Studies on the gonococcal IgA1 protease II

Journal oflmmunological Methods, 144 (1991) 215-221

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© 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50

JIM 06102

Studies on the gonococcal IgA1 protease II Improved methods of enzyme purification and production of monoclonal antibodies to the enzyme M.S. Blake and C. Eastby Laboratory of Bacteriology and Immunology, The Rockefeller University, New York, N~, U.S.A. (Received 3 April 1991, revised received 12 July 1991, accepted 15 July 1991)

Two types of extremely active proteases that cleave human IgA1 are produced by pathogenic Neisseria in minute concentrations. To study the antigenicity of these enzymes, a simplified method is described to purify these enzymes from large batch cultures to obtain a sufficient quantity of these IgA1 proteases to study these characteristics. In addition, we describe the production of both rabbit polyclonal and mouse monoclonal antibodies to one of these enzymes. One such monoclonal antibody seemed directed toward the active site of the IgA1 protease and inhibited its enzymatic activity. Key words: Neisseria; IgA1 protease; IgA1; Monoclonal antibody; Polyclonal serum; (Human)

Introduction Although many bacteria inhabit the human mucosa, only relatively few have the potential to become pathogenic. Among some pathogenic characteristics that have been defined to these few is the production of IgA cleaving enzymes, known collectively as IgA1 proteases. Among the Neisserial species, only N. gonorrheae and N. meningitidis have been found to produce these enzymes. The presence of these proteases in a growing liquid culture was first described by Miiller (Miiller, 1971) and then more specifically by Plaut et al. (Mehta et al., 1973; Genco et al., 1975; Plaut et al., 1975; Plaut, 1978, 1983; Mulks and Plaut, 1978) for many other bacterial species

Correspondence to: M.S. Blake, The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A.

including the gonococcal and meningococcal IgA1 proteases. Plaut et al. showed that these enzymes cleave the IgA1 molecule in a specific area within the hinge region producing Fab and Fc fragments. Furthermore, that the specific area effected by these enzymes was absent from the IgA2 molecules. Many of the genes for the IgA1 proteases from several different bacterial species have been cloned and sequenced (Koomey et al., 1982; Halter et al., 1984). Evidence is also given within these reports on how these proteases are expressed and processed (Halter et al., 1984). The data would suggest that in Neisseria two separate IgA1 proteases exist indicated both from cleavage patterns of the IgA1 enzymes and the endonuclease cleavage patterns of the cloned genes (Mulks and Knapp, 1987; Mulks et al., 1980). Yet, very little is known about the antigenic differences between these enzymes and if an immune re-

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sponse is elicited in humans upon exposure to these proteases. Also, there is little direct evidence for their involvement into the pathogenic mechanisms of the organisms that express them except their presence during infection (Blake et al., 1979). To answer some of these questions, we sought a way to purify several of these enzymes to study their antigenicity and immunogenicity. Several papers describe methods to purify the IgA1 protease from spent culture supernatants (Blake and Swanson, 1978; Plaut et al., 1978; Halter et al., 1984; Simpson et al., 1988). But, most of these techniques are time consuming and labor intensive. The difficulty in purifying these enzymes lies in the fact that they are produced in such low amounts that large cultures are necessary to produce relatively small amounts of the enzyme for further study. Thus, we sought easier ways to process the large volume of culture supernatant needed. Herein, we describe the use of phenylSepharose, a hydrophobic matrix, as the first step in the purification of gonococcal and meningococcal IgA1 proteases. In addition, the production of polyclonal and monoclonal antibodies to one of these enzymes is described.

Materials and methods

Bacteria and culture conditions Gonococci were maintained by single colony transfers every 18-24 h on solid typing media (Swanson, 1978) and grown in an incubator maintained at 37°C with 5% CO 2. Meningococci were grown similarly with the exception that they were maintained at 30°C in a candle extinction jar. The bacteria were verified as N. gonorrhoeae or N. rneningitidis by Gram's staining, and oxidase and fermentation reactions. The growing of the gonococci or meningococci in a fermentor was performed as previously described (Blake and Gotschlich, 1982) with these exceptions. The fermentor was a 100 liter automated fermentor (New Brunswick Scientific, New Brunswick, NJ). Typically, 16 liters of 5 × growth media were pumped into the fermentor through a Pellicon Cassette System with a PLGC Packet (Millipore, Bedford, MA). An additional 64 liters

of distilled water were added and the mixture sterilized in the fermentor. After the media cooled to 37°C, 1 liter of growth supplements identical in composition to Isovitalex (BBL, Baltimore, MD) was added sterily. The starting inoculum was as previously described (Blake and Gotschlich, 1982). The fermentor was maintained with an air flow rate of 5 liters/min, an agitation of 100 rpm, and a pressure of 10 psi for 12 h. The culture was then harvested using a Pellicon Cassette System with a GVLP packet (Millipore) and the supernatant collected, brought to 100 mM EDTA, 0.2 M (NH4)2SO 4, pH 9.0 and cooled to 4°C.

ELISA assay for IgA1 protease The functional assay used for detecting the IgA1 protease in column fractions, etc., has been previously described (Russell-Jones et al., 1984). The IgA binding protein isolated from group B streptococci (Russell-Jones et al., 1984) and used in the detection of IgA1 protease, is commercially available under the registered trade mark name of protein B (Blake Laboratories, Cambridge, MA). Use of protein B in ELISA assays has been described by others (Faulmann et al., 1991). Briefly, microtiter plates (Nunc-Immuno Plate MaxiSorp, Laboratory Disposable Products, N. Haledon, N J) were sensitized with protein B (100 /zl/well of a 2 ~ g / m l solution in 0.1 M carbonate buffer pH 9.8) (Blake Laboratories). The plates were washed six times with a solution of Brij 35 (0.05%) in saline (Brij-NaC1). Samples (50 /xl) to be tested for IgA1 protease were then added to the wells. An IgA1 myeloma protein (50 ~1) (Organon Teknika-Cappel, Malvern, PA) at a concentration of 2 / ~ g / m l in 0.2 M Tris HC1, 0.4 M NaCI, 40 mM MgCI z, 40 mM CaCI 2, pH 8.0 was added and the mixture incubated in a shaking incubator at 37°C for 1 h. The plates were then washed as before and an alkaline-phosphatase conjugated rabbit anti-human kappa chain (Tago, Burlingame, CA) was added. After incubating at room temperature for 2 h, the plates were rewashed as described and developed using p-nitrophenyl phosphate (Sigma, St. Louis, MO) in 10% diethanolamine for 1 h at 37°C. The absorbance at 405 nm was then read in a Elida-5 automated reader (Physica Biomedical Inst., New York, NY).

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IgA1 protease purification The purity of the enzyme was determined by SDS-PAGE analysis. The culture supernatant from above was pumped directly on to a K 50/30 FF column loaded with phenyl-Sepharose Fast Flow (Pharmacia LKB Biotechnology, Piscataway, N J), which had been previously equilibrated with cold 0.05 M Tris, 100 mM EDTA, 0.2 M (NH4)2SO4, pH 9.0, at a flow rate of approximately 2 liters/min. The column was then washed with 4 column volumes of equilibration buffer. After that, the column was eluted with 2 column volume gradient between 0.05 M Tris, 100 mM EDTA, 0.2 M (NH4)2SO4, pH 9.0 and 0.05 M sodium acetate buffer with 100 mM EDTA, pH 6.0. Protein elution was monitored by 280 nm absorption and SDS-PAGE, and the fractions assayed for IgA1 protease. Fractions showing enzyme activity were pooled and rechromatographed to final purity on a FPLC (Pharmacia LKB Biotechnology) Mono S column equilibrated with the sodium acetate buffer. The Mono S column was then washed with 4 column volumes of the sodium acetate buffer and eluted with a NaCI gradient between 0 and 0.5 M in a volume of 200 ml with 4 ml fractions being collected. The protein and enzyme elution was monitored as before. SDS-PAGE and Western blot The SDS-PAGE was a variation of Laemmli's method (Laemmli, 1970) as described previously (Blake and Gotschlich, 1984). Electrophoretic transfer to Immobilon P (Millipore) was performed according to the methods of Towbin et al. 1979) with the exception that the paper was first wetted in methanol. The Western blots were probed with phosphatase conjugated reagents (Blake et al., 1984). Production of polyclonal antibodies New Zealand White rabbits were used to produce polyclonal antibodies. The rabbits were initially immunized subscapularly with 100 ~g of purified IgA1 protease mixed with Freund's complete adjuvant. Booster immunizations were similar except Freund's incomplete adjuvant was used. Each animal was immunized four times and bled

three weeks after the last injection for analysis of antibody production by ELISA assay.

Production of monoclonal antibodies BALB/c mice were used for the production of monoclonal antibodies. The mice were immunized intraperitoneally with 100 /xg of purified IgA1 protease preparation emulsified in incomplete Freund's adjuvant in a total volume of 0.1 ml. The mice received two booster injections of 0.1 ml of the protease in saline. The serum titer for each animal was measured by ELISA. For production of hybrid clones, we employed the non-secreting mouse myeloma cell line P3NS1/1-Ag4-1. Three mice with the highest titer were killed and their spleens aseptically removed, lymphocyte suspension prepared and fused with the myeloma cells using polyethylene glycol (Kwan et al., 1980). The hybrid cells were selected by using the HAT selective media (Littlefield, 1964). The cells were suspended in HAT at a density of 2.5-5 x 106 myeloma cells/ml and plated at approximately 0.1 ml/well in 96 microculture plates. The plates were incubated in a 6.5% CO z atmosphere at 37°C. The wells were fed 100 /xl of HAT medium on the 4th day. Thereafter, 100/xl from each of the wells was removed every other day and replaced by 100/zl of fresh HAT media. The 100/zl spent media of each well was assayed for antibodies reactive to the IgA1 protease by ELISA and Western blot analysis. Cells from those microwells exhibiting positive reactions were cloned by limiting dilution and stabilized.

Results

Bacteria and culture conditions The initial growth of the bacteria and the expression of the IgA1 protease was monitored by OD 600, the protease ELISA assay, and SDSPAGE of the culture supernatant. Most of the IgA1 protease was produced in late log phase of bacterial growth (Blake and Swanson, 1978; Simpson et al., 1988). Once the monoclonal and polyclonal antibodies to the protease were made, we also monitored the culture supernatant by Western blot analyses using these antibodies. These results showed that the major contaminant

218 in the culture supernatant after removal of membrane associated material was a 37 kDa protein. The concentration of this 37 kDa protein increased rapidly in late log into stationary phase of each culture. Using a monoclonal antibody kindly provided by Timothy Mietzner, this protein was identified as the major iron regulated protein of Neisseria (Mietzner et al., 1987). Additional iron added to the growth media tended to suppress the amount of this protein that contaminated the culture supernatant. With the use of the monoclonal antibodies in a Western blot analysis, attempts were made to decide if the expression of the IgA1 protease was regulated by decreasing amounts of iron present in the media. Increasing or decreasing amounts of iron made no difference of expression of the IgA1 proteases (data not shown).

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30 IgA1 protease purification Starting with the spent culture supernatant, a desirable method to purify the IgA1 protease would be one that would require very few initial adjustments to the spent culture supernatant and serve both to concentrate and eliminate several contaminants. The addition of the 100 mM EDTA-tetrasodium to type 2 gonococcal IgA1 protease cultures was to eliminate other proteolytic enzymes that were present and to inactivate the self degradation that has been shown for the gonococcal IgA1 protease (Halter et al., 1984). The pH of the culture was then adjusted to a pH of 9 to increase the hydrophobic interactions between the protease and the phenyl-Sepharose column. With the type 2 IgA1 proteases these adjustments to the broth were sufficient to cause the enzyme to bind to the hydrophobic matrix while the type 1 enzyme required the addition of the ammonium sulfate. Initially with the use of the enzyme E L I S A assay and later using Western blot analysis, it was demonstrated that the phenyl-Sepharose column removed the IgA1 proteases quantitativily from the culture supernatant. Neither enzymatic activity nor immunologically reactive enzyme could be detected in the flow thru of the phenyl-Sepharose column. The IgA1 protease activity eluted in one peak at the very end of the gradient. This was convenient because the eluted enzyme could be loaded directly onto

21 Fig. 1. SDS-PAGE analysis of a type 2 IgA1 protease eluted from FPLC Mono-S column. The molecular weight standards are: /3-galactosidase (135 kDa), human transferrin (80 kDa), catalase (60 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), and soy bean trypsin inhibitor (21 kDa). Note that besides the most prominent protein, migrating ahead of the /3-galactosidase, two other minor bands migrate at approximately 65 kDa and 45 kDa. the ionic exchange column for further purification without additional adjustments. The major contaminating protein eluting with the IgA1 proteases was the 37 kDa major iron regulated protein mentioned previously. The IgA1 protease adhered to the CM-Sepharose column and both enzyme activity and immunological reactivity eluted from the column between 6-8 m O - 1 . The contaminating major iron regulated protein eluted in later fractions according to previous reported data (Mietzner et al., 1987). The SDS-PAGE analysis of one of the purified IgA1 proteases is shown in Fig. 1. If further purification was necessary, the preparation was diluted 1 / 2 with distilled water and rerun under the same condition

219 o n a F P L C M o n o - S c o l u m n . T h e u s u a l yield of purified p r o t e a s e was a p p r o x i m a t e l y 0.4 m g / 1 .

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Production o f polyclonal and monoclonal antibodies

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T h e polyclonal r a b b i t sera raised to the type 2 gonococcal IgA1 p r o t e a s e was e v a l u a t e d by E L I S A a n d W e s t e r n blots. By W e s t e r n blot analysis, these sera cross-reacted with b o t h type 1 a n d type 2 Neisserial e n z y m e s (data n o t shown). T h e s e

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Fig. 2. A Western blot analysis of concentrated culture supernatant from strain R10 N. gonorrhoeae, using three monoclonal antibodies (lanes A, B, and C) and a rabbit polyclonal serum (lane D). 10 ml of culture supernate was concentrated by 10% TCA precipitation. The precipitate was washed once in ethanol, once acetone, and resuspended in 0.5 ml of SDS-PAGE sample preparation buffer. The total 0.5 ml mixture was applied to a fiat topped SDS-PAGE gel (12 cm x 12 cm x 0,2 cm), electrophoresed, and blotted. The Immobilon P was cut into strips, one strip incubated with each of the monoclonal antibodies and developed as described in the materials and methods section. Note that each of the three monoclonal antibodies 9H9/C6 (lane A), 9E3/B10 (lane B), and 6C7/F4 (lane C) react with not only the uppermost intact IgA1 protease, but also various other smaller molecular weight species. This would suggest the presence of proteolytie enzymes in the culture which tend to digest the holoenzyme into smaller molecular weight species. Of particular interest is the monoclonal antibody 9H9/C6 which inhibits the IgA1 protease activity. This antibody seems to react with both the intact enzyme and with the fragment which migrates at 65 kDa. This would suggest that the enzymes active site is located within this fragment.

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Fig. 3. A Western blot analysis of a concentrated culture supernatant from N. gonorrhoeae strain R10 and a E. coli carrying the plasmid pVD 105 containing the IgAl protease gene. 1 ml of culture supernate from each bacterial culture was concentrated by a 10% TCA precipitation. The precipitate was washed once in ethanol, once acetone, and resuspended in 25 /zl of SDS-PAGE sample preparation buffer. The total 25 /.tl mixture of each was loaded in a lane of a SDS-PAGE gel, electrophoresed, and blotted. The monoclonal antibody used in the analysis was 9E3/B10.

sera have recently b e e n tested m o r e extensively a n d have b e e n shown to react with the IgA1 p r o t e a s e s p r o d u c e d by all the strains tested (G.F. Brooks, C.J. L a m m e l a n d M.S. Blake, m a n u s c r i p t in p r e p a r a t i o n ) . T h e initial e v a l u a t i o n for m o n o clonal a n t i b o d i e s reactive to purified type 2 gonococcal IgA1 p r o t e a s e was m a d e using a n E L I S A assay. T h r e e m o n o c l o n a l antibodies, reactive in E L I S A with the purified IgA1 enzyme, were selected for f u r t h e r e x a m i n a t i o n . W e s t u d i e d their ability to react in a W e s t e r n blot assay with (1) the purified IgA1 protease, (2) s u p e r n a t a n t s from cultures of gonococci, (3) the s u p e r n a t a n t from a gonococcal m u t a n t lacking the IgA1 protease, a n d (4) s u p e r n a t a n t from a n E. coli into which the g e n e for the gonococcal type 2 p r o t e a s e g e n e h a d b e e n cloned. T h e last two samples were kindly p r o v i d e d by J. Michael Koomey. I n addi-

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Inhibition of IgA protease

Discussion

by Monoclonal Antibodies

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Fig. 4. 5/xg of each of the three monoclonal antibodies raised against the type 2 gonococcal IgA1 protease was added to the IgA1 protease ELISA assay (described in the materials and methods section) to examine if any of the antibodies inhibited the proteolytic activity of the enzyme. The monoclonal antibody 9H9/C6 inhibited the IgA1 protease significantly better than the other two antibodies in this assay.

tion, each of the three monoclonal antibodies were examined for their ability to inhibit an active enzyme in the ELISA enzyme assay. Fig. 2 shows the reactivity of these three monoclonal antibodies with concentrated culture supernatants from gonococci expressing the type 2 protease. As can be seen from the figure, the monoclonal antibody reacts with primarily one band at approximately 110 kDa but also shows reactivity with several other minor constituents, a 65 kDa peptide in particular. This is consistent with the degradation of the protease that occurs within the culture, causing these fragments of the IgA1 protease. In Fig. 3, a comparison is made between the type 2 IgA1 protease produced by gonococcal strain R10 and that produced by E. coli carrying the pVD 105 plasmid containing the IgA1 protease gene (Koomey and Falkow, 1984). Only one, monoclonal 9H9/C6, of the three monoclonal antibodies was able to substantially inhibit the IgA1 protease as seen in Fig. 4. This monoclonal antibody has been used in screening studies to differentiate between type 2 and type 1 IgA1 proteases produced by both N. gonorrheae and N. meningitidis (G.F. Brooks, C.J. Lammel and M.S. Blake, manuscript in preparation).

The expression of IgA1 proteases has been reported for several different pathogenic bacteria (see for review Plaut, 1983). Although much is known about their enzymatic activity and the sites within the IgA1 molecule where cleavage occurs, very little is known about the antigenicity and immunogenicity of these enzymes. It has been shown by cleavage site specificity and restriction endonuclease cleavage of the cloned genes that two types of enzymes are each independently expressed by different Neisserial organisms. From the reported biochemical and genetic studies, these two enzymes are similar to each other as regard to p I and primary sequence. Yet, little is known about the antigenic differences between these two enzymes. Because these proteases are produced in very small amounts, are autocatalytic, and are subject to digestion by other enzymes produced by the organisms, the ability to isolate suffieient amounts of these enzymes to study them more closely has been labor intensive and resulted in very low yields. We have described a simpler method for isolating the IgA1 proteases in amounts large enough to study their immunological properties. The polyclonal rabbit sera elicited with the type 2 gonococcal protease reacted with a high molecular weight band in all strains of N. gonorrhoeae and N. meningitidis tested, suggesting that all IgA1 proteases have some antigenic similarity. But, the monoclonal antibody 9H9/C6 that seemed to be directed at the enzyme active site of the type 2 gonococcal protease reacted strictly with the type 2 enzyme and not with the type 1. This would confirm that the two enzymes differ somewhat in this region and cleave at differing sites on the IgA1 molecule. Further analysis of the enzyme active region on the IgA1 proteases is necessary to evaluate the activity. The monoclonal antibody 9H9/C6 will help in locating this area of the enzyme.

Acknowledgements

This work was supported by PHS grants AI 19469 and AI 18367.

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Fc fragment by an enteric microbial proteolytic enzyme. J. Immunol. 111, 1274. Mietzner, T.A., Bolan, G., Schoolnik, G.K. and Morse, S.A. (1987) Purification and characterization of the major ironregulated protein expressed by pathogenic Neisseriae. J. Exp. Med. 165, 1041. Mulks, M.H. and Knapp, J.S. (1987) Immunoglobulin A1 protease types of Neisseria gonorrhoeae and their relationship to auxotype and serovar. Infect. Immun. 55, 931. Mulks, M.H. and Plaut, A.G. (1978) IgA protease production as a characteristic distinguishing pathogenic from harmless Neisseriaceae. New Engl. J. Med. 299, 973. Mulks, M.H., Plaut, A.G., Feldman, H.A. and Frangione, B. (1980) IgA proteases of two distinct specificities are released by Neisseria meningitidis. J. Exp. Med. 152, 1442. M[iller, H.E. (1971) Immunelektrophoretische Untersuchungen zur Einwirkung bakterieler Enzyme auf Menschliche Plasmaproteine. Zbl. Bakt., Hyg., I. Abt. Orig. A 217, 254. Plaut, A.G. (1978) Microbial IgA proteases. New Eng. J. Med. 298, 1459. Plaut, A.G. (1983) The IgA1 proteases of pathogenic bacteria. Ann. Rev. Microbiol. 37, 603. Plaut, A.G., Gilbert, J.V., Artenstein, M.S. and Capra, J.D. (1975) Neisseria gonorrhoeae and Neisseria meningitidis: Extracellular enzyme cleaves human immunoglobulin A. Science 190, 1103. Plaut, A.G., Gilbert, J.V. and Rule, A.H. (1978) Isolation and properties of the immunoglobulin A proteases of Neisseria gonorrhoeae and Streptococcus sanguis. In: G.F. Brooks, E.C. Gotschlich, K.K. Holmes, W.D. Sawyer and F.E. Young (Eds.), Immunobiology of Neisseria gonorrhoeae. American Society for Microbiology, Washington, DC, p. 279. Russell-Jones, G.J., Gotschlich, E.C. and Blake, M.S. (1984) A surface receptor specific for human IgA on group B streptococci possessing the Ibc protein antigen. J. Exp. Med. 160, 1467. Simpson, D.A., Hausinger, R.P. and Mulks, M.H. (1988) Purification, characterization, and comparison of the immunoglobulin A1 proteases of Neisseria gonorrhoeae. J. Bacteriol. 170, 1866. Swanson, J.L. (1978) Studies on gonococcus infection. XIV. Cell wall protein differences among color/opacity colony variants of N. gonorrhoeae. Infect. Immun. 21, 292. Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350,