Vol. 26, No. 1
INFECTION AND IMMUNITY, Oct. 1979, p. 254-261 0019-9567/79/10-0254/08$02.00/0
Immunoglobulin Al Protease Production by Haemophilus influenza and Streptococcus pneumoniae CAROLYN J. MALE and Department of Microbiology Immunology, University of Colorado Medical School, Denver, Colorado 80262
Received for publication 7 May 1979
Bacterial strains of Haemophilus species and Streptococcus pneumoniae were examined for synthesis of the enzyme immunoglobulin Al (IgAl) protease. Of 36 H. influenzae strains examined, 35 produced IgAl protease; strains included all six capsular types, unencapsulated variants of types b and d, and untypable H. influenza. Eight Haemophilus strains (non-H. influenza) were studied, and two produced IgAl protease. All 10 strains of S. pneumoniae produced IgAl protease; these strains included 9 different capsular polysaccharide types and 1 untypable strain. Both IgAl proteases cleaved myeloma IgAl and secretary IgA but not myeloma IgA2, IgM, or IgG as determined by immunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes cleaved IgAl myeloma sera, but not IgA2, into two fragments. The apparent molecular weight of the cleaved fragments was dependent both on the specific IgAl protease assayed and the specific IgAl substrate utilized. It is postulated that both carbohydrate variation between the IgAl substrates studied and the ability of S. pneumoniae glycosidases to cleave carbohydrates from glycoprotein offer an explanation for the different fragment sizes observed.
Bacterial immunoglobulin A (IgA) protease was first described by Mehta et al. (14). This enzyme specifically cleaves only human serum IgAl and secretary IgA; IgA2, IgG, IgM, and IgE are resistant to enzymatic cleavage (14, 17). Cleavage of myeloma IgAl at a proline-threonine bond in the hinge region results in the formation of Fc and Fab fragments; the Fab fragments of serum IgAl are unable to bind antigen (18, 20, 21). A wide variety of microorganisms have been examined for IgA protease production and found to be negative (10). The enzyme is synthesized by many strains of Streptococcus sanguis and group H streptococci (10), by virtually all tested strains of Neisseria gonorrheae (18), and by typable and nontypable Neisseria meningitidis strains but not by normal commensal Neisseria species (15). Secretory IgA is the predominant immunoglobulin in colostrum and parotid fluid, and it is felt that IgA may be the predominant immunoglobulin in genital and nasal secretions. Plaut and his co-workers have suggested that IgA protease cleavage of IgAl in mucosal secretions may be very important in the pathogenesis of infections caused by bacteria which synthesize IgA protease (21). Recently, it has been shown that N. gonorrheae IgAl protease can be detected in the vaginal secretions of women with gonorrhea
(5).
Both Haemophilus influenza and Streptococcus pneumoniae are major pathogens, primarily of children, and it is believed that initiation of systemic infection follows nasopharyngeal colonization. For this reason we elected to examine strains of these organisms for IgAl protease production. (This paper was presented in part at the 79th Annual Meeting of the American Society for Microbiology, 4 to 8 May 1979, Los Angeles, Calif. [Abstr. no. B83, p. 29].) MATERLALS AND METHODS Bacteria. A known positive IgA protease strain, S. sanguis ATCC 10556, was provided by R. Eisenberg (University of Pennsylvania). A laboratory strain of group A streptococcus was confirmed as not producing IgA protease and was used as a negative control. The pneumococcal strains were provided by Barth Reller. Haemophilus strains were provided by the Colorado State Health Laboratories, Barth Reller, Kenneth McIntosh, and Mimi Glode (Children's Hospital, Denver, Co.). In addition to providing numerous Haemophilus strains, M. Glode also furnished the Escherichia coli K100 strains. The clinical origin of most, but not all, of the H. influenza strains was known and included cerebrospinal, blood, and nasopharyngeal specimens. Most of the specimens were from children with invasive H. influenza disease, but some specimens were from "asymptomatic" carriers. Two of the H. aphrophilus isolates were presumed to be from patients with endocarditis. The origin of the other Haemophilus
254
VOL. 26, 1979 strains (non-H. influenza) is not known. The pneumococcal strains were from patients with systemic pneumococcal disease. Stock cultures of bacteria were maintained in ToddHewitt broth with 30% glycerol at -70oC. Bacterial strains to be tested for IgA protease were grown on blood agar or chocolate agar in a C02 jar at 360C for 16 to 18 h. Immunoglobulins and antisera. Whole IgAl and IgA2 myeloma sera were provided by Carlos Abel and Howard Grey (National Jewish Hospital, Denver, Co.), Peter Kohler, Aroop Mangalik, and several private physicians in the Denver area. Purified IgA myeloma and colostral IgA were provided by William Brown and R. Eisenberg. Whole colostrum was received from C. Abel, W. Fuller, and M. Fishut. Concentrated parotid fluid was received from George Revis. The IgA concentrations of the whole myeloma and purified samples ranged from 2 to 15 mg/ml. Routine screening for IgA protease production was done with an IgAl kappa myeloma serum generously provided by Carlos Abel. This serum had a high IgAl titer, had no detectable IgG or IgM (by immunoelectrophoresis), and was available in large amounts. The light chains of the IgAl and IgA2 substrates were typed with antiserum to kappa and lambda light chains. IgM and IgG human immunoglobulins were also assayed for susceptibility to the Haemophilus and S. pneumoniae enzymes. IgM myeloma sera were provided by P. Kohler and R. Eisenberg, and the IgG was Cohn fraction II. Rabbit antisera to alpha, gamma, mu, kappa, and lambda chains were purchased from Biorad Laboratories. IgA protease assay. Several loopfuls of bacteria to be tested were taken from solid media and evenly suspended in 100 IL of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 8.1). Usually 10 pl of bacterial suspension was mixed with 10 microcentrifuge tube pl of IgA substrate in a 400-pl with attached cap, and the mixture was incubated in a 370C water bath for 3 to 4 h; longer incubations were used when secretary IgA was the substrate. Slides for electrophoresis were prepared with 2% granulated agar (BBL Microbiology Systems). General procedure, buffers, etc. for immunoelectrophoresis were those of Crowle (7). A 3- to 4-pl portion of the incubated bacteria and IgA mixture was placed in a well, and the sample was electrophoresed for 1 h (2 mA/slide, 100 V). Troughs were cut and 50 1pl of the appropriate antiserum was added. The slides were allowed to develop at 40C for 2 to 3 days. Slides were processed by the method of Crowle (personal communication) and stained with Crocein scarlet-brilliant blue R (8). For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), acrylamide slab gels (12.5% gel, 0.3% SDS) with a 5% stacking gel were prepared by the general method of Laemmli (13). After incubation of myeloma proteins with enzyme or buffer, samples were boiled for 3 min in the presence of 2% SDS and 1% mercaptoethanol. Samples were applied to the slab gel and electrophoresed at 10 mA until the dye front reached the bottom of the gel. The gels were stained for 1 h with Coomassie brilliant blue.
IgAl PROTEASE PRODUCTION
255
RESULTS Incidence of IgA protease production. The incidence of IgAl protease production among the Haemophilus and pneumococcal strains is shown in Table 1. Protease activity was determined by immunoelectrophoresis. Clearly, IgAl protease synthesis is a common feature of the H. influenza strains studied; only one type a strain was negative for protease synthesis. Although the number. of other Haemophilus species available for testing was limited, the majority of these strains did not make detectable IgAl protease. Suspensions of all pneumococci tested, including one untypable strain, contained IgAl protease. Because of the recently recognized association between invasive H. influenzae disease and gut colonization with E. coli K100 strains (11, 27), two E. coli K100 strains isolated from those patients were tested for IgA protease activity; both strains were negative. Evidence for IgAl protease cleavage of IgAl as determined by immunoelectrophoresis. The data presented in Fig. 1 indicate that the IgAl proteases produced by both H. influTABLE 1. Incidence of IgAI protease production by Haemophilus and S. pneumoniae strains No. No. positive (%) Bacterial strains studied Haemophilus strains 2 1 (50) H. influenza type a 15 (100) 15 H. influenzae type b 2 2 (100) H. influenzae type c 2 (100) 2 H. influenza type d 3 (100) 3 H. influenza type e 3 (100) 3 H. influenza type f 1 (100) 1 Unencapsulated type b 2 (100) 2 Unencapsulated type d
Untypable Total H. parainfluenzae H. parahaemolyticus H. aphrophilus Total S. pneumoniae strains S. pneumoniae type 1 S. pneumoniae type 3 S. pneumoniae type 4 S. pneumoniae type 5 S. pneumoniae type 6 S. pneumoniae type 8 S. pneumoniae type 14 S. pneumoniae type 17 S. pneumoniae type 19 Untypable Total
6 36
6 (100) 35 (97)
4 1 3
1 (25) 1 (100)
8
0 (0) 2 (25)
2 2 2 1 1 1 1 1 1
2 (100) 2 (100) 2 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100)
1
13
1
(100)
13 (100)
256
MALE
INFECT. IMMUN.
FIG. 1. Immunoelectrophoresis of IgAl and IgA2 myeloma immunoglobulins and colostrum incubated with H. influenzae or S. pneumoniae IgAl protease. (A) Well 1, IgAl My + Tris buffer (0.05 M, pH 8.1); well 2, IgAl My + S. pneumoniae type 1 cells; well 3, IgA2Ke + S. pneumoniae type 1; well 4, IgA2Ke + Tris buffer. Troughs a, b, and c: anti-alpha + anti-kappa mixed. (B) Well 1, IgAl My + Tris buffer; wells 2 and 3, IgAl My + S. pneumoniae type 1; well 4, IgA2Ke + S. pneumoniae type 1. Trough a, anti-alpha + anti-kappa, mixed; trough b, anti-kappa; trough c, anti-alpha. (C) Well 1, IgAl My + Tris buffer; wells 2 and 3, IgAl My + H. influenzae type b; well 4, IgA2Ke + H. influenzae type b. Trough a, anti-alpha + anti-kappa, mixed; trough b, anti-kappa; trough c, anti-alpha. (D) Well 1, colostrum Ca + Tris buffer; well 2, colostrum Ca + H. influenzae type b; well 3, colostrum Ca + S. pneumoniae type 1; well 4, colostrum Ab + H. influenzae type b. Troughs a, b, and c: anti-alpha, anti-kappa, anti-lambda, mixed. (B) Arrows indicate Fab and Fc fragments. Bacteria were incubated with myeloma substrates for 6 h, and colostrum substrates were incubated for 16 h. Anode is to the left.
enzae and S. pneumoniae specifically cleaved IgAl but not IgA2 immunoglobulins. Use of monospecific antisera (Fig. 1B and C) suggested that incomplete cleavage of IgAl into Fab and Fc fragments had occurred. Anti-kappa antiserum reacts only with the Fab fragment, and antialpha chain antiserum reacts only with the Fc fragment, as demonstrated by Mehta et al. (14). Note in slide A that the IgA2 myeloma substrate has IgG and IgM immunoglobulins which are detected with the anti-kappa antiserum. These precipitin lines must be distinguished from the precipitin lines which result from true protease cleavage; the contaminant precipitin lines are qualitatively different from the Fab precipitin lines and are also present in the buffer control. We emphasize this point because if an IgAl substrate with detectable IgG and IgM is employed, and unequal amounts of substrate are placed in the wells, it is possible to get no contaminant lines in the buffer control and contaminant lines in the incubated mixture. Haemophilus IgAl protease seldom cleaved all the IgAl substrate to completion regardless of the length of incubation. Partial cleavage (Fig. 1C) was more commonly seen, although occa-
sionally complete cleavage into Fab and Fc fragments was observed as evidenced by crossed precipitin lines of nonidentity (14). Pneumococcal IgAl protease routinely appeared to cleave IgAl substrates more rapidly than Haemophilus IgAl protease. Since no attempt was made to quantitate the protease assay, it is not known if, with respect to Haemophilus IgAl protease, pneumococci synthesize more protease or the protease has greater specific activity or there are more pneumococci in the assay tube. The pneumococcal and Haemophilus cleavage products differed with respect to both electrophoretic mobility and diffusion, as a comparison of slides B and C illustrates. We believe that these electrophoretic and diffusion differences may be related to the ability of pneumococcal enzymes to cleave carbohydrate from the heavy chains of IgAl. The rapid diffusion of pneumococcal fragments made it difficult, methodologically, to produce slides for publication which showed separate Fab and Fc fragments which had not diffused into the troughs; use of hyperimmune antisera would have been beneficial. Both bacterial proteases cleaved secretary IgA (whole clostrum or parotid fluid; purified colos-
VOL. 26, 1979
tral IgAl), but not all secretary IgA substrates were cleaved by both enzymes. Cleavage of secretory IgA required a longer incubation period than that with myeloma sera. Comparison of apparent cleavage of colostrum by both IgAl proteases is shown in Fig. iD. The pneumococcal IgAl protease appeared to cleave all secretory IgA substrates most rapidly; protease from H. influenza was the least active against a variety of secretary IgA substrates and S. sanguis was somewhat intermediate (data not shown). We have not yet identified the specific cleavage products of secretary IgA. Identification of secretary IgA cleavage products by immunoelectrophoresis alone is difficult because of the presence of large amounts of IgAl protease-resistant IgA2 immunoglobulin. Our cleavage patterns are siibilar to those shown by Genco et al. in slide B of their paper (10). These authors also showed complete cleavage of secretory IgA substrate into Fc and Fab fragments; however, their buffer control showed two precipitin bands referred to as "an experimental artifact." With fresh, structurally intact secretary IgA, which gives a single precipitin band against anti-alpha antiserum, we have never seen cleavage which results in separate Fab and Fc fragments. Similar to the results of Genco et al., we have seen such a pattern with a parotid saliva IgA substrate which was partially degraded and showed two separate precipitin bands when incubated with buffer only. Neither the H. influenza nor S. pneumoniae IgAl proteases cleaved human serum IgG or IgM (data not shown). IgAl protease cleavage as determined by SDS-PAGE. As further confirmation that H. influenza and S. pneumoniae produced IgAl protease, we ran polyacrylamide gels to demonstrate the two expected cleavage products from IgAl myeloma substrates. We were surprised to find the following: (i) the light chains of various myeloma proteins had different mobilities; (ii) assaying a single myeloma substrate, the cleavage products of the Haemophilus and pneumococcal IgAl proteases appeared to be different; (iii) the cleavage products resulting from either Haemophilus or pneumococcal IgAl protease varied among the IgAl myeloma sera tested; and (iv) the heavy chains of IgA2 proteins had a faster mobility after incubation with pneumococci. Blake and Swanson reported that the cleavage products of N. gonorrheae IgAl protease were 30,000 and 33,000 molecular weight as determined by SDS-PAGE (6). Since it is known that the gonococcal IgAl protease cleaves in the hinge region of the IgAl immunoglobulin (18), presumably the two cleavage
257
IgAl PROTEASE PRODUCTION
products on SDS-PAGE are the specific Fc and Fd fragments which result from cleavage of the IgAl alpha chain. Although we have not yet determined the exact site of cleavage for either of our IgAl proteases, our immunoelectrophoretic and gel electrophoresis data are consistent with cleavage in the hinge region. The data in Fig. 2 and 3 indicate that the pneumococcal IgAl protease cleaves IgAl substrates with the appearance of two bands; one band is very heterogeneous, with an apparent average molecular weight of about 36,000, whereas the faster moving fragment is more homogeneous and has an apparent molecular weight of about 30,000 (this second band is not discernible from the light chains in panel I of Fig. 3). In Fig. 2C one can see that the pneumococcal IgAl protease has completely cleaved the substrate with the disappearance of the heavy chains; the band above the heavy chains comigrates with the 67,000-molecular-weight
.H K.
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FIG. 2. SDS-PAGE of IgAl and IgA2 myeloma immunoglobulins incubated with H. influenza type b or S. pneumoniae type 1 IgAl protease. (A) Pharmacia low-molecular-weight standards; (B) IgAlSa (20 Ag) + Tris buffer; (C) IgAlSa (20 pg + S. pneumoniae; (D) IgAlSa (20 g) + H. influenza; (E) IgA2He (about 40 Ag) + Tris buffer; (F) IgA2He (about 40 pig) + S. pneumoniae, (0) IgA2He (about 40 pg) + H. influenza; (H) S. pneumoniae + Tris buffer; (I) H. influenza + Tris buffer; (J) IgA2Ke (about 40 pg) + Tris buffer; (K) IgA2Ke (about 40 pg) + S. pneumoniae; (L) IgA2Ke (about 40 pg) + H. influenza;
(M) Pharmacia standards. Arrows indicate H (heavy or alpha) and L (light) chains. Bacteria and myeloma substrates were incubated for 16 h.
258
MALE
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FIG. 3. SDS-PAGE of IgAl and IgA2 myeloma immunoglobulins incubated with H. influenzae type b or S. pneumoniae type I IgAl protease. (A) Pharmacia standards; (B) IgAlSa (15 pg) + Tris buffer; (C) IgAlSa (15 pg.) + S. pneumoniae; (D) S. pneumoniae + Tris buffer; (E) IgAlSa (15 pg) + H. influenzae; (F) IgAlSa (30 pg) + H. influenzae; (G) H. influenzae + Tris buffer; (H) IgAITr (30 pgJ + H. influenzae; (I) IgAlTr (15 ) + S. pneumoniae; (J) + Tris buffer; (K) IgA2Ke (about 20 IgAlTr (15 pg) + Tris buffer; (L) IgA2Ke (about 20 pg) + S. pneumoniae; (M) IgA2Ke (about 20 pg) + H. influenzae. Bacteria and myeloma substrates were incubated for 16 h.
marker bovine serum albumin and is presumably human serum albumin. The interaction of pneumococcal IgAl protease with the IgA2 substrates is particularly interesting. The heavy chains remain intact, but migrate faster than the heavy chains which have been incubated with buffer alone or with Haemophilus IgAl protease. It is possible that this shift in molecular weight is due to the cleavage of oligosaccharide side chains from the IgA2 proteins by S. pneumoniae glycosidases. In addition to the altered mobility of the heavy chains, two bands can be seen just above the light chain band in Fig. 2F and K; the two bands are not resolved in Fig. 3L, although it is the same substrate as that shown in Fig. 2K. The bands are 32,000 and 33,000 molecular weight with IgA2He and 31,000 and 32,500 molecular weight with IgA2Ke. We suggest that these bands might be the Fc and Fd cleavage fragments which result from the action of pneumo-
INFECT. IMMUN.
coccal IgAl protease on normal IgAl present in the IgA2 myeloma sera. The Haemophilus IgAl protease, as noted earlier, does not cleave IgAl substrates as well as the pneumococcal enzyme. In Fig. 2D, one can barely detect the cleaved fragments at molecular weights 33,000 and 36,500. Note that the cleavage fragments obtained with the pneumococcal and the Haemophilus enzyme from the same IgAl substrate have different apparent molecular weights. When Haemophilus IgAl protease is incubated with IgA2 immunoglobulin, there is no shift in the molecular weight of the heavy chains. Incubation of Haemophilus IgAl protease with IgA2 substrates does not lead to cleavage of postulated normal serum IgAl in the IgA2 myeloma substrates as was seen with the pneumococcal IgAl protease. In Fig. 3E and F one can more readily see the Haemophilus cleavage products which were so faint in Fig. 2D (same IgAl substrate). In addition, it can be seen that when Haemophilus IgAl protease cleaves a different IgAl myeloma substrate the cleavage products are different. Clearly the fragment in Fig. 3E and F with an apparent molecular weight of about 38,000 is not seen in Fig. 3H, although the 33,000-molecular-weight protein is present. From the increased density of staining below the light chain band in Fig. 3H, it is possible that the second cleavage fragment may be about 29,000 molecular weight. Note that this IgAl substrate (Tr) is partially degraded and that a band at 33,000 and material moving faster than the light chains can be seen in the buffer control sample (Fig. 3J); spontaneous cleavage of IgAl myeloma proteins in the hinge region is known to occur (C. Abel, personal communication). DISCUSSION The results reported here extend the reported incidence of IgAl protease-binding bacteria to two very important human pathogens, H. influenzae and S. pneumoniae. The incidence and severity of infection with these pathogens in both the pediatric and adult populations are amply documented. IgAl protease production appears to be associated with the majority of H. influenzae strains tested. Thus IgAl protease production cannot, by itself, explain either the increased pathogenicity observed with type b strains (25) or the high rate of nasopharyngeal colonization observed with untypable strains versus the very low rate of colonization seen with encapsulated strains (25). It is not excluded that in vivo interaction between IgAl proteases from type b and untypable H. influenzae strains with secretary or serum IgAl or both may be different from the
VOL. 26, 1979
interaction observed with myeloma sera. All strains of S. pneumoniae studied produced IgAl protease. Because S. pneumoniae strains also have several other enzymes which apparently can alter immunoglobulins by cleaving off all or part of their carbohydrates, we are currently examining the possible effects that carbohydrate cleavage might have on antibody function. The results obtained with electrophoresis of cleaved myeloma substrates and use of monospecific antisera are consistent with the conclusion that H. influenzae and S. pneumoniae synthesize IgAl proteases which cleave only IgAl substrates into Fab and Fc fragments. SDS-PAGE was initially performed to confirm the immunoelectrophoretic results. We had not expected to find (i) variations in light chain mobility which influenced the ability to detect cleavage fragments, (ii) alterations in the mobility of IgA2 alpha chains after incubation with the pneumococcus, and (iii) variations in the apparent molecular weights of IgAl proteasecleaved fragments. It is known that IgA2 myeloma proteins have complex oligosaccharides attached to asparagine residues in both the Fc and Fd portions of the alpha chain (22). IgAl myeloma proteins have five N-acetylgalactosamines attached to serine residues in the hinge region; four of the five galactosamines have an attached ,8-1,3-linked galactose (3). IgAl myeloma proteins also have complex oligosaccharides attached to asparagine residues; the oligosaccharides are usually found only in the Fc portion of the alpha chain, but can be found in both the Fd and Fc regions, depending on the specific myeloma protein studied (2, 22). It has been shown that IgG myeloma proteins can vary widely with respect to the quantity and quality of carbohydrates found on the light chains and in the Fd region; carbohydrate content of the Fc fragment is quite constant (1). Although IgA myeloma proteins have not been as extensively studied as the IgG myeloma proteins and such carbohydrate variations have not been reported, our SDS-PAGE data are consistent with carbohydrate variations on the alpha chains. Complex oligosaccharides of the type found on IgAl molecules can be enzymatically cleaved from glycoproproteins by the sequential enzymatic action of neuraminidase, 8l-galactosidase, exoglucosaminidase, and endoglucosaminidase (12, 23). The pneumococcus synthesizes all four enzymes (12) and might be expected to cleave carbohydrates from the IgAl substrates studied here, whereas H. influenzae is known not to synthesize neuraminidase (16) and therefore
IgAl PROTEASE PRODUCTION
259
could not initiate cleavage of carbohydrates. Since our SDS-PAGE data showed that the alpha chains of IgA2 proteins incubated with pneumococci had a faster mobility than those
incubated with buffer or H. influenzae alone, we
suggest that pneumococcal glycosidases can also cleave complex oligosaccharides from IgA2 proteins. Glycosidase cleavage of carbohydrate from various immunoglobulins is being studied. In light of the above discussion, our data could be interpreted in the following manner. Pneumococcal IgAl protease cleaved myeloma IgAl Sa into a homogeneous fragment of 29,000 molecular weight and a very heterogeneous band with an average apparent molecular weight of
36,500, whereas H. influenzae IgAl protease
cleaved the same substrate into relatively homogeneous fragments of 33,000 and 37,000 molecular weight. Although it is recognized that mobilities of glycoproteins in SDS-PAGE gels do not always relate to their carbohydrate content, the faster mobility of the pneumococcusderived fragments is consistent with cleavage of carbohydrates from these fragments. By analogy with the IgG myeloma data cited above, which showed little carbohydrate variation in the Fc region of heavy chains, we suggest that the faster moving, more homogeneous band might be the Fc fragments. The very heterogeneous, slower moving pneumococcal-derived band may be composed of a family of Fd fragments which contain variable amounts of carbohydrate as a result of cleavage by pneumococcal glycosidases. Alternatively, the reverse situation is perhaps even more plausible. That is, the heterogeneous band could be composed of Fc fragments containing variable amounts of carbohydrate. All IgAl myeloma proteins studied so far have oligosaccharides in the Fc region (2, 9, 22); carbohydrate in the Fd region is much rarer (22). Interpretation of the data obtained with myeloma IgAl Tr is more difficult because the buffer control shows that spontaneous fragmentation of the substrate had occurred, a buffer control containing the same amount of Tr protein as that used for H. influenzae was not included, and certain of the protease-cleaved fragments are obscured by the slow moving light chains. Nevertheless, it is clear that the Haemophilus and pneumococcus-derived fragments from the same substrate are different, as was seen with IgAlSa. Comparison of the pneumococcus-derived fragments from both IgAl substrates shows differences. The faster migrating fragments from both substrates do not have the same mobility; it is possible that the faster migrating fragment from IgAlTr may be comigrating with the light
260
MALE
chains. The broad, slow-moving heterogeneous band is a feature common to cleavage of both substrates. Comparison of the Haemophilus-derived fragments from both IgAl substrates indicates that the 33,000-molecular-weight fragment is common, but the 37,000-molecularweight fragment from IgAlSa is not observable with IgAlTr. We tentatively ascribe these apparent differences in the fragment sizes obtained by the same IgAl protease (Haemophilus or pneumococcus) acting on different IgAl substrates to carbohydrate variations of the alpha chains. We suggested earlier that the two homogeneous fragments observed above the light chains with both IgA2 substrates were the result of pneumococcal IgAl protease cleavage of normal serum IgAl in the IgA2 myeloma sera. Patients with IgA myeloma do not suppress synthesis of other immunoglobulins as severely as do patients with IgG myeloma (24). Considering the variations in fragment sizes obtained with IgAl myeloma sera, it is interesting that the fragments obtained from cleavage of presumed normal serum IgAl are quite homogeneous and practically identical in both IgA2 sera. It is necessary to exclude the possibility that the fragments detected in the IgA2 myeloma sera are derived from some other component rather than from normal serum IgAl before speculating on the possible reasons for the observed homogeneity. Two additional points should be made concerning the data presented in Fig. 3. First, with proteins Sa and Tr, the presumed human serum albumin band is not resolved from the heavy chains. Although several methodological explanations are possible, the validity of data concerning the cleavage products is not an issue. Second, it is quite apparent that the light chains of all three myeloma proteins have different mobilities. Altered mobility of various myeloma light chains has previously been reported (26). It is possible, analogous to some IgG myeloma proteins (1), that some IgA light chains have carbohydrate and some do not. If this were true, we might have expected the pneumococcal glycosidases to alter the mobility of the light chains; no obvious alteration in mobility was seen. However, contamination with IgG and IgM light chains may be obscuring small alterations. Alternatively, it has been reported that lambdatype light chains associated with alpha chains are often unusual with respect to primary structure (22). Proteins Ke and Sa have kappa light chains, and protein Tr has lamba chains. Whatever the explanations) for variation in light chain mobility, it is important to choose an IgAl substrate whose light chains will not comigrate
INFECT. IMMUN.
with IgAl protease-cleaved fragments. Plaut et al. have noted that two thirds of their myeloma IgAl substrates had Fab fragments whose mobility in cellulose acetate electrophoresis was similar to the Fc fragment or uncleaved substrate or both (19). It was mentioned earlier that pneumococcal IgAl protease appeared to cleave substrates more rapidly than Haemophilus protease. It was also stated that quantitative data are needed to substantiate this qualitative observation. In addition to the variables mentioned earlier, and because we feel the observation is potentially significant, there are at least two other variables that we wish to briefly mention. First, antibodies to Haemophilus protease which inhibit enzyme activity may be abundant in all IgAl substrates, whereas antibodies to S. pneumoniae protease are uniformly absent or present at very low levels in all IgAl substrates. We find this explanation unlikely. Second, pneumococcal glycosidases may expose the IgAl protease cleavage site by enzymatically cleaving the N-acetylgalactosamine residues in the hinge region, or the complex oligosaccharides found elsewhere on the alpha chain, or both. Although this hypothesis is attractive, it remains highly speculative. It was mentioned in Results that pneumococcal IgAl protease appeared to rapidly cleave all secretary IgA substrates examined. Several variables have been mentioned above and in Results to help explain the consistently observed increased cleavage of substrates by pneumococcal enzyme(s). S. sanguis and H. influenzae IgAl proteases showed varying degrees of activity against a variety of secretary IgA substrates. This differential activity may indeed be due to different levels of antibody (possibly IgA2) against the cell-bound or free Haemophilus and S. sanguis IgAl proteases or both in the different secretary IgA specimens. We are particularly interested in determining the possible in vivo functions) of S. pneumoniae and H. influenzae IgAl proteases. Systemic or local disease or both with both organisms is thought to be preceded by nasopharyngeal colonization. The complex host and microbial factors involved in the establishment and duration of carriage and the transition from the carrier state to systemic or local invasion or both are not well defined. These events no doubt include initial microbial interactions with secretary IgA, other microbes and host cells. The host's local and systemic cellular and humoral response to nasopharyngeal or gut colonization or both with haemophili, streptococci, and various cross-reacting microbes may well influence the transition from the carrier state to disease. It is possible that IgAl protease may modulate coloni-
VOL. 26, 1979
IgAl PROTEASE PRODUCTION
zation and may be involved in the transition to local or systemic disease or both. We are currently testing this hypothesis. ACKNOWLEDGMENTS I gratefully acknowledge Kenneth McIntosh's suggestion that S. pneumoniae be assayed for IgAl protease production. I would like to thank B. Reller, M. Glode, and K. McIntosh for bacterial strains and C. Abel and W. Brown and numerous others for immunoglobulin preparations. Meredith Peake's instruction in and assistance with the SDS-PAGE analyses was excellent. For their helpful discussions and enthusiastic support I thank M. Glode, A. Crowle, C. Abel, K. McIntosh, and A. Zlotnik. This project was supported in whole or part by Public Health Service BRSG RR-05357 awarded by the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health.
LITERATURE CITED 1. Abel, C. A., H. L. Spiegelberg, and H. M. Grey. 1968. The carbohydrate content of fragments and polypeptide chains of human yG-myeloma proteins of different heavy-chain subclasses. Biochemistry 7:1271-1278. 2. Baenziger, J., and S. Kornfeld. 1974. Structure of the carbohydrate units of IgAl immunoglobulin. I. Composition, glycopeptide isolation, and structure of the asparagine-linked oligosaccharide units. J. Biol. Chem. 249:7260-7269. 3. Baenziger, J., and S. Kornfeld. 1974. Structure of the carbohydrate units of IgAl Immunoglobulin. II. Structure of the O-glycosidically linked oligosaccharide units. J. Biol. Chem. 249:7270-7281. 4. Bergsagel, D. E. 1977. Plasma cell myeloma, p. 10991125. In W. J. Williams, E. Beutler, A. Erslev, and R. W. Rundles (ed.), Hematology, 2nd ed. McGraw-Hill Book Co., New York. 5. Blake, M., K. K. Holmes, and J. Swanson. 1979. Studies on gonococcus infection. XVII. IgAl-cleaving protease in vaginal washings from women with gonorrhea. J. Infect. Dis. 139:89-92. 6. Blake, M. S., and J. Swanson. 1978. Studies on gonoccus infection. XVI. Purification of Neisseria gonorrhoeae immunoglobulin A, protease. Infect. Immun. 22:350358. 7. Crowle, A. J. 1973. Immunodiffusion. Academic Press Inc., New York. 8. Crowle, A. J., and L. J. Cline. 1977. An improved stain for immunodiffusion tests. J. Immunol. Methods 17: 379-381. 9. Despont, J. J., and C. A. Abel. 1974. Glycopeptides of heavy chains from human IgA myeloma proteins. J. Immunol. 112:1623-1627. 10. Genco, R. J., A. G. Plaut, and R. C. Moellering, Jr. 1975. Evaluation of human oral organisms and pathogenic Streptococcus for production of IgA protease. J. Infect. Dis. 131:S17-S21. 11. Ginsburg, C. M., G. H. McCraken, Jr., R. Schneerson, J. B. Robbins, and J. C. Parke, Jr. 1978. Association between cross-reacting Escherichia coli K100 and disease caused by Haemophilus influenzae type b. Infect.
261
Immun. 22:339-342. 12. Koide, N., and T. Muramatsu. 1974. Endo-f?-N-acetylglucosaminidase acting on carbohydrate moieties of glycoproteins. J. Biol. Chem. 249:4897-4904. 13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 14. 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. 15. Mulks, M. H., and A. G. Plaut. 1978. IgA protease production as a characteristic distinguishing pathogenic from harmless neisseriaceae. N. Engl. J. Med. 299:973976. 16. O'Toole, R. D., L. Goode, and C. Howe. 1971. Neuraminidase activity in bacterial meningitis. J. Clin. Invest. 50:979-985. 17. Plaut, A. G., R. J. Genco, and T. B. Tomasi, Jr., 1974. Isolation of an enzyme from Streptococcus sanguis which specifically cleaves IgA. J. Immunol. 113:289291. 18. Plaut, A. G., J. V. Gilbert, M. S. Artenstein, and J. D. Capra. 1975. Neisseria gonorrhoeae and Neisseria meningitidis: extracellular enzyme cleaves human immunoglobulin A. Science 190:1103-1105. 19. Plaut, A. G., J. V. Gilbert and I. Heller. 1978. Assay and properties of IgA protease of Streptococcus sanguis, p. 489-495. In J. R. McGhee, J. Mestecky and J. L. Babb (ed.), Secretory immunity and infection. Plenum Publishing Corp., New York. 20. 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. 21. Plaut, A. G., R. W. Wistar, Jr., and J. D. Capra. 1974. Differential susceptibility of human IgA immunoglobulins to streptococcal IgA protease. J. Clin. Invest. 54: 1295-1300. 22. Putnam, F. W., A Torano, Y. Tsuzukida, A. Infante, and Y.-S. V. Liu. 1977. Structure of human IgAl and IgA2 immunoglobulins, p. 803-810. In H. Peeters (ed.), Protides of the biological fluids. Pergamon Press, New York. 23. Reading, C. L., E. E. Penhoet, and C. E. Ballou. 1978. Carbohydrate structure of vesicular stomatitis virus glycoprotein. J. Biol. Chem. 253:5600-5612. 24. Torano, A., Y. Tsuzukida, Y.-S. V. Liu, and F. W. Putnam. 1977. Location and structural significance of the oligo-saccharides in human IgAl and IgA2 immunoglobulins. Proc. Natl. Acad. Sci. U. S. A. 74:23012305. 25. Turk, D. C., and J. R. May. 1967. Haemophilus influenzae, p. 13-23. The English Universities Press, Ltd.,
London. 26. Virella, G., and I. M. Coelho. 1974. Unexpected mobility of human lambda chains in sodium dodecyl sulfate-
polyacrylamide gel electrophoresis. Immunochemistry
11:157-160. 27. Ward, J. I., G. Gorman, C. Phillips, and D. W. Fraser. 1978. Hemophilus influenzae type b disease in a daycare center. J. Pediatr. 92:713-717.
INFECTION AND IMMUNITY, Oct. 1979, p. 254-261 0019-9567/79/10-0254/08$02.00/0
Immunoglobulin Al Protease Production by Haemophilus influenza and Streptococcus pneumoniae CAROLYN J. MALE and Department of Microbiology Immunology, University of Colorado Medical School, Denver, Colorado 80262
Received for publication 7 May 1979
Bacterial strains of Haemophilus species and Streptococcus pneumoniae were examined for synthesis of the enzyme immunoglobulin Al (IgAl) protease. Of 36 H. influenzae strains examined, 35 produced IgAl protease; strains included all six capsular types, unencapsulated variants of types b and d, and untypable H. influenza. Eight Haemophilus strains (non-H. influenza) were studied, and two produced IgAl protease. All 10 strains of S. pneumoniae produced IgAl protease; these strains included 9 different capsular polysaccharide types and 1 untypable strain. Both IgAl proteases cleaved myeloma IgAl and secretary IgA but not myeloma IgA2, IgM, or IgG as determined by immunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes cleaved IgAl myeloma sera, but not IgA2, into two fragments. The apparent molecular weight of the cleaved fragments was dependent both on the specific IgAl protease assayed and the specific IgAl substrate utilized. It is postulated that both carbohydrate variation between the IgAl substrates studied and the ability of S. pneumoniae glycosidases to cleave carbohydrates from glycoprotein offer an explanation for the different fragment sizes observed.
Bacterial immunoglobulin A (IgA) protease was first described by Mehta et al. (14). This enzyme specifically cleaves only human serum IgAl and secretary IgA; IgA2, IgG, IgM, and IgE are resistant to enzymatic cleavage (14, 17). Cleavage of myeloma IgAl at a proline-threonine bond in the hinge region results in the formation of Fc and Fab fragments; the Fab fragments of serum IgAl are unable to bind antigen (18, 20, 21). A wide variety of microorganisms have been examined for IgA protease production and found to be negative (10). The enzyme is synthesized by many strains of Streptococcus sanguis and group H streptococci (10), by virtually all tested strains of Neisseria gonorrheae (18), and by typable and nontypable Neisseria meningitidis strains but not by normal commensal Neisseria species (15). Secretory IgA is the predominant immunoglobulin in colostrum and parotid fluid, and it is felt that IgA may be the predominant immunoglobulin in genital and nasal secretions. Plaut and his co-workers have suggested that IgA protease cleavage of IgAl in mucosal secretions may be very important in the pathogenesis of infections caused by bacteria which synthesize IgA protease (21). Recently, it has been shown that N. gonorrheae IgAl protease can be detected in the vaginal secretions of women with gonorrhea
(5).
Both Haemophilus influenza and Streptococcus pneumoniae are major pathogens, primarily of children, and it is believed that initiation of systemic infection follows nasopharyngeal colonization. For this reason we elected to examine strains of these organisms for IgAl protease production. (This paper was presented in part at the 79th Annual Meeting of the American Society for Microbiology, 4 to 8 May 1979, Los Angeles, Calif. [Abstr. no. B83, p. 29].) MATERLALS AND METHODS Bacteria. A known positive IgA protease strain, S. sanguis ATCC 10556, was provided by R. Eisenberg (University of Pennsylvania). A laboratory strain of group A streptococcus was confirmed as not producing IgA protease and was used as a negative control. The pneumococcal strains were provided by Barth Reller. Haemophilus strains were provided by the Colorado State Health Laboratories, Barth Reller, Kenneth McIntosh, and Mimi Glode (Children's Hospital, Denver, Co.). In addition to providing numerous Haemophilus strains, M. Glode also furnished the Escherichia coli K100 strains. The clinical origin of most, but not all, of the H. influenza strains was known and included cerebrospinal, blood, and nasopharyngeal specimens. Most of the specimens were from children with invasive H. influenza disease, but some specimens were from "asymptomatic" carriers. Two of the H. aphrophilus isolates were presumed to be from patients with endocarditis. The origin of the other Haemophilus
254
VOL. 26, 1979 strains (non-H. influenza) is not known. The pneumococcal strains were from patients with systemic pneumococcal disease. Stock cultures of bacteria were maintained in ToddHewitt broth with 30% glycerol at -70oC. Bacterial strains to be tested for IgA protease were grown on blood agar or chocolate agar in a C02 jar at 360C for 16 to 18 h. Immunoglobulins and antisera. Whole IgAl and IgA2 myeloma sera were provided by Carlos Abel and Howard Grey (National Jewish Hospital, Denver, Co.), Peter Kohler, Aroop Mangalik, and several private physicians in the Denver area. Purified IgA myeloma and colostral IgA were provided by William Brown and R. Eisenberg. Whole colostrum was received from C. Abel, W. Fuller, and M. Fishut. Concentrated parotid fluid was received from George Revis. The IgA concentrations of the whole myeloma and purified samples ranged from 2 to 15 mg/ml. Routine screening for IgA protease production was done with an IgAl kappa myeloma serum generously provided by Carlos Abel. This serum had a high IgAl titer, had no detectable IgG or IgM (by immunoelectrophoresis), and was available in large amounts. The light chains of the IgAl and IgA2 substrates were typed with antiserum to kappa and lambda light chains. IgM and IgG human immunoglobulins were also assayed for susceptibility to the Haemophilus and S. pneumoniae enzymes. IgM myeloma sera were provided by P. Kohler and R. Eisenberg, and the IgG was Cohn fraction II. Rabbit antisera to alpha, gamma, mu, kappa, and lambda chains were purchased from Biorad Laboratories. IgA protease assay. Several loopfuls of bacteria to be tested were taken from solid media and evenly suspended in 100 IL of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 8.1). Usually 10 pl of bacterial suspension was mixed with 10 microcentrifuge tube pl of IgA substrate in a 400-pl with attached cap, and the mixture was incubated in a 370C water bath for 3 to 4 h; longer incubations were used when secretary IgA was the substrate. Slides for electrophoresis were prepared with 2% granulated agar (BBL Microbiology Systems). General procedure, buffers, etc. for immunoelectrophoresis were those of Crowle (7). A 3- to 4-pl portion of the incubated bacteria and IgA mixture was placed in a well, and the sample was electrophoresed for 1 h (2 mA/slide, 100 V). Troughs were cut and 50 1pl of the appropriate antiserum was added. The slides were allowed to develop at 40C for 2 to 3 days. Slides were processed by the method of Crowle (personal communication) and stained with Crocein scarlet-brilliant blue R (8). For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), acrylamide slab gels (12.5% gel, 0.3% SDS) with a 5% stacking gel were prepared by the general method of Laemmli (13). After incubation of myeloma proteins with enzyme or buffer, samples were boiled for 3 min in the presence of 2% SDS and 1% mercaptoethanol. Samples were applied to the slab gel and electrophoresed at 10 mA until the dye front reached the bottom of the gel. The gels were stained for 1 h with Coomassie brilliant blue.
IgAl PROTEASE PRODUCTION
255
RESULTS Incidence of IgA protease production. The incidence of IgAl protease production among the Haemophilus and pneumococcal strains is shown in Table 1. Protease activity was determined by immunoelectrophoresis. Clearly, IgAl protease synthesis is a common feature of the H. influenza strains studied; only one type a strain was negative for protease synthesis. Although the number. of other Haemophilus species available for testing was limited, the majority of these strains did not make detectable IgAl protease. Suspensions of all pneumococci tested, including one untypable strain, contained IgAl protease. Because of the recently recognized association between invasive H. influenzae disease and gut colonization with E. coli K100 strains (11, 27), two E. coli K100 strains isolated from those patients were tested for IgA protease activity; both strains were negative. Evidence for IgAl protease cleavage of IgAl as determined by immunoelectrophoresis. The data presented in Fig. 1 indicate that the IgAl proteases produced by both H. influTABLE 1. Incidence of IgAI protease production by Haemophilus and S. pneumoniae strains No. No. positive (%) Bacterial strains studied Haemophilus strains 2 1 (50) H. influenza type a 15 (100) 15 H. influenzae type b 2 2 (100) H. influenzae type c 2 (100) 2 H. influenza type d 3 (100) 3 H. influenza type e 3 (100) 3 H. influenza type f 1 (100) 1 Unencapsulated type b 2 (100) 2 Unencapsulated type d
Untypable Total H. parainfluenzae H. parahaemolyticus H. aphrophilus Total S. pneumoniae strains S. pneumoniae type 1 S. pneumoniae type 3 S. pneumoniae type 4 S. pneumoniae type 5 S. pneumoniae type 6 S. pneumoniae type 8 S. pneumoniae type 14 S. pneumoniae type 17 S. pneumoniae type 19 Untypable Total
6 36
6 (100) 35 (97)
4 1 3
1 (25) 1 (100)
8
0 (0) 2 (25)
2 2 2 1 1 1 1 1 1
2 (100) 2 (100) 2 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100) 1 (100)
1
13
1
(100)
13 (100)
256
MALE
INFECT. IMMUN.
FIG. 1. Immunoelectrophoresis of IgAl and IgA2 myeloma immunoglobulins and colostrum incubated with H. influenzae or S. pneumoniae IgAl protease. (A) Well 1, IgAl My + Tris buffer (0.05 M, pH 8.1); well 2, IgAl My + S. pneumoniae type 1 cells; well 3, IgA2Ke + S. pneumoniae type 1; well 4, IgA2Ke + Tris buffer. Troughs a, b, and c: anti-alpha + anti-kappa mixed. (B) Well 1, IgAl My + Tris buffer; wells 2 and 3, IgAl My + S. pneumoniae type 1; well 4, IgA2Ke + S. pneumoniae type 1. Trough a, anti-alpha + anti-kappa, mixed; trough b, anti-kappa; trough c, anti-alpha. (C) Well 1, IgAl My + Tris buffer; wells 2 and 3, IgAl My + H. influenzae type b; well 4, IgA2Ke + H. influenzae type b. Trough a, anti-alpha + anti-kappa, mixed; trough b, anti-kappa; trough c, anti-alpha. (D) Well 1, colostrum Ca + Tris buffer; well 2, colostrum Ca + H. influenzae type b; well 3, colostrum Ca + S. pneumoniae type 1; well 4, colostrum Ab + H. influenzae type b. Troughs a, b, and c: anti-alpha, anti-kappa, anti-lambda, mixed. (B) Arrows indicate Fab and Fc fragments. Bacteria were incubated with myeloma substrates for 6 h, and colostrum substrates were incubated for 16 h. Anode is to the left.
enzae and S. pneumoniae specifically cleaved IgAl but not IgA2 immunoglobulins. Use of monospecific antisera (Fig. 1B and C) suggested that incomplete cleavage of IgAl into Fab and Fc fragments had occurred. Anti-kappa antiserum reacts only with the Fab fragment, and antialpha chain antiserum reacts only with the Fc fragment, as demonstrated by Mehta et al. (14). Note in slide A that the IgA2 myeloma substrate has IgG and IgM immunoglobulins which are detected with the anti-kappa antiserum. These precipitin lines must be distinguished from the precipitin lines which result from true protease cleavage; the contaminant precipitin lines are qualitatively different from the Fab precipitin lines and are also present in the buffer control. We emphasize this point because if an IgAl substrate with detectable IgG and IgM is employed, and unequal amounts of substrate are placed in the wells, it is possible to get no contaminant lines in the buffer control and contaminant lines in the incubated mixture. Haemophilus IgAl protease seldom cleaved all the IgAl substrate to completion regardless of the length of incubation. Partial cleavage (Fig. 1C) was more commonly seen, although occa-
sionally complete cleavage into Fab and Fc fragments was observed as evidenced by crossed precipitin lines of nonidentity (14). Pneumococcal IgAl protease routinely appeared to cleave IgAl substrates more rapidly than Haemophilus IgAl protease. Since no attempt was made to quantitate the protease assay, it is not known if, with respect to Haemophilus IgAl protease, pneumococci synthesize more protease or the protease has greater specific activity or there are more pneumococci in the assay tube. The pneumococcal and Haemophilus cleavage products differed with respect to both electrophoretic mobility and diffusion, as a comparison of slides B and C illustrates. We believe that these electrophoretic and diffusion differences may be related to the ability of pneumococcal enzymes to cleave carbohydrate from the heavy chains of IgAl. The rapid diffusion of pneumococcal fragments made it difficult, methodologically, to produce slides for publication which showed separate Fab and Fc fragments which had not diffused into the troughs; use of hyperimmune antisera would have been beneficial. Both bacterial proteases cleaved secretary IgA (whole clostrum or parotid fluid; purified colos-
VOL. 26, 1979
tral IgAl), but not all secretary IgA substrates were cleaved by both enzymes. Cleavage of secretory IgA required a longer incubation period than that with myeloma sera. Comparison of apparent cleavage of colostrum by both IgAl proteases is shown in Fig. iD. The pneumococcal IgAl protease appeared to cleave all secretory IgA substrates most rapidly; protease from H. influenza was the least active against a variety of secretary IgA substrates and S. sanguis was somewhat intermediate (data not shown). We have not yet identified the specific cleavage products of secretary IgA. Identification of secretary IgA cleavage products by immunoelectrophoresis alone is difficult because of the presence of large amounts of IgAl protease-resistant IgA2 immunoglobulin. Our cleavage patterns are siibilar to those shown by Genco et al. in slide B of their paper (10). These authors also showed complete cleavage of secretory IgA substrate into Fc and Fab fragments; however, their buffer control showed two precipitin bands referred to as "an experimental artifact." With fresh, structurally intact secretary IgA, which gives a single precipitin band against anti-alpha antiserum, we have never seen cleavage which results in separate Fab and Fc fragments. Similar to the results of Genco et al., we have seen such a pattern with a parotid saliva IgA substrate which was partially degraded and showed two separate precipitin bands when incubated with buffer only. Neither the H. influenza nor S. pneumoniae IgAl proteases cleaved human serum IgG or IgM (data not shown). IgAl protease cleavage as determined by SDS-PAGE. As further confirmation that H. influenza and S. pneumoniae produced IgAl protease, we ran polyacrylamide gels to demonstrate the two expected cleavage products from IgAl myeloma substrates. We were surprised to find the following: (i) the light chains of various myeloma proteins had different mobilities; (ii) assaying a single myeloma substrate, the cleavage products of the Haemophilus and pneumococcal IgAl proteases appeared to be different; (iii) the cleavage products resulting from either Haemophilus or pneumococcal IgAl protease varied among the IgAl myeloma sera tested; and (iv) the heavy chains of IgA2 proteins had a faster mobility after incubation with pneumococci. Blake and Swanson reported that the cleavage products of N. gonorrheae IgAl protease were 30,000 and 33,000 molecular weight as determined by SDS-PAGE (6). Since it is known that the gonococcal IgAl protease cleaves in the hinge region of the IgAl immunoglobulin (18), presumably the two cleavage
257
IgAl PROTEASE PRODUCTION
products on SDS-PAGE are the specific Fc and Fd fragments which result from cleavage of the IgAl alpha chain. Although we have not yet determined the exact site of cleavage for either of our IgAl proteases, our immunoelectrophoretic and gel electrophoresis data are consistent with cleavage in the hinge region. The data in Fig. 2 and 3 indicate that the pneumococcal IgAl protease cleaves IgAl substrates with the appearance of two bands; one band is very heterogeneous, with an apparent average molecular weight of about 36,000, whereas the faster moving fragment is more homogeneous and has an apparent molecular weight of about 30,000 (this second band is not discernible from the light chains in panel I of Fig. 3). In Fig. 2C one can see that the pneumococcal IgAl protease has completely cleaved the substrate with the disappearance of the heavy chains; the band above the heavy chains comigrates with the 67,000-molecular-weight
.H K.
A B CD E 94K
J K L M .
PG H
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FIG. 2. SDS-PAGE of IgAl and IgA2 myeloma immunoglobulins incubated with H. influenza type b or S. pneumoniae type 1 IgAl protease. (A) Pharmacia low-molecular-weight standards; (B) IgAlSa (20 Ag) + Tris buffer; (C) IgAlSa (20 pg + S. pneumoniae; (D) IgAlSa (20 g) + H. influenza; (E) IgA2He (about 40 Ag) + Tris buffer; (F) IgA2He (about 40 pig) + S. pneumoniae, (0) IgA2He (about 40 pg) + H. influenza; (H) S. pneumoniae + Tris buffer; (I) H. influenza + Tris buffer; (J) IgA2Ke (about 40 pg) + Tris buffer; (K) IgA2Ke (about 40 pg) + S. pneumoniae; (L) IgA2Ke (about 40 pg) + H. influenza;
(M) Pharmacia standards. Arrows indicate H (heavy or alpha) and L (light) chains. Bacteria and myeloma substrates were incubated for 16 h.
258
MALE
. -. . ;
*
X}q
..
t
..
D :E t G 1 |
..
j K L
L.w>,,
FIG. 3. SDS-PAGE of IgAl and IgA2 myeloma immunoglobulins incubated with H. influenzae type b or S. pneumoniae type I IgAl protease. (A) Pharmacia standards; (B) IgAlSa (15 pg) + Tris buffer; (C) IgAlSa (15 pg.) + S. pneumoniae; (D) S. pneumoniae + Tris buffer; (E) IgAlSa (15 pg) + H. influenzae; (F) IgAlSa (30 pg) + H. influenzae; (G) H. influenzae + Tris buffer; (H) IgAITr (30 pgJ + H. influenzae; (I) IgAlTr (15 ) + S. pneumoniae; (J) + Tris buffer; (K) IgA2Ke (about 20 IgAlTr (15 pg) + Tris buffer; (L) IgA2Ke (about 20 pg) + S. pneumoniae; (M) IgA2Ke (about 20 pg) + H. influenzae. Bacteria and myeloma substrates were incubated for 16 h.
marker bovine serum albumin and is presumably human serum albumin. The interaction of pneumococcal IgAl protease with the IgA2 substrates is particularly interesting. The heavy chains remain intact, but migrate faster than the heavy chains which have been incubated with buffer alone or with Haemophilus IgAl protease. It is possible that this shift in molecular weight is due to the cleavage of oligosaccharide side chains from the IgA2 proteins by S. pneumoniae glycosidases. In addition to the altered mobility of the heavy chains, two bands can be seen just above the light chain band in Fig. 2F and K; the two bands are not resolved in Fig. 3L, although it is the same substrate as that shown in Fig. 2K. The bands are 32,000 and 33,000 molecular weight with IgA2He and 31,000 and 32,500 molecular weight with IgA2Ke. We suggest that these bands might be the Fc and Fd cleavage fragments which result from the action of pneumo-
INFECT. IMMUN.
coccal IgAl protease on normal IgAl present in the IgA2 myeloma sera. The Haemophilus IgAl protease, as noted earlier, does not cleave IgAl substrates as well as the pneumococcal enzyme. In Fig. 2D, one can barely detect the cleaved fragments at molecular weights 33,000 and 36,500. Note that the cleavage fragments obtained with the pneumococcal and the Haemophilus enzyme from the same IgAl substrate have different apparent molecular weights. When Haemophilus IgAl protease is incubated with IgA2 immunoglobulin, there is no shift in the molecular weight of the heavy chains. Incubation of Haemophilus IgAl protease with IgA2 substrates does not lead to cleavage of postulated normal serum IgAl in the IgA2 myeloma substrates as was seen with the pneumococcal IgAl protease. In Fig. 3E and F one can more readily see the Haemophilus cleavage products which were so faint in Fig. 2D (same IgAl substrate). In addition, it can be seen that when Haemophilus IgAl protease cleaves a different IgAl myeloma substrate the cleavage products are different. Clearly the fragment in Fig. 3E and F with an apparent molecular weight of about 38,000 is not seen in Fig. 3H, although the 33,000-molecular-weight protein is present. From the increased density of staining below the light chain band in Fig. 3H, it is possible that the second cleavage fragment may be about 29,000 molecular weight. Note that this IgAl substrate (Tr) is partially degraded and that a band at 33,000 and material moving faster than the light chains can be seen in the buffer control sample (Fig. 3J); spontaneous cleavage of IgAl myeloma proteins in the hinge region is known to occur (C. Abel, personal communication). DISCUSSION The results reported here extend the reported incidence of IgAl protease-binding bacteria to two very important human pathogens, H. influenzae and S. pneumoniae. The incidence and severity of infection with these pathogens in both the pediatric and adult populations are amply documented. IgAl protease production appears to be associated with the majority of H. influenzae strains tested. Thus IgAl protease production cannot, by itself, explain either the increased pathogenicity observed with type b strains (25) or the high rate of nasopharyngeal colonization observed with untypable strains versus the very low rate of colonization seen with encapsulated strains (25). It is not excluded that in vivo interaction between IgAl proteases from type b and untypable H. influenzae strains with secretary or serum IgAl or both may be different from the
VOL. 26, 1979
interaction observed with myeloma sera. All strains of S. pneumoniae studied produced IgAl protease. Because S. pneumoniae strains also have several other enzymes which apparently can alter immunoglobulins by cleaving off all or part of their carbohydrates, we are currently examining the possible effects that carbohydrate cleavage might have on antibody function. The results obtained with electrophoresis of cleaved myeloma substrates and use of monospecific antisera are consistent with the conclusion that H. influenzae and S. pneumoniae synthesize IgAl proteases which cleave only IgAl substrates into Fab and Fc fragments. SDS-PAGE was initially performed to confirm the immunoelectrophoretic results. We had not expected to find (i) variations in light chain mobility which influenced the ability to detect cleavage fragments, (ii) alterations in the mobility of IgA2 alpha chains after incubation with the pneumococcus, and (iii) variations in the apparent molecular weights of IgAl proteasecleaved fragments. It is known that IgA2 myeloma proteins have complex oligosaccharides attached to asparagine residues in both the Fc and Fd portions of the alpha chain (22). IgAl myeloma proteins have five N-acetylgalactosamines attached to serine residues in the hinge region; four of the five galactosamines have an attached ,8-1,3-linked galactose (3). IgAl myeloma proteins also have complex oligosaccharides attached to asparagine residues; the oligosaccharides are usually found only in the Fc portion of the alpha chain, but can be found in both the Fd and Fc regions, depending on the specific myeloma protein studied (2, 22). It has been shown that IgG myeloma proteins can vary widely with respect to the quantity and quality of carbohydrates found on the light chains and in the Fd region; carbohydrate content of the Fc fragment is quite constant (1). Although IgA myeloma proteins have not been as extensively studied as the IgG myeloma proteins and such carbohydrate variations have not been reported, our SDS-PAGE data are consistent with carbohydrate variations on the alpha chains. Complex oligosaccharides of the type found on IgAl molecules can be enzymatically cleaved from glycoproproteins by the sequential enzymatic action of neuraminidase, 8l-galactosidase, exoglucosaminidase, and endoglucosaminidase (12, 23). The pneumococcus synthesizes all four enzymes (12) and might be expected to cleave carbohydrates from the IgAl substrates studied here, whereas H. influenzae is known not to synthesize neuraminidase (16) and therefore
IgAl PROTEASE PRODUCTION
259
could not initiate cleavage of carbohydrates. Since our SDS-PAGE data showed that the alpha chains of IgA2 proteins incubated with pneumococci had a faster mobility than those
incubated with buffer or H. influenzae alone, we
suggest that pneumococcal glycosidases can also cleave complex oligosaccharides from IgA2 proteins. Glycosidase cleavage of carbohydrate from various immunoglobulins is being studied. In light of the above discussion, our data could be interpreted in the following manner. Pneumococcal IgAl protease cleaved myeloma IgAl Sa into a homogeneous fragment of 29,000 molecular weight and a very heterogeneous band with an average apparent molecular weight of
36,500, whereas H. influenzae IgAl protease
cleaved the same substrate into relatively homogeneous fragments of 33,000 and 37,000 molecular weight. Although it is recognized that mobilities of glycoproteins in SDS-PAGE gels do not always relate to their carbohydrate content, the faster mobility of the pneumococcusderived fragments is consistent with cleavage of carbohydrates from these fragments. By analogy with the IgG myeloma data cited above, which showed little carbohydrate variation in the Fc region of heavy chains, we suggest that the faster moving, more homogeneous band might be the Fc fragments. The very heterogeneous, slower moving pneumococcal-derived band may be composed of a family of Fd fragments which contain variable amounts of carbohydrate as a result of cleavage by pneumococcal glycosidases. Alternatively, the reverse situation is perhaps even more plausible. That is, the heterogeneous band could be composed of Fc fragments containing variable amounts of carbohydrate. All IgAl myeloma proteins studied so far have oligosaccharides in the Fc region (2, 9, 22); carbohydrate in the Fd region is much rarer (22). Interpretation of the data obtained with myeloma IgAl Tr is more difficult because the buffer control shows that spontaneous fragmentation of the substrate had occurred, a buffer control containing the same amount of Tr protein as that used for H. influenzae was not included, and certain of the protease-cleaved fragments are obscured by the slow moving light chains. Nevertheless, it is clear that the Haemophilus and pneumococcus-derived fragments from the same substrate are different, as was seen with IgAlSa. Comparison of the pneumococcus-derived fragments from both IgAl substrates shows differences. The faster migrating fragments from both substrates do not have the same mobility; it is possible that the faster migrating fragment from IgAlTr may be comigrating with the light
260
MALE
chains. The broad, slow-moving heterogeneous band is a feature common to cleavage of both substrates. Comparison of the Haemophilus-derived fragments from both IgAl substrates indicates that the 33,000-molecular-weight fragment is common, but the 37,000-molecularweight fragment from IgAlSa is not observable with IgAlTr. We tentatively ascribe these apparent differences in the fragment sizes obtained by the same IgAl protease (Haemophilus or pneumococcus) acting on different IgAl substrates to carbohydrate variations of the alpha chains. We suggested earlier that the two homogeneous fragments observed above the light chains with both IgA2 substrates were the result of pneumococcal IgAl protease cleavage of normal serum IgAl in the IgA2 myeloma sera. Patients with IgA myeloma do not suppress synthesis of other immunoglobulins as severely as do patients with IgG myeloma (24). Considering the variations in fragment sizes obtained with IgAl myeloma sera, it is interesting that the fragments obtained from cleavage of presumed normal serum IgAl are quite homogeneous and practically identical in both IgA2 sera. It is necessary to exclude the possibility that the fragments detected in the IgA2 myeloma sera are derived from some other component rather than from normal serum IgAl before speculating on the possible reasons for the observed homogeneity. Two additional points should be made concerning the data presented in Fig. 3. First, with proteins Sa and Tr, the presumed human serum albumin band is not resolved from the heavy chains. Although several methodological explanations are possible, the validity of data concerning the cleavage products is not an issue. Second, it is quite apparent that the light chains of all three myeloma proteins have different mobilities. Altered mobility of various myeloma light chains has previously been reported (26). It is possible, analogous to some IgG myeloma proteins (1), that some IgA light chains have carbohydrate and some do not. If this were true, we might have expected the pneumococcal glycosidases to alter the mobility of the light chains; no obvious alteration in mobility was seen. However, contamination with IgG and IgM light chains may be obscuring small alterations. Alternatively, it has been reported that lambdatype light chains associated with alpha chains are often unusual with respect to primary structure (22). Proteins Ke and Sa have kappa light chains, and protein Tr has lamba chains. Whatever the explanations) for variation in light chain mobility, it is important to choose an IgAl substrate whose light chains will not comigrate
INFECT. IMMUN.
with IgAl protease-cleaved fragments. Plaut et al. have noted that two thirds of their myeloma IgAl substrates had Fab fragments whose mobility in cellulose acetate electrophoresis was similar to the Fc fragment or uncleaved substrate or both (19). It was mentioned earlier that pneumococcal IgAl protease appeared to cleave substrates more rapidly than Haemophilus protease. It was also stated that quantitative data are needed to substantiate this qualitative observation. In addition to the variables mentioned earlier, and because we feel the observation is potentially significant, there are at least two other variables that we wish to briefly mention. First, antibodies to Haemophilus protease which inhibit enzyme activity may be abundant in all IgAl substrates, whereas antibodies to S. pneumoniae protease are uniformly absent or present at very low levels in all IgAl substrates. We find this explanation unlikely. Second, pneumococcal glycosidases may expose the IgAl protease cleavage site by enzymatically cleaving the N-acetylgalactosamine residues in the hinge region, or the complex oligosaccharides found elsewhere on the alpha chain, or both. Although this hypothesis is attractive, it remains highly speculative. It was mentioned in Results that pneumococcal IgAl protease appeared to rapidly cleave all secretary IgA substrates examined. Several variables have been mentioned above and in Results to help explain the consistently observed increased cleavage of substrates by pneumococcal enzyme(s). S. sanguis and H. influenzae IgAl proteases showed varying degrees of activity against a variety of secretary IgA substrates. This differential activity may indeed be due to different levels of antibody (possibly IgA2) against the cell-bound or free Haemophilus and S. sanguis IgAl proteases or both in the different secretary IgA specimens. We are particularly interested in determining the possible in vivo functions) of S. pneumoniae and H. influenzae IgAl proteases. Systemic or local disease or both with both organisms is thought to be preceded by nasopharyngeal colonization. The complex host and microbial factors involved in the establishment and duration of carriage and the transition from the carrier state to systemic or local invasion or both are not well defined. These events no doubt include initial microbial interactions with secretary IgA, other microbes and host cells. The host's local and systemic cellular and humoral response to nasopharyngeal or gut colonization or both with haemophili, streptococci, and various cross-reacting microbes may well influence the transition from the carrier state to disease. It is possible that IgAl protease may modulate coloni-
VOL. 26, 1979
IgAl PROTEASE PRODUCTION
zation and may be involved in the transition to local or systemic disease or both. We are currently testing this hypothesis. ACKNOWLEDGMENTS I gratefully acknowledge Kenneth McIntosh's suggestion that S. pneumoniae be assayed for IgAl protease production. I would like to thank B. Reller, M. Glode, and K. McIntosh for bacterial strains and C. Abel and W. Brown and numerous others for immunoglobulin preparations. Meredith Peake's instruction in and assistance with the SDS-PAGE analyses was excellent. For their helpful discussions and enthusiastic support I thank M. Glode, A. Crowle, C. Abel, K. McIntosh, and A. Zlotnik. This project was supported in whole or part by Public Health Service BRSG RR-05357 awarded by the Biomedical Research Grant Program, Division of Research Resources, National Institutes of Health.
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