin, and egg albumin were also resistant toproteolysis. Enzyme preparations from twostrains of H. pleuropneumoniae isolated fromfatal cases of pleuropneumonia in pigs cleavedporcine S-IgA, but did not affect any of thehuman immunoglobulins, including IgAl.Properties of cleavage products. Immu-

noelectrophoresis of digested IgAl and S-IgArevealed two separate fragments with differentelectrophoretic mobilities (Fig. 2). A fragmentwhich had a slow electrophoretic mobility simi-lar to that of the undigested protein reactedstrongly with anti-L-chain sera but not withanti-a-chain serum. Conversely, the second frag-ment reacted only with anti-a-chain serum. Onthe basis of these properties, the two fragmentswere identified as Fab and Fc, respectively.

All proteins used in the study, including im-munoglobulins of IgA, IgG, and IgM classes andalbumins, were subjected to SDS-polyacryl-amide gel electrophoresis before and after incu-bation with IgAl protease preparations (Fig. 3).Reduced samples of undigested monomeric andpolymeric IgAl migrated as two bands with ap-

parent molecular weights of21,000 (L-chain) and52,000 (H-chains); S-IgA contained, in additionto H- and L-chains, a protein with an apparent

IgA

... ..;.A:.....,>:..

Fab*g

IgA

1

*

IgA

*%Vi./

2'

.SlgA

FIG. 2. Antigenic analyses of polymeric IgAl-Kmyeloma protein (Car) and S-IgA. Samples digestedwith IgAl protease from H. influenza are labeledwith an asterisk. Antisera: (1) rabbit anti-S-IgA, notadsorbed for L-chain reactivity, (2) rabbit anti-K-chain, (3) rabbit anti-a chain. Anode to the right.

..~~~~~~~~~~~~~~~~~....~~~~~~~~~~~~..'Nip

21,

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IMMUNOGLOBULIN Al PROTEASE 147

a1

B.

A B CDFIG. 3. SDS-polyacrylamide gel electrophoresis

patterns of IgA proteins. Gel A shows human oligo-meric IgAl-K protein before cleavage (arrow). Arrowon gel B indicates principal cleavage product of thesame myeloma IgA protein after digestion with IgAlprotease from H. influenza. Porcine S-IgA before (C)and after (D) digestion with IgA protease from H.pleuropneumoniae.

from H. influenzae HK 393, to a Sephadex G-200 column. The fragments of the polymericIgAl protein were eluted in two major peaksthat were divided into four fractions as indicatedin Fig. 4. From subsequent examinations byimmunoelectrophoresis and SDS-polyacryl-amide gel electrophoresis, it was concluded thatfraction 4 corresponded to the Fab fragment: itreacted with anti-L-chain serum, had an appar-

ent molecular weight of 65,000, and resolved,after reduction, into two polypeptides (molecu-lar weights of 21,000 and 26,000). Fractions 1through 3 contained proteins with apparent mo-lecular weights of 137,000, 76,000, and 62,000. Onimmunoelectrophoresis, they reacted with an

anti-a-chain but not with an anti-L-chain serum.After reduction, a major band with a molecularweight of 27,000 was observed. Based on thesedata, we concluded that fractions 1 through 3contained Fc fragments in various molecularconfigurations.Undigested and digested (18 h at 370C) mon-

omeric and polymeric IgAl proteins and S-IgAwere also examined by analytical ultracentrifu-gation (Fig. 5). The results supported the obser-vations described above. Fragments of digestedmonomeric IgAl protein (Pet) sedimented at therates of 3.OS (major fragment) and 4.5S (minorfragment) (Fig. 5A). Polymeric IgAl protein

EC0

*0.4C

.0

0.2

1 23 4

50 100 150Effluent volume (ml)

FIG. 4. Gel filtrationpattern ofIgAl myelomapro-tein (Car) (20 mg), digested with IgAl protease fromH. influenza. Column: Sephadex G-200 in 1% am-monium bicarbonate buffer, 1.6 by 90 cm, downwardflow.

(Kni), which consisted principally of compo-nents sedimenting at 9, 11, and 13S plus smallamounts of 7, 14, and 17S material, was cleavedinto a major fragment with a sedimentation rateof 3.7S and a heterogeneous fragment of 7S (Fig.5B). S-IgA (initially composed of 11 and 15SIgA) remained largely undigested although afragment with a sedimentation rate of 3.1S wasdetected (Fig. 5C). The reported sedimentationrates were not corrected by extrapolating toinfinite protein dilution and, accordingly, maybe influenced by the concentration of the indi-vidual components.

DISCUSSIONIn this study we demonstrated that strains of

H. influenzae, H. aegyptius, and S. pneumoniaereleased a protease which specifically cleavedhuman IgAl and partially cleaved S-IgA, buthad no effect on a variety of other proteins,including human IgA2, IgG, and IgM, as well asporcine and bovine S-IgA. The immunochemicaland ultracentrifugation analyses indicated thatthe protease(s) from both of these organismseffectively cleaved the IgAl molecule at thehinge region of the a-chain, thus releasing Faband Fc fragments. In regard to substrate speci-ficity and cleavage products generated, the en-zymes from H. influenzae, H. aegyptius, and S.pneumoniae are analogous to those produced byN. meningitidis, N. gonorrhoeae, and S. sanguis(11-15). It has been established that enzymesfrom the latter two organisms cleave a prolyl-threonyl bond in the hinge region of the al-

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148 KILIAN, MESTECKY, AND SCHROHENLOHER

FIG. 5. Ultracentrifugal patterns of (A) monomeric IgAl-K myeloma protein (Pet), (B) polymeric IgAl-Kmyeloma protein (Kni), and (C) S-IgA digested with H. influenzae IgA1 protease for 18 h at 370C. Undigestedsample in the lower field; digested sample above. Protein concentrations were 10 mg/ml in all samples. Thepatterns were recorded after 48 min (A) and 32 min (B and C) of centrifugation. Arrows indicate fragments ofthese IgA proteins (see text).

chain (12, 13). The immunoelectrophoretic mo-bilities ofthe Fc fragments released by the actionof proteases from H. influenzae, N. meningiti-dis, S. pneumoniae, and S. sanguis suggest thatthe former two species cleave the IgAl moleculeat a site which differs from that of the latter twoorganisms. Nevertheless, the fact that IgA2 pro-tein was resistant to proteases produced by allfour organisms indicates that the susceptiblebond is located within the 13-amino acid seg-ment that distinguishes the a-chain of IgAl sub-class. Amino acid sequence analyses of the Fcfragments will be required to establish the exactcleavage sites of IgAl protease derived from H.influenzae and S. pneumoniae.Both H. influenzae and S. pneumoniae may

be found as part of the normal flora of the upperrespiratory tract. However, these species are alsoimplicated in a variety of infectious diseaseswhich originate at the mucous membranes ofthe respiratory tract. H. aegyptius, which isclosely related to H. influenza (5), is implicatedin cases of acute conjunctivitis. The possibilitythat IgAl protease production constitutes a sig-nificant virulence factor is supported by the factthat the enzyme was absent in the species H.parainfluenzae, H. aphrophilus, H. parapro-philus, and H. segnis, all of which may be con-sidered opportunistic pathogens (5, 6). A similarrelationship between pathogenic potential andIgAl protease activity has recently been dem-onstrated in the genus Neisseria (11). With ourobservation that both H. influenza and S.pneumoniae produce IgAl proteases, this char-acteristic has now been established for all of thethree major etiological agents of bacterial men-ingitis.The species H. influenza has been subdi-

vided into five biotypes which correlate with

specific disease entities (5). For example, biotypeI is almost exclusively implicated in meningitisand epiglottitis, whereas biotype III is rarelyisolated from acute infections and is more oftenfound as a part of the normal flora in the upperrespiratory tract. Since IgAl protease was foundin all of these five biotypes it is apparent thatthis property may be only one of the parametersdetermining pathogenicity.

It is of considerable interest that two strainsof the species H. pleuropneumoniae digestedporcine S-IgA but not the human immunoglob-ulins. This species is the cause of a highly con-tagious and often fatal pleuropneumonic infec-tion in pigs, but human infections due to thisorganism have not been reported. Conversely,H. influenza rarely, if ever, produces naturalinfections in animal species. The ability of theseorganisms to cleave IgA of their specific hostsmay be one of the factors that would explain thepoorly understood species specificity of certaininfectious diseases.Although the function of IgAl protease has

not been clearly established, the detection of Fcfragment in stools (8) and in secretions of N.gonorrhoeae-infected vaginal tracts (1) indicatesthat cleavage of IgA occurs in vivo. Furtherstudies are required to determine the biologicalconsequences of S-IgA and serum IgAl cleavageon inhibitions of bacterial adherence (18, 22), ofbactericidal effect (2, 3), and of phagocytosis (7,17, 19, 20).

ACKNOWLEDGMENTSWe thank Rose Kulhavy and Kenneth L. Roland for excel-

lent technical assistance, Philip Porter for providing us withpurified porcine S-IgA and corresponding antisera, and F. W.Kraus, S. Jackson, and J. L. Babb for their comments.

This work was supported by Public Health Service contractDE-52456 and grant AI 10854 from the National Institutes ofHealth.

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VOL. 26, 1979

LITERATURE CITED

1. Blake, M., K. K. Holmes, and J. Swanson. 1979. Stud-ies of gonococcus infection. XVII. IgAl-cleaving pro-tease in vaginal washings from women with gonorrhea.J. Infect. Dis. 139:89-92.

2. Griffiss, J. M. 1975. Bactericidal activity of meningococ-cal antisera. Blocking by IgA of lytic antibody in humanconvalescent sera. J. Immunol. 114:1779-1784.

3. Griffiss, J. M., and M. A. Bertram. 1977. Immunoepi-demiology of meningococcal disease in military recruits.II. Blocking of serum bactericidal activity by circulatingIgA early in the course of invasive disease. J. Infect.Dis. 136:733-739.

4. Higerd, T. B., G. Virella, R. Cardenas, J. Koistinen,and J. W. Fett. 1977. New method for obtaining IgA-specific protease. J. Immunol. Methods 18:245-249.

5. Kilian, M. 1976. A taxonomic study of the genus Hae-mophilus with the proposal of a new species. J. Gen.Microbiol. 93:9-62.

6. Kilian, M., and C. R. Schi0tt. 1975. Haemophili andrelated bacteria in the human oral cavity. Arch. OralBiol. 20:791-796.

7. Magnusson, K.-E., 0. Stendahl, I. Stjernstrdm, andL Edebo. 1979. Reduction of phagocytosis, surfacehydrophobicity and charge of Sabnonella typhimurium395 MR1O by reaction with secretary IgA (SIgA). Im-munology 36:439-447.

8. Mehta, S. K., A. G. Plaut, N. J. Calvanico, and T. B.Tomasi, Jr. 1973. Human immunoglobulin A. Produc-tion of an Fc fragment by an enteric microbial proteo-lytic enzyme. J. Immunol. 111:1274-1276.

9. Mestecky, J., W. J. Hammack, R. Kulhavy, G. P.Wright, and M. Tomana. 1977. Properties of IgAmyeloma proteins isolated from sera of patients withthe hyperviscosity syndrome. J. Lab. Clin. Med. 89:919-927.

10. Mestecky, J., RK Kulhavy, and F. W. Kraus. 1972.Studies on human secretary immunoglobulin A. II. Sub-unit structure. J. Immunol. 108:738-747.

11. Mulks, M. H., and A. G. Plaut. 1978. IgA proteaseproduction as a characteristic distinguishing pathogenicfrom harmless Neisseriaceae. N. Engl. J. Med. 299:

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973-976.12. Plaut, A. G. 1978. Microbial IgA protease. N. Engl. J.

Med. 298:1459-1463.13. Plaut, A. G., J. V. Gilbert, M. S. Artenstein, and J. D.

Capra. 1975. Neisseria gonorrhoeae and Neisseriameningitidies: extracellular enzyme cleaves human im-munoglobulin A. Science 190:1103-1105.

14. Plaut, A. G., J. V. Gilbert, and R. Wistar, Jr. 1977.Loss of antibody activity in human immunoglobulin Aexposed to extracellular immunoglobulin A proteases ofNeisseria gonorrhoeae and Streptococcus sanguis. In-fect. Immun. 17:130-135.

15. Plaut, A. G., R. Wistar, Jr., and J. D. Capra. 1974.Differential susceptibility of human IgA immunoglob-ulins to streptococcal IgA protease. J. Clin. Invest. 54:1295-1300.

16. Schachman, H. K. 1959. Ultracentrifugation in biochem-istry, p. 63-180. Academic Press Inc., New York.

17. Spiegelberg, H. L., D. A. Lawrence, and P. Hanson.1974. Cytophilic properties of IgA to human neutro-phils. Adv. Exp. Med. Biol. 45:67-74.

18. Svanborg-Eden, C., and A.-M. Svennerholm. 1978.Secretory immunoglobulin A and G antibodies preventadhesion of Escherichia coli to human urinary tractepithelial cells. Infect. Immun. 22:790-797.

19. Van Epps, D. E., K. Reed, and R. C. Williams, Jr.1978. Suppression of human PMN bactericidal activityby human IgA paraproteins. Cell. Immunol. 36:363-376.

20. Van Epps, D. E., and R. C. Williams, Jr. 1976. Sup-pression of leukocyte chemotaxis by human IgA mye-loma components. J. Exp. Med. 144:1227-1242.

21. Weber, K., and M. Osborn. 1969. The reliability ofmolecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.

22. Williams, R. C., Jr., and R. J. Gibbons. 1972. Inhibitionof bacterial adherence by secretory immunoglobulin A:a mechanism of antigen disposal. Science 177:697-699.

23. Zikan, J., J. Mestecky, R. E. Schrohenloher, M. To-mana, and R. Kulhavy. 1972. Studies on human se-cretory immunoglobulin A. V. Trypsin hydrolysis atelevated temperatures. Immunochemistry 9:1185-1193.

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