INFECTION AND IMMUNITY, Oct. 1979, p. 143-149 0019-9567/79/10-0143/07$02.00/0
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Pathogenic Species of the Genus Haemophilus and Streptococcus pneumoniae Produce Immunoglobulin Al Protease MOGENS KILIAN,t* JIRI MESTECKY, AND RALPH E. SCHROHENLOHER Department of Microbiology, Institute of Dental Research and Division of Clinical Immunology and Rheumatology, University ofAlabama in Birmingham, Birmingham, Alabama 35294 Received for publication 4 May 1979
Thirty-seven strains of the genus Haemophilus and five strains of Streptococcus pneumoniae were examined for their ability to produce extracellular enzyme that cleaves immunoglobulin molecules. All strains of H. influenza, H. aegyptius, and S. pneumoniae elaborated enzyme that selectively cleaved human immunoglobulin Al (IgAl) myeloma proteins but was inactive against a variety of other proteins including human IgA2, IgG, and IgM, porcine and bovine secretary IgA, human and bovine serum albumins, and ovalbumin. Although susceptible, human secretary IgA remained largely undigested. Two strains of H. pleuropneumoniae isolated from fatally infected pigs cleaved porcine secretary IgA, but had no effect on human IgA proteins. None of 16 strains that belonged to nonpathogenic Haemophilus species produced IgA protease. Analyses of the cleavage products of human IgAl and secretary IgA proteins by immunochemical methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analytical ultracentrifugation revealed that Fab and Fc fragments were produced. Since the production of IgAl protease by Neisseria meningitidis has been reported previously, our finding that H. influenzae and S. pneumoniae produce an IgAl protease indicates that 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 the pathogenesis of this disease. Mucosal surfaces of the upper respiratory resulted in a considerable loss of antibody activtract are inhabited by a broad spectrum of bac- ity (14), it appears probable that these IgA proteria some of which may, under conditions that teases represent a significant virulence factor. are poorly understood, invade mucosal tissues A classic example of a systemic infection and produce systemic infection. Certain bacteria which is initiated by bacteria that gain access which colonize mucosal surfaces, such as Neis- into the circulation through the mucous memseria meningitidis, N. gonorrhoeae, and Strep- branes of the respiratory tract is bacterial mentococcus sanguis, were shown to produce an ingitis. N. nningitidis, Haemophilus influextracellular enzyme that cleaves serum immu- enzae, and S. pneumoniae represent the princinoglobulins belonging to immunoglobulin Al pal bacterial species responsible for this disease. (IgAl) but not IgA2, IgG, or IgM classes (11-15). It was previously reported that pathogenic Secretory IgA (S-IgA), the principal carrier of strains of N. meningitidis produce IgAl protease specific humoral defense on mucous surfaces, is (11), and we report in this communication that only partly susceptible to digestion by bacterial the other two leading causative agents of bacIgA proteases. This relative insusceptibility may terial meningitis, H. influenzae and S. pneumobe explained by the presence of a higher propor- niae, also produce an enzyme which cleaves tion of IgA2 in S-IgA than in serum IgA (12), by IgAl and to a limited extent S-IgA, but has no the association of IgA with secretary component effect on IgA2, IgM, and IgG. which renders S-IgA resistant to various proMATERIALS AND METHODS teases, and by the presence of S-IgA-associated Bacterial strains. Thirty-seven human and porantibodies that may neutralize IgA protease activity (A. G. Plaut et al., Fed. Proc. 38:5275, cine isolates of the genus Haemophilus and five strains blood, cerebro1979). Since cleavage of IgAl by such enzymes of S. pneumoniae isolated from human spinal fluid, and sputum were included in the study. t Present address: Department of Microbiology, Royal The Haemophilus strains that represent each of five Dental College, DK-8000 Aarhus, Denmark. biotypes of H. influenza and the species H. aegyptius, 143
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H. parainfluenzae, H. aphrophilus, H. paraphrophi- (ii) sodium dodecyl sulfate (SDS)-polyacrylamide gel lus, H. segnis, and H. pleuropneumoniae have been electrophoresis, and (iii) analytical ultracentrifugation. Immunoelectrophoresis. Immunoelectrophoresis previously described in detail (5). The Haemophilus and S. pneumoniae strains are listed in Table 1, which of digested proteins and undigested controls was perprovides information on serotype and site of isolation. formed in 2% agar in Veronal buffer, pH 8.6. Antisera S. sanguis strain ATCC 10556 obtained from the used for development of immunoelectrophoresis were American Type Culture Collection, Rockville, Md., unabsorbed rabbit anti-human S-IgA (10), and comand a clinical isolate of N. meningitidis (strain VK 4) mercial, monospecific antisera against K- or A-light served as positive controls for IgAl protease activity. chains (Meloy, Springfield, Va.) and a-chain of IgA Haemophilus, S. pneumoniae, and Neisseria strains (Hyland Laboratories, Costa Mesa, Calif., and Behring Diagnostics, Somerville, N.J.). were cultivated on chocolate agar (blood agar base SDS-polyacrylamide gel electrophoresis. Sam[Difco Laboratories, Detroit, Mich.] with 10% [vol/ vol] heated, defibrinated horse blood). Agar plates ples were electrophoresed in sodium phosphate buffer, pH 7.2, in the presence of 0.1% SDS and 7 M urea by were incubated at 37°C in air with an additional 5% CO2 in accordance with the growth requirements of the method of Weber and Osborn (21). To cleave the individual strains. S. sanguis was cultivated on disulfide bridges, immunoglobulins were reduced by brain heart infusion agar (Difco) incubated under mi- mixing samples with an equal volume of a solution of 8 M urea, 2% SDS, and 0.2 M 2-mercaptoethanol for croaerophilic conditions. Immunoglobulin preparations. IgA, IgM, and 30 to 60 min prior to gel electrophoresis. Apparent IgG paraproteins were isolated from plasma of patients molecular weights of fragments observed in SDS-polywith multiple myeloma or Waldenstrom's macroglob- acrylamide gels were calculated as described by Weber ulinemia. Details of the purification procedures, which and Osborn (21), with the following proteins with included ammonium sulfate precipitation, gel filtration known molecular weights used as standards: human IgG paraprotein (150,000), ovalbumin (45,000), chyon Sephadex G-200 and Sepharose 6B, and ion exchange chromatography on diethylaminoethyl-Seph- motrypsinogen (25,000), and chicken egg white lysoadex, have been described (9, 10). The properties of zyme (14,400). The molecular weights of fragments in the IgA myeloma proteins such as sedimentation con- reduced samples were determined with reference to stant, carbohydrate composition, L-chain type, pres- reduced and purified L- and H-chains of IgG. Analytical ultracentrifugation. Digested and ence of J chain, and IgA subclass (determined by monospecific antisera and carbohydrate compositions) undigested proteins were centrifuged concurrently in have been reported (9). Polyclonal IgG was isolated a Spinco model E analytical ultracentrifuge (Beckman from normal human serum. S-IgA was isolated from Instruments, Inc.) at 56,000 rpm and 20'C. Samples, pooled colostrum and processed as described previ- which contained 10 mg of protein per ml, were dialyzed ously (10). Immunochemical and ultracentrifugation against PBS prior to analysis. A 600 phase-plate angle analyses revealed that colostral S-IgA consisted of 15S was used for photographs taken at 8-min intervals. and llS forms, with a preponderance of the latter type Sedimentation rates were calculated by the method of (23). Purified S-IgA isolated from pig milk was a gift Schachman (16) on the basis of measurements carried from P. Porter, Unilever Research, Bedford, U.K. out with a Nikon microcomparator (Nippon Kogaku, Bovine S-IgA, partly purified by the above-described Tokyo, Japan). methods (10), was a gift from A. Bhown, University of Gel filtration. A 20-mg amount of polymeric IgAl Alabama in Birmingham. paraprotein (Car) was digested with an IgAl protease Preparation of IgA protease. IgA protease was preparation from H. influenzae strain HK 393 for 16 prepared as described by Higerd et al. (4). Suspensions h at 37°C. The digested protein was chromatographed of the respective bacterial strains in sterile saline were on a Sephadex G-200 column (1.6 by 90 cm) in 1% inoculated onto the surface of presterilized dialysis ammonium bicarbonate buffer. Pooled fractions (see membranes placed on chocolate or brain heart infusion below) were lyophilized, redissolved in Veronal buffer, agar plates. After incubation for 2 days at 37°C the pH 8.6, and used for antigenic and molecular-weight membranes were removed from the agar surface and analyses. washed in a minimal volume of 0.05 M potassium phosphate buffer, pH 7.5, with 0.85% (wt/vol) NaCl RESULTS (PBS). The wash was clarified by centrifugation at IgAl protease-producing bacteria. Ex30,000 x g for 15 min at 4°C and was concentrated 20 times by positive-pressure ultrafiltration using an amination ofthe bacterial strains (listed in Table Amicon PM 10 membrane. 1) for IgA protease activity revealed that all Detection of IgA protease activity. Concen- strains of H. influenzae, H. aegyptius, and S. trated protease preparations were incubated for 3 h at pneumoniae cleaved IgAl proteins. The IgAl 37°C with equal volumes of the respective immuno- protease-producing strains of H. influenzae repglobulins dissolved in PB3S at a concentration of 5 mg/ ml. Controls contained buffer instead of enzyme prep- resented all five biotypes of this species and aration. For the initial screening of strains for IgA included encapsulated strains isolated from paprotease activity, individual colonies from 1- to 2-day tients with meningitis and other infections as chocolate agar cultures were suspended in 50 p1 of a 5 well as nonencapsulated strains from upper resmg/ml immunoglobulin solution which was then in- piratory tracts of healthy individuals. None of cubated for 15 to 18 h at 37°C. Proteolysis was de- the 16 strains of other Haemophilus species tected by three methods: (i) immunoelectrophoresis, produced an enzyme with the ability to cleave
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TABLE 1. Bacterial stroains used in the study, their origin and ability to cleave human IgAl and S-IgAa Species/biotype and strain Serotype Origin IgA 1 protease activity H. influenzae I HK 66 Throat HK 193, 194, 195 b Meningitis + HK 393 - NCTC 8467 b HK 395 = CIP 5423 H. influenza II HK 21, 24, 25, 30 Throat + HK 208 b Meningitis HK 402 = NCTC 7918 f Pus, mastoid H. influenza III HK 387 = NCTC 4560 + HK 57 Throat H. influenza IV HK 368 = CIP 5424 + HK 397 = NCTC 8470 d Throat H. influenza V HK 223, 238, 240 Otitis media + H. aegyptius = HK 368 ATCC 11116 Conjunctivitis + HK 270 Conjunctivitis H. parainfluenzae HK 47 Throat HK 82, 90, 133 Saliva HK 409 Septic finger H. aphrophilus HK 308, 315 Dental plaque HK 371 = NCTC 5886 Endocarditis H. paraphrophilus HK 83 Saliva HK 414 = NCTC 10556 Abscess HK 415 = NCTC 10557 Paronychia H. segnis HK 84 Saliva HK 307 Dental plaque HK 316 = NCTC 10977 Dental plaque H. pleuropneumoniae HK 405 = ATCC 27088 Pleuropneumonia, pig Cleave porcine s-IgA HK 407 Pleuropneumonia, pig but not human IgA S. pneumoniae VK 3 Sputum VK 5 3 Blood VK 6 1 Meningitis VK 7,8 ND Blood 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 the assay were cleaved by S. sanguis ATCC 10556 and N. meningitidis VK 4, both of which are known producers of IgAl protease (12). IgAl protein (Car) digested by the two latter strains was compared, by immunoelectrophoresis, with the same protein incubated with H. influenzae HK 393 and S. pneumoniae VK 7. Apparent resemblance was noted in the immunoelectrophoresis pattern of IgAl protein digested by S. sanguis and S. pneumoniae. IgAl digested by H. influenza and N. meningitidis resulted in a mutually similar immunoelectrophoresis pattern
which, however, differed from that observed with the two streptococcal species (Fig. 1). Substrate specificity. Enzyme preparations from strains of the species H. influenza, H. aegyptius, and S. pneumoniae cleaved each of three IgAl paraproteins including polymeric and monomeric IgA of both kappa and lambda types. SDS-polyacrylamide gel electrophoresis and analytical ultracentrifugation (see below) indicated that fully active enzyme preparations caused virtually complete cleavage of IgAl within 3 h of incubation. The same enzyme preparations were also capable of cleaving S-IgA, but only a
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~ ~ ~.
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KILIAN, MESTECKY, AND SCHROHENLOHER
molecular weight of 84,000. This protein was previously identified as secretary component (10). After incubation with IgA protease, mono..~~~~ ~~~~~~~~~~~~~.... meric IgAl was cleaved into two fragments with apparent molecular weights of 65,000 (major component) and 54,000 (minor component). Digested polymeric IgAl protein showed two bands with molecular weights of 65,000 (major band) and 127,000. In contrast, the major portion of S-IgA remained undigested. However, two 'Nip bands with molecular weights of 65,000 and 193,000 were also observed. Pig S-IgA incubated with strains of H. pleuropneumoniae was cleaved into several components (Fig. 3C and D). Because of the limited amount of pig S-IgA available, further analyses were not performed. To purify the fragments of cleaved IgAl protein, we applied 20 mg of a polymeric IgAl-K paraprotein (Car), digested by protease derived FIG. 1. Immunoelectrophoresis of IgAl myeloma protein (Car) incubated with: (A) PBS, (B) H. influenzae HK 393, (C) S. pneumoniae VK 7, (D) N. meningitidis VK 4, and (E) S. sanguis ATCC 10556. Antiserum: rabbit anti-S-IgA, not adsorbed for Lchain reactivity. Anode to the left.
IgA ... .. ;.A :.....,>:..
part of the available protein was digested after incubation for up to 18 h. Proteolysis was not observed with IgA2, IgG, and IgM paraproteins, or with normal serum IgG and porcine or bovine S-IgA. Human serum albumin, bovine serum albumin, and egg albumin were also resistant to proteolysis. Enzyme preparations from two strains of H. pleuropneumoniae isolated from fatal cases of pleuropneumonia in pigs cleaved porcine S-IgA, but did not affect any of the human immunoglobulins, including IgAl. Properties of cleavage products. Immunoelectrophoresis of digested IgAl and S-IgA revealed two separate fragments with different electrophoretic mobilities (Fig. 2). A fragment which had a slow electrophoretic mobility similar to that of the undigested protein reacted strongly with anti-L-chain sera but not with anti-a-chain serum. Conversely, the second fragment reacted only with anti-a-chain serum. On the basis of these properties, the two fragments were identified as Fab and Fc, respectively. All proteins used in the study, including immunoglobulins of IgA, IgG, and IgM classes and albumins, were subjected to SDS-polyacrylamide gel electrophoresis before and after incubation with IgAl protease preparations (Fig. 3). Reduced samples of undigested monomeric and polymeric IgAl migrated as two bands with apparent molecular weights of 21,000 (L-chain) and 52,000 (H-chains); S-IgA contained, in addition to H- and L-chains, a protein with an apparent
1
Fab g
*
IgA
21, *
IgA
I 2' *%Vi./
.SlgA
FIG. 2. Antigenic analyses of polymeric IgAl-K myeloma protein (Car) and S-IgA. Samples digested with IgAl protease from H. influenza are labeled with an asterisk. Antisera: (1) rabbit anti-S-IgA, not adsorbed for L-chain reactivity, (2) rabbit anti-Kchain, (3) rabbit anti-a chain. Anode to the right.
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a1 EC
0
*0.4 C
.0 0.2
1 23 50
100
150
Effluent volume (ml)
B.
A B
4
patterns of IgA proteins. Gel A shows human oligo-
FIG. 4. Gel filtration pattern of IgAl myeloma protein (Car) (20 mg), digested with IgAl protease from H. influenza. Column: Sephadex G-200 in 1% ammonium bicarbonate buffer, 1.6 by 90 cm, downward
meric IgAl-K protein before cleavage (arrow). Arrow
flow.
CD
FIG. 3. SDS-polyacrylamide gel electrophoresis on gel B indicates principal cleavage product of the same myeloma IgA protein after digestion with IgAl
protease 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 G200 column. The fragments of the polymeric IgAl protein were eluted in two major peaks that were divided into four fractions as indicated in Fig. 4. From subsequent examinations by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis, it was concluded that fraction 4 corresponded to the Fab fragment: it reacted with anti-L-chain serum, had an apparent molecular weight of 65,000, and resolved, after reduction, into two polypeptides (molecular weights of 21,000 and 26,000). Fractions 1 through 3 contained proteins with apparent molecular weights of 137,000, 76,000, and 62,000. On immunoelectrophoresis, they reacted with an anti-a-chain but not with an anti-L-chain serum. After reduction, a major band with a molecular weight of 27,000 was observed. Based on these data, we concluded that fractions 1 through 3 contained Fc fragments in various molecular configurations. Undigested and digested (18 h at 370C) monomeric and polymeric IgAl proteins and S-IgA were also examined by analytical ultracentrifugation (Fig. 5). The results supported the observations described above. Fragments of digested monomeric IgAl protein (Pet) sedimented at the rates of 3.OS (major fragment) and 4.5S (minor fragment) (Fig. 5A). Polymeric IgAl protein
(Kni), which consisted principally of components sedimenting at 9, 11, and 13S plus small amounts of 7, 14, and 17S material, was cleaved into a major fragment with a sedimentation rate of 3.7S and a heterogeneous fragment of 7S (Fig. 5B). S-IgA (initially composed of 11 and 15S IgA) remained largely undigested although a fragment with a sedimentation rate of 3.1S was detected (Fig. 5C). The reported sedimentation rates were not corrected by extrapolating to infinite protein dilution and, accordingly, may be influenced by the concentration of the individual components.
DISCUSSION In this study we demonstrated that strains of H. influenzae, H. aegyptius, and S. pneumoniae released a protease which specifically cleaved human IgAl and partially cleaved S-IgA, but had no effect on a variety of other proteins, including human IgA2, IgG, and IgM, as well as porcine and bovine S-IgA. The immunochemical and ultracentrifugation analyses indicated that the protease(s) from both of these organisms effectively cleaved the IgAl molecule at the hinge region of the a-chain, thus releasing Fab and Fc fragments. In regard to substrate specificity and cleavage products generated, the enzymes from H. influenzae, H. aegyptius, and S. pneumoniae are analogous to those produced by N. meningitidis, N. gonorrhoeae, and S. sanguis (11-15). It has been established that enzymes from the latter two organisms cleave a prolylthreonyl bond in the hinge region of the al-
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FIG. 5. Ultracentrifugal patterns of (A) monomeric IgAl-K myeloma protein (Pet), (B) polymeric IgAl-K myeloma protein (Kni), and (C) S-IgA digested with H. influenzae IgA1 protease for 18 h at 370C. Undigested sample in the lower field; digested sample above. Protein concentrations were 10 mg/ml in all samples. The patterns were recorded after 48 min (A) and 32 min (B and C) of centrifugation. Arrows indicate fragments of these IgA proteins (see text).
chain (12, 13). The immunoelectrophoretic mobilities ofthe Fc fragments released by the action of proteases from H. influenzae, N. meningitidis, S. pneumoniae, and S. sanguis suggest that the former two species cleave the IgAl molecule at a site which differs from that of the latter two organisms. Nevertheless, the fact that IgA2 protein was resistant to proteases produced by all four organisms indicates that the susceptible bond is located within the 13-amino acid segment that distinguishes the a-chain of IgAl subclass. Amino acid sequence analyses of the Fc fragments will be required to establish the exact cleavage 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 upper respiratory tract. However, these species are also implicated in a variety of infectious diseases which originate at the mucous membranes of the respiratory tract. H. aegyptius, which is closely related to H. influenza (5), is implicated in cases of acute conjunctivitis. The possibility that IgAl protease production constitutes a significant virulence factor is supported by the fact that the enzyme was absent in the species H. parainfluenzae, H. aphrophilus, H. paraprophilus, and H. segnis, all of which may be considered opportunistic pathogens (5, 6). A similar relationship between pathogenic potential and IgAl protease activity has recently been demonstrated in the genus Neisseria (11). With our observation that both H. influenza and S. pneumoniae produce IgAl proteases, this characteristic has now been established for all of the three major etiological agents of bacterial meningitis.
The species H. influenza has been subdi-
vided into five biotypes which correlate with
specific disease entities (5). For example, biotype I is almost exclusively implicated in meningitis and epiglottitis, whereas biotype III is rarely isolated from acute infections and is more often found as a part of the normal flora in the upper respiratory tract. Since IgAl protease was found in all of these five biotypes it is apparent that this property may be only one of the parameters determining pathogenicity. It is of considerable interest that two strains of the species H. pleuropneumoniae digested porcine S-IgA but not the human immunoglobulins. This species is the cause of a highly contagious and often fatal pleuropneumonic infection in pigs, but human infections due to this organism have not been reported. Conversely, H. influenza rarely, if ever, produces natural infections in animal species. The ability of these organisms to cleave IgA of their specific hosts may be one of the factors that would explain the poorly understood species specificity of certain infectious diseases. Although the function of IgAl protease has not been clearly established, the detection of Fc fragment in stools (8) and in secretions of N. gonorrhoeae-infected vaginal tracts (1) indicates that cleavage of IgA occurs in vivo. Further studies are required to determine the biological consequences of S-IgA and serum IgAl cleavage on inhibitions of bacterial adherence (18, 22), of bactericidal effect (2, 3), and of phagocytosis (7, 17, 19, 20). ACKNOWLEDGMENTS We thank Rose Kulhavy and Kenneth L. Roland for excellent technical assistance, Philip Porter for providing us with purified 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 contract DE-52456 and grant AI 10854 from the National Institutes of Health.
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LITERATURE CITED 1. Blake, M., K. K. Holmes, and J. Swanson. 1979. Studies of gonococcus infection. XVII. IgAl-cleaving protease in vaginal washings from women with gonorrhea. J. Infect. Dis. 139:89-92. 2. Griffiss, J. M. 1975. Bactericidal activity of meningococcal antisera. Blocking by IgA of lytic antibody in human convalescent sera. J. Immunol. 114:1779-1784. 3. Griffiss, J. M., and M. A. Bertram. 1977. Immunoepidemiology of meningococcal disease in military recruits. II. Blocking of serum bactericidal activity by circulating IgA 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 IgAspecific protease. J. Immunol. Methods 18:245-249. 5. Kilian, M. 1976. A taxonomic study of the genus Haemophilus with the proposal of a new species. J. Gen. Microbiol. 93:9-62. 6. Kilian, M., and C. R. Schi0tt. 1975. Haemophili and related bacteria in the human oral cavity. Arch. Oral Biol. 20:791-796. 7. Magnusson, K.-E., 0. Stendahl, I. Stjernstrdm, and L Edebo. 1979. Reduction of phagocytosis, surface hydrophobicity and charge of Sabnonella typhimurium 395 MR1O by reaction with secretary IgA (SIgA). Immunology 36:439-447. 8. Mehta, S. K., A. G. Plaut, N. J. Calvanico, and T. B. Tomasi, Jr. 1973. Human immunoglobulin A. Production of an Fc fragment by an enteric microbial proteolytic enzyme. J. Immunol. 111:1274-1276. 9. Mestecky, J., W. J. Hammack, R. Kulhavy, G. P. Wright, and M. Tomana. 1977. Properties of IgA myeloma proteins isolated from sera of patients with the 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. Subunit structure. J. Immunol. 108:738-747. 11. Mulks, M. H., and A. G. Plaut. 1978. IgA protease production as a characteristic distinguishing pathogenic from harmless Neisseriaceae. N. Engl. J. Med. 299:
149
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 Neisseria meningitidies: extracellular enzyme cleaves human immunoglobulin A. Science 190:1103-1105. 14. Plaut, A. G., J. V. Gilbert, and R. Wistar, Jr. 1977. Loss of antibody activity in human immunoglobulin A exposed to extracellular immunoglobulin A proteases of Neisseria gonorrhoeae and Streptococcus sanguis. Infect. Immun. 17:130-135. 15. Plaut, A. G., R. Wistar, Jr., and J. D. Capra. 1974. Differential susceptibility of human IgA immunoglobulins to streptococcal IgA protease. J. Clin. Invest. 54: 1295-1300. 16. Schachman, H. K. 1959. Ultracentrifugation in biochemistry, 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 neutrophils. Adv. Exp. Med. Biol. 45:67-74. 18. Svanborg-Eden, C., and A.-M. Svennerholm. 1978. Secretory immunoglobulin A and G antibodies prevent adhesion of Escherichia coli to human urinary tract epithelial cells. Infect. Immun. 22:790-797. 19. Van Epps, D. E., K. Reed, and R. C. Williams, Jr. 1978. Suppression of human PMN bactericidal activity by human IgA paraproteins. Cell. Immunol. 36:363376. 20. Van Epps, D. E., and R. C. Williams, Jr. 1976. Suppression of leukocyte chemotaxis by human IgA myeloma components. J. Exp. Med. 144:1227-1242. 21. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412. 22. Williams, R. C., Jr., and R. J. Gibbons. 1972. Inhibition of bacterial adherence by secretory immunoglobulin A: a mechanism of antigen disposal. Science 177:697-699. 23. Zikan, J., J. Mestecky, R. E. Schrohenloher, M. Tomana, and R. Kulhavy. 1972. Studies on human secretory immunoglobulin A. V. Trypsin hydrolysis at elevated temperatures. Immunochemistry 9:1185-1193.
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Pathogenic Species of the Genus Haemophilus and Streptococcus pneumoniae Produce Immunoglobulin Al Protease MOGENS KILIAN,t* JIRI MESTECKY, AND RALPH E. SCHROHENLOHER Department of Microbiology, Institute of Dental Research and Division of Clinical Immunology and Rheumatology, University ofAlabama in Birmingham, Birmingham, Alabama 35294 Received for publication 4 May 1979
Thirty-seven strains of the genus Haemophilus and five strains of Streptococcus pneumoniae were examined for their ability to produce extracellular enzyme that cleaves immunoglobulin molecules. All strains of H. influenza, H. aegyptius, and S. pneumoniae elaborated enzyme that selectively cleaved human immunoglobulin Al (IgAl) myeloma proteins but was inactive against a variety of other proteins including human IgA2, IgG, and IgM, porcine and bovine secretary IgA, human and bovine serum albumins, and ovalbumin. Although susceptible, human secretary IgA remained largely undigested. Two strains of H. pleuropneumoniae isolated from fatally infected pigs cleaved porcine secretary IgA, but had no effect on human IgA proteins. None of 16 strains that belonged to nonpathogenic Haemophilus species produced IgA protease. Analyses of the cleavage products of human IgAl and secretary IgA proteins by immunochemical methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analytical ultracentrifugation revealed that Fab and Fc fragments were produced. Since the production of IgAl protease by Neisseria meningitidis has been reported previously, our finding that H. influenzae and S. pneumoniae produce an IgAl protease indicates that 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 the pathogenesis of this disease. Mucosal surfaces of the upper respiratory resulted in a considerable loss of antibody activtract are inhabited by a broad spectrum of bac- ity (14), it appears probable that these IgA proteria some of which may, under conditions that teases represent a significant virulence factor. are poorly understood, invade mucosal tissues A classic example of a systemic infection and produce systemic infection. Certain bacteria which is initiated by bacteria that gain access which colonize mucosal surfaces, such as Neis- into the circulation through the mucous memseria meningitidis, N. gonorrhoeae, and Strep- branes of the respiratory tract is bacterial mentococcus sanguis, were shown to produce an ingitis. N. nningitidis, Haemophilus influextracellular enzyme that cleaves serum immu- enzae, and S. pneumoniae represent the princinoglobulins belonging to immunoglobulin Al pal bacterial species responsible for this disease. (IgAl) but not IgA2, IgG, or IgM classes (11-15). It was previously reported that pathogenic Secretory IgA (S-IgA), the principal carrier of strains of N. meningitidis produce IgAl protease specific humoral defense on mucous surfaces, is (11), and we report in this communication that only partly susceptible to digestion by bacterial the other two leading causative agents of bacIgA proteases. This relative insusceptibility may terial meningitis, H. influenzae and S. pneumobe explained by the presence of a higher propor- niae, also produce an enzyme which cleaves tion of IgA2 in S-IgA than in serum IgA (12), by IgAl and to a limited extent S-IgA, but has no the association of IgA with secretary component effect on IgA2, IgM, and IgG. which renders S-IgA resistant to various proMATERIALS AND METHODS teases, and by the presence of S-IgA-associated Bacterial strains. Thirty-seven human and porantibodies that may neutralize IgA protease activity (A. G. Plaut et al., Fed. Proc. 38:5275, cine isolates of the genus Haemophilus and five strains blood, cerebro1979). Since cleavage of IgAl by such enzymes of S. pneumoniae isolated from human spinal fluid, and sputum were included in the study. t Present address: Department of Microbiology, Royal The Haemophilus strains that represent each of five Dental College, DK-8000 Aarhus, Denmark. biotypes of H. influenza and the species H. aegyptius, 143
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H. parainfluenzae, H. aphrophilus, H. paraphrophi- (ii) sodium dodecyl sulfate (SDS)-polyacrylamide gel lus, H. segnis, and H. pleuropneumoniae have been electrophoresis, and (iii) analytical ultracentrifugation. Immunoelectrophoresis. Immunoelectrophoresis previously described in detail (5). The Haemophilus and S. pneumoniae strains are listed in Table 1, which of digested proteins and undigested controls was perprovides information on serotype and site of isolation. formed in 2% agar in Veronal buffer, pH 8.6. Antisera S. sanguis strain ATCC 10556 obtained from the used for development of immunoelectrophoresis were American Type Culture Collection, Rockville, Md., unabsorbed rabbit anti-human S-IgA (10), and comand a clinical isolate of N. meningitidis (strain VK 4) mercial, monospecific antisera against K- or A-light served as positive controls for IgAl protease activity. chains (Meloy, Springfield, Va.) and a-chain of IgA Haemophilus, S. pneumoniae, and Neisseria strains (Hyland Laboratories, Costa Mesa, Calif., and Behring Diagnostics, Somerville, N.J.). were cultivated on chocolate agar (blood agar base SDS-polyacrylamide gel electrophoresis. Sam[Difco Laboratories, Detroit, Mich.] with 10% [vol/ vol] heated, defibrinated horse blood). Agar plates ples were electrophoresed in sodium phosphate buffer, pH 7.2, in the presence of 0.1% SDS and 7 M urea by were incubated at 37°C in air with an additional 5% CO2 in accordance with the growth requirements of the method of Weber and Osborn (21). To cleave the individual strains. S. sanguis was cultivated on disulfide bridges, immunoglobulins were reduced by brain heart infusion agar (Difco) incubated under mi- mixing samples with an equal volume of a solution of 8 M urea, 2% SDS, and 0.2 M 2-mercaptoethanol for croaerophilic conditions. Immunoglobulin preparations. IgA, IgM, and 30 to 60 min prior to gel electrophoresis. Apparent IgG paraproteins were isolated from plasma of patients molecular weights of fragments observed in SDS-polywith multiple myeloma or Waldenstrom's macroglob- acrylamide gels were calculated as described by Weber ulinemia. Details of the purification procedures, which and Osborn (21), with the following proteins with included ammonium sulfate precipitation, gel filtration known molecular weights used as standards: human IgG paraprotein (150,000), ovalbumin (45,000), chyon Sephadex G-200 and Sepharose 6B, and ion exchange chromatography on diethylaminoethyl-Seph- motrypsinogen (25,000), and chicken egg white lysoadex, have been described (9, 10). The properties of zyme (14,400). The molecular weights of fragments in the IgA myeloma proteins such as sedimentation con- reduced samples were determined with reference to stant, carbohydrate composition, L-chain type, pres- reduced and purified L- and H-chains of IgG. Analytical ultracentrifugation. Digested and ence of J chain, and IgA subclass (determined by monospecific antisera and carbohydrate compositions) undigested proteins were centrifuged concurrently in have been reported (9). Polyclonal IgG was isolated a Spinco model E analytical ultracentrifuge (Beckman from normal human serum. S-IgA was isolated from Instruments, Inc.) at 56,000 rpm and 20'C. Samples, pooled colostrum and processed as described previ- which contained 10 mg of protein per ml, were dialyzed ously (10). Immunochemical and ultracentrifugation against PBS prior to analysis. A 600 phase-plate angle analyses revealed that colostral S-IgA consisted of 15S was used for photographs taken at 8-min intervals. and llS forms, with a preponderance of the latter type Sedimentation rates were calculated by the method of (23). Purified S-IgA isolated from pig milk was a gift Schachman (16) on the basis of measurements carried from P. Porter, Unilever Research, Bedford, U.K. out with a Nikon microcomparator (Nippon Kogaku, Bovine S-IgA, partly purified by the above-described Tokyo, Japan). methods (10), was a gift from A. Bhown, University of Gel filtration. A 20-mg amount of polymeric IgAl Alabama in Birmingham. paraprotein (Car) was digested with an IgAl protease Preparation of IgA protease. IgA protease was preparation from H. influenzae strain HK 393 for 16 prepared as described by Higerd et al. (4). Suspensions h at 37°C. The digested protein was chromatographed of the respective bacterial strains in sterile saline were on a Sephadex G-200 column (1.6 by 90 cm) in 1% inoculated onto the surface of presterilized dialysis ammonium bicarbonate buffer. Pooled fractions (see membranes placed on chocolate or brain heart infusion below) were lyophilized, redissolved in Veronal buffer, agar plates. After incubation for 2 days at 37°C the pH 8.6, and used for antigenic and molecular-weight membranes were removed from the agar surface and analyses. washed in a minimal volume of 0.05 M potassium phosphate buffer, pH 7.5, with 0.85% (wt/vol) NaCl RESULTS (PBS). The wash was clarified by centrifugation at IgAl protease-producing bacteria. Ex30,000 x g for 15 min at 4°C and was concentrated 20 times by positive-pressure ultrafiltration using an amination ofthe bacterial strains (listed in Table Amicon PM 10 membrane. 1) for IgA protease activity revealed that all Detection of IgA protease activity. Concen- strains of H. influenzae, H. aegyptius, and S. trated protease preparations were incubated for 3 h at pneumoniae cleaved IgAl proteins. The IgAl 37°C with equal volumes of the respective immuno- protease-producing strains of H. influenzae repglobulins dissolved in PB3S at a concentration of 5 mg/ ml. Controls contained buffer instead of enzyme prep- resented all five biotypes of this species and aration. For the initial screening of strains for IgA included encapsulated strains isolated from paprotease activity, individual colonies from 1- to 2-day tients with meningitis and other infections as chocolate agar cultures were suspended in 50 p1 of a 5 well as nonencapsulated strains from upper resmg/ml immunoglobulin solution which was then in- piratory tracts of healthy individuals. None of cubated for 15 to 18 h at 37°C. Proteolysis was de- the 16 strains of other Haemophilus species tected by three methods: (i) immunoelectrophoresis, produced an enzyme with the ability to cleave
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TABLE 1. Bacterial stroains used in the study, their origin and ability to cleave human IgAl and S-IgAa Species/biotype and strain Serotype Origin IgA 1 protease activity H. influenzae I HK 66 Throat HK 193, 194, 195 b Meningitis + HK 393 - NCTC 8467 b HK 395 = CIP 5423 H. influenza II HK 21, 24, 25, 30 Throat + HK 208 b Meningitis HK 402 = NCTC 7918 f Pus, mastoid H. influenza III HK 387 = NCTC 4560 + HK 57 Throat H. influenza IV HK 368 = CIP 5424 + HK 397 = NCTC 8470 d Throat H. influenza V HK 223, 238, 240 Otitis media + H. aegyptius = HK 368 ATCC 11116 Conjunctivitis + HK 270 Conjunctivitis H. parainfluenzae HK 47 Throat HK 82, 90, 133 Saliva HK 409 Septic finger H. aphrophilus HK 308, 315 Dental plaque HK 371 = NCTC 5886 Endocarditis H. paraphrophilus HK 83 Saliva HK 414 = NCTC 10556 Abscess HK 415 = NCTC 10557 Paronychia H. segnis HK 84 Saliva HK 307 Dental plaque HK 316 = NCTC 10977 Dental plaque H. pleuropneumoniae HK 405 = ATCC 27088 Pleuropneumonia, pig Cleave porcine s-IgA HK 407 Pleuropneumonia, pig but not human IgA S. pneumoniae VK 3 Sputum VK 5 3 Blood VK 6 1 Meningitis VK 7,8 ND Blood 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 the assay were cleaved by S. sanguis ATCC 10556 and N. meningitidis VK 4, both of which are known producers of IgAl protease (12). IgAl protein (Car) digested by the two latter strains was compared, by immunoelectrophoresis, with the same protein incubated with H. influenzae HK 393 and S. pneumoniae VK 7. Apparent resemblance was noted in the immunoelectrophoresis pattern of IgAl protein digested by S. sanguis and S. pneumoniae. IgAl digested by H. influenza and N. meningitidis resulted in a mutually similar immunoelectrophoresis pattern
which, however, differed from that observed with the two streptococcal species (Fig. 1). Substrate specificity. Enzyme preparations from strains of the species H. influenza, H. aegyptius, and S. pneumoniae cleaved each of three IgAl paraproteins including polymeric and monomeric IgA of both kappa and lambda types. SDS-polyacrylamide gel electrophoresis and analytical ultracentrifugation (see below) indicated that fully active enzyme preparations caused virtually complete cleavage of IgAl within 3 h of incubation. The same enzyme preparations were also capable of cleaving S-IgA, but only a
146
~ ~ ~.
INFECT. IMMUN.
KILIAN, MESTECKY, AND SCHROHENLOHER
molecular weight of 84,000. This protein was previously identified as secretary component (10). After incubation with IgA protease, mono..~~~~ ~~~~~~~~~~~~~.... meric IgAl was cleaved into two fragments with apparent molecular weights of 65,000 (major component) and 54,000 (minor component). Digested polymeric IgAl protein showed two bands with molecular weights of 65,000 (major band) and 127,000. In contrast, the major portion of S-IgA remained undigested. However, two 'Nip bands with molecular weights of 65,000 and 193,000 were also observed. Pig S-IgA incubated with strains of H. pleuropneumoniae was cleaved into several components (Fig. 3C and D). Because of the limited amount of pig S-IgA available, further analyses were not performed. To purify the fragments of cleaved IgAl protein, we applied 20 mg of a polymeric IgAl-K paraprotein (Car), digested by protease derived FIG. 1. Immunoelectrophoresis of IgAl myeloma protein (Car) incubated with: (A) PBS, (B) H. influenzae HK 393, (C) S. pneumoniae VK 7, (D) N. meningitidis VK 4, and (E) S. sanguis ATCC 10556. Antiserum: rabbit anti-S-IgA, not adsorbed for Lchain reactivity. Anode to the left.
IgA ... .. ;.A :.....,>:..
part of the available protein was digested after incubation for up to 18 h. Proteolysis was not observed with IgA2, IgG, and IgM paraproteins, or with normal serum IgG and porcine or bovine S-IgA. Human serum albumin, bovine serum albumin, and egg albumin were also resistant to proteolysis. Enzyme preparations from two strains of H. pleuropneumoniae isolated from fatal cases of pleuropneumonia in pigs cleaved porcine S-IgA, but did not affect any of the human immunoglobulins, including IgAl. Properties of cleavage products. Immunoelectrophoresis of digested IgAl and S-IgA revealed two separate fragments with different electrophoretic mobilities (Fig. 2). A fragment which had a slow electrophoretic mobility similar to that of the undigested protein reacted strongly with anti-L-chain sera but not with anti-a-chain serum. Conversely, the second fragment reacted only with anti-a-chain serum. On the basis of these properties, the two fragments were identified as Fab and Fc, respectively. All proteins used in the study, including immunoglobulins of IgA, IgG, and IgM classes and albumins, were subjected to SDS-polyacrylamide gel electrophoresis before and after incubation with IgAl protease preparations (Fig. 3). Reduced samples of undigested monomeric and polymeric IgAl migrated as two bands with apparent molecular weights of 21,000 (L-chain) and 52,000 (H-chains); S-IgA contained, in addition to H- and L-chains, a protein with an apparent
1
Fab g
*
IgA
21, *
IgA
I 2' *%Vi./
.SlgA
FIG. 2. Antigenic analyses of polymeric IgAl-K myeloma protein (Car) and S-IgA. Samples digested with IgAl protease from H. influenza are labeled with an asterisk. Antisera: (1) rabbit anti-S-IgA, not adsorbed for L-chain reactivity, (2) rabbit anti-Kchain, (3) rabbit anti-a chain. Anode to the right.
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IMMUNOGLOBULIN Al PROTEASE
147
a1 EC
0
*0.4 C
.0 0.2
1 23 50
100
150
Effluent volume (ml)
B.
A B
4
patterns of IgA proteins. Gel A shows human oligo-
FIG. 4. Gel filtration pattern of IgAl myeloma protein (Car) (20 mg), digested with IgAl protease from H. influenza. Column: Sephadex G-200 in 1% ammonium bicarbonate buffer, 1.6 by 90 cm, downward
meric IgAl-K protein before cleavage (arrow). Arrow
flow.
CD
FIG. 3. SDS-polyacrylamide gel electrophoresis on gel B indicates principal cleavage product of the same myeloma IgA protein after digestion with IgAl
protease 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 G200 column. The fragments of the polymeric IgAl protein were eluted in two major peaks that were divided into four fractions as indicated in Fig. 4. From subsequent examinations by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis, it was concluded that fraction 4 corresponded to the Fab fragment: it reacted with anti-L-chain serum, had an apparent molecular weight of 65,000, and resolved, after reduction, into two polypeptides (molecular weights of 21,000 and 26,000). Fractions 1 through 3 contained proteins with apparent molecular weights of 137,000, 76,000, and 62,000. On immunoelectrophoresis, they reacted with an anti-a-chain but not with an anti-L-chain serum. After reduction, a major band with a molecular weight of 27,000 was observed. Based on these data, we concluded that fractions 1 through 3 contained Fc fragments in various molecular configurations. Undigested and digested (18 h at 370C) monomeric and polymeric IgAl proteins and S-IgA were also examined by analytical ultracentrifugation (Fig. 5). The results supported the observations described above. Fragments of digested monomeric IgAl protein (Pet) sedimented at the rates of 3.OS (major fragment) and 4.5S (minor fragment) (Fig. 5A). Polymeric IgAl protein
(Kni), which consisted principally of components sedimenting at 9, 11, and 13S plus small amounts of 7, 14, and 17S material, was cleaved into a major fragment with a sedimentation rate of 3.7S and a heterogeneous fragment of 7S (Fig. 5B). S-IgA (initially composed of 11 and 15S IgA) remained largely undigested although a fragment with a sedimentation rate of 3.1S was detected (Fig. 5C). The reported sedimentation rates were not corrected by extrapolating to infinite protein dilution and, accordingly, may be influenced by the concentration of the individual components.
DISCUSSION In this study we demonstrated that strains of H. influenzae, H. aegyptius, and S. pneumoniae released a protease which specifically cleaved human IgAl and partially cleaved S-IgA, but had no effect on a variety of other proteins, including human IgA2, IgG, and IgM, as well as porcine and bovine S-IgA. The immunochemical and ultracentrifugation analyses indicated that the protease(s) from both of these organisms effectively cleaved the IgAl molecule at the hinge region of the a-chain, thus releasing Fab and Fc fragments. In regard to substrate specificity and cleavage products generated, the enzymes from H. influenzae, H. aegyptius, and S. pneumoniae are analogous to those produced by N. meningitidis, N. gonorrhoeae, and S. sanguis (11-15). It has been established that enzymes from the latter two organisms cleave a prolylthreonyl bond in the hinge region of the al-
148
KILIAN, MESTECKY, AND SCHROHENLOHER
INFECT. IMMUN.
FIG. 5. Ultracentrifugal patterns of (A) monomeric IgAl-K myeloma protein (Pet), (B) polymeric IgAl-K myeloma protein (Kni), and (C) S-IgA digested with H. influenzae IgA1 protease for 18 h at 370C. Undigested sample in the lower field; digested sample above. Protein concentrations were 10 mg/ml in all samples. The patterns were recorded after 48 min (A) and 32 min (B and C) of centrifugation. Arrows indicate fragments of these IgA proteins (see text).
chain (12, 13). The immunoelectrophoretic mobilities ofthe Fc fragments released by the action of proteases from H. influenzae, N. meningitidis, S. pneumoniae, and S. sanguis suggest that the former two species cleave the IgAl molecule at a site which differs from that of the latter two organisms. Nevertheless, the fact that IgA2 protein was resistant to proteases produced by all four organisms indicates that the susceptible bond is located within the 13-amino acid segment that distinguishes the a-chain of IgAl subclass. Amino acid sequence analyses of the Fc fragments will be required to establish the exact cleavage 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 upper respiratory tract. However, these species are also implicated in a variety of infectious diseases which originate at the mucous membranes of the respiratory tract. H. aegyptius, which is closely related to H. influenza (5), is implicated in cases of acute conjunctivitis. The possibility that IgAl protease production constitutes a significant virulence factor is supported by the fact that the enzyme was absent in the species H. parainfluenzae, H. aphrophilus, H. paraprophilus, and H. segnis, all of which may be considered opportunistic pathogens (5, 6). A similar relationship between pathogenic potential and IgAl protease activity has recently been demonstrated in the genus Neisseria (11). With our observation that both H. influenza and S. pneumoniae produce IgAl proteases, this characteristic has now been established for all of the three major etiological agents of bacterial meningitis.
The species H. influenza has been subdi-
vided into five biotypes which correlate with
specific disease entities (5). For example, biotype I is almost exclusively implicated in meningitis and epiglottitis, whereas biotype III is rarely isolated from acute infections and is more often found as a part of the normal flora in the upper respiratory tract. Since IgAl protease was found in all of these five biotypes it is apparent that this property may be only one of the parameters determining pathogenicity. It is of considerable interest that two strains of the species H. pleuropneumoniae digested porcine S-IgA but not the human immunoglobulins. This species is the cause of a highly contagious and often fatal pleuropneumonic infection in pigs, but human infections due to this organism have not been reported. Conversely, H. influenza rarely, if ever, produces natural infections in animal species. The ability of these organisms to cleave IgA of their specific hosts may be one of the factors that would explain the poorly understood species specificity of certain infectious diseases. Although the function of IgAl protease has not been clearly established, the detection of Fc fragment in stools (8) and in secretions of N. gonorrhoeae-infected vaginal tracts (1) indicates that cleavage of IgA occurs in vivo. Further studies are required to determine the biological consequences of S-IgA and serum IgAl cleavage on inhibitions of bacterial adherence (18, 22), of bactericidal effect (2, 3), and of phagocytosis (7, 17, 19, 20). ACKNOWLEDGMENTS We thank Rose Kulhavy and Kenneth L. Roland for excellent technical assistance, Philip Porter for providing us with purified 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 contract DE-52456 and grant AI 10854 from the National Institutes of Health.
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LITERATURE CITED 1. Blake, M., K. K. Holmes, and J. Swanson. 1979. Studies of gonococcus infection. XVII. IgAl-cleaving protease in vaginal washings from women with gonorrhea. J. Infect. Dis. 139:89-92. 2. Griffiss, J. M. 1975. Bactericidal activity of meningococcal antisera. Blocking by IgA of lytic antibody in human convalescent sera. J. Immunol. 114:1779-1784. 3. Griffiss, J. M., and M. A. Bertram. 1977. Immunoepidemiology of meningococcal disease in military recruits. II. Blocking of serum bactericidal activity by circulating IgA 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 IgAspecific protease. J. Immunol. Methods 18:245-249. 5. Kilian, M. 1976. A taxonomic study of the genus Haemophilus with the proposal of a new species. J. Gen. Microbiol. 93:9-62. 6. Kilian, M., and C. R. Schi0tt. 1975. Haemophili and related bacteria in the human oral cavity. Arch. Oral Biol. 20:791-796. 7. Magnusson, K.-E., 0. Stendahl, I. Stjernstrdm, and L Edebo. 1979. Reduction of phagocytosis, surface hydrophobicity and charge of Sabnonella typhimurium 395 MR1O by reaction with secretary IgA (SIgA). Immunology 36:439-447. 8. Mehta, S. K., A. G. Plaut, N. J. Calvanico, and T. B. Tomasi, Jr. 1973. Human immunoglobulin A. Production of an Fc fragment by an enteric microbial proteolytic enzyme. J. Immunol. 111:1274-1276. 9. Mestecky, J., W. J. Hammack, R. Kulhavy, G. P. Wright, and M. Tomana. 1977. Properties of IgA myeloma proteins isolated from sera of patients with the 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. Subunit structure. J. Immunol. 108:738-747. 11. Mulks, M. H., and A. G. Plaut. 1978. IgA protease production as a characteristic distinguishing pathogenic from harmless Neisseriaceae. N. Engl. J. Med. 299:
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