Immunoglobulin Al Protease Types of Neisseria gonorrhoeae and

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1 NFECTON AND MMUNTY, Apr. 1987, p /87/ $02.OO/0 Copyright 1987, American Society for Microbiology Vol. 55, No. 4 mmunoglobulin Al Protease Types of Neisseria gonorrhoeae and Their Relationship to Auxotype and Serovar MARTHA H. MULKS1* AND JOAN S. KNAPP2 Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824,1 and Sexually Transmitted Diseases Laboratory Program, Center for nfectious Diseases, Centers for Disease Control, Atlanta, Georgia Received 19 September 1986/Accepted 4 January 1987 mmunoglobulin Al (gal) proteases are extracellular bacterial proteolytic enzymes which correlate with virulence in several species of human pathogens. We report that Neisseria gonorrhoeae produced two distinct types of gal protease, each of which cleaved a different peptide bond in the hinge region of human gal. The type of gal protease produced correlated with both nutritional auxotype and outer membrane protein serovar in this organism. Gonococcal type 1 gal protease was produced primarily by N. gonorrhoeae strains which require arginine, hypoxanthine, and uracil (AHU) and which belong to the protein A-1 or A-2 serovar. Most isolates of other auxotypes and serovars produced type 2 gal protease. Although both the AHU auxotype and protein A serogroup were found to be associated with disseminated gonococcal infection, there was no direct correlation of gal protease type with disseminated or with uncomplicated gonorrhea. The immunoglobulin Al (gal) proteases are extracellular proteolytic enzymes produced by several species of pathogenic bacteria, both gram positive and gram negative (6, 11, 12, 18). There are several lines of evidence which suggest that these enzymes play a role in bacterial virulence. (i) They are uniquely specific for ga, the class of immunoglobulin which predominates in human mucosal secretions, and indeed, cleavage of ga by these enzymes destroys its functional integrity (12, 17-19). (ii) The enzymes are produced primarily by medically important bacteria which infect human mucosal epithelial tissues, including Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae (11, 12, 18). (iii) Closely related species of bacteria which are common commensal inhabitants of the human nasopharynx do not produce these enzymes (6, 12, 15, 18). (iv) ga proteases are elaborated in vivo during the course of infection with these organisms, and antiprotease antibodies are elicited during these infections (12, 18). Polymorphism in gal protease type has been reported in both N. meningitidis (16) and H. influenzae (6, 12, 14, 18), with assignment of enzyme type based on specificity for different peptide bonds in the hinge region of human gal (see Fig. 2). n both species, there is a correlation between the type of enzyme produced and surface markers associated with virulence. n H. influenzae, protease type correlates closely with capsular serotype (2, 14) and there is evidence that genes encoding these characteristics are linked (M. H. Mulks, A. Zwahlen, E. R. Moxon, and A. G. Plaut, unpublished data). n N. meningitidis, there is some phenotypic linkage of gal protease type with both capsular serogroup and outer membrane protein serotype (12, 16). Previous studies in several laboratories have suggested that all isolates of N. gonorrhoeae produce the same type of gal protease (11, 15, 18). We report here that there are two distinct phenotypes of gal protease produced by N. gonorrhoeae and that these protease phenotypes correlate with * Corresponding author. 931 both nutritional auxotype and outer menbrane protein serovar in this organism. MATERALS AND METHODS Bacterial isolates. All isolates examined in this study were patient isolates in the strain collection of the Neisseria Reference Laboratory in Seattle, Wash. solates were grown on GC medium base (Difco Laboratories, Detroit, Mich.) plus 1% Kellogg supplement (7) or on chocolate agar (BBL Microbiology Systems, Cockeysville, Md.) and were stored at -70 C in either 50% (vol/vol) gamma globulin-free horse serum in tryptic soy broth (Difco) or 20% glycerol in 2% tryptone (Difco). gal protease assays. gal protease type was determined by analysis of gal cleavage products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 14, 16). Colonies of each isolate were incubated with 30,ul of 125-labeled purified human gal (50,ug/ml in 0.05 M Tris hydrochloride [ph 7.5]-0.01 M MgCl M CaCl2) for 16 h at 37 C. These samples were subjected to SDS-PAGE on a 9% polyacrylamide gel with a discontinuous buffer system, and the separated [1251]gAl cleavage products were visualized by autoradiography on Kodak XAR-5 film. Protease type was assigned based on the mobility of the cleavage products compared with controls of known specificities. Auxotyping. Each isolate was auxotyped on defined medium (7) and scored for requirements for arginine, hypoxanthine, methionine, proline, and uracil. All strains required half cystine or cysteine for growth. Serologic classification. solates were classified into serovars by a coagglutination test by using six anti-protein A and six anti-protein B monoclonal antibodies as previously described (9). Amino acid sequence studies. Human gal protein (10 mg), purified from the serum of a patient with multiple myeloma, was digested with gonococcal type 1 gal protease for 48 h at 37 C. Fca fragments were purified from these digestion mixtures by chromatography over an FPLC Superose-6 molecular-sieve column (Pharmacia, nc., Piscataway,

2 932 MULKS AND KNAPP NFECT. MMUN. Fd FG. 1. Autoradiograph of gal protease digests of 1251-labeled human gal examined by SDS-PAGE under reducing conditions. The lanes show human gal incubated with buffer (1), N. gonorrhoeae type 2 protease (2), N. gonorrhoeae type 1 protease (3), N. meningitidis type 1 protease (4), and N. meningitidis type 2 protease (5). H, ga heavy chain; Fc, Fca fragment; Fd, heavy chain component of Faba fragment. Gonococcal and meningococcal type 1 enzymes produce identical cleavage fragments, as do the neisserial type 2 proteases. N.J.). Purified Fcot material, identified by Ouchterlony analysis, was dialyzed against distilled deionized water and lyophilized. Amino acid sequence determination was by automated Edman degradation using a liquid-phase sequencer (890M; Beckman nstruments, nc., Fullerton, Calif.). Phenylthiohydantoin amino acids were identified by two independent high-performance liquid chromatography reverse-phase columns. Sequencing was performed by the macromolecular Structure Facility, Department of Biochemistry, Michigan State University, East Lansing. RESULTS Two types of gal protease were produced by N. gonorrhoeae, each isolate elaborating one or the other enzyme but not both. SDS-PAGE of human gal digested with gonococcal protease preparations revealed two distinct patterns of cleavage, which were indistinguishable from those produced by the two meningococcal gal proteases (Fig. 1). The exact peptide bonds in the human gal hinge region cleaved by both meningococcal enzymes and by one of the gonococcal enzymes have been previously reported (16). Limited aminoterminal sequence analysis of purified Fca fragments produced by the second type of gonococcal enzyme yielded the sequence -Ser-Pro-Ser-(Cys)-(Cys)-His-Pro-Arg-Leu-Ser-. When this sequence is aligned with that of the human gal hinge region (Fig. 2), it can be seen that this enzyme cleaved the proline-serine bond at position 237 to 238. There was no evidence in either this sequence analysis or in gels from SDS-PAGE of six different human gal myeloma proteins cleaved with both gonococcal gal proteases (data not shown) that these enzymes cleave more than one peptide bond in the gal molecule. Each enzyme is apparently completely specific for its own particular peptide bond as a substrate. The peptide bonds in the hinge region of human gal which are cleaved by each of the four neisserial enzymes are shown in Fig. 2. The gonococcal enzymes which cleave the same peptide bonds as the previously described (16) meningococcal type 1 and type 2 enzymes we designated as types 1 and 2. Type 1 neisserial gal proteases cleaved the proline-serine peptide bond at position 237 to 238 in the hinge region of the human gal heavy chain, yielding an Fda fragment of -28,500 daltons and an Fca fragment of -30,500 daltons. Type 2 neisserial enzymes cleaved the prolinethreonine bond at position 235 to 236 and yielded Fda and Fca fragments of -28,000 and -31,000 daltons, respectively. Similar cleavage patterns on SDS-PAGE were seen when human gal(k) or gal(a) myeloma protein or human secretory ga was used as a substrate, although the exact sizes of the gal fragments produced may vary with the specific substrate used and a portion of the secretory ga, presumably sga2, remained uncleaved. Human ga2 myeloma proteins were not cleaved by either type 1 or type 2 gonococcal gal proteases. The first gonococcal isolate which we examined that produced type 1 gal protease was an arginine-, hypoxanthine-, and uracil-requiring (AHU), serogroup protein A (W) strain isolated from a patient with disseminated disease. Because of the correlation of the AHU auxotype (1, 7) or the protein serogroup (1, 3) or both with disseminated disease and because of evidence of linkage in both N. meningitidis and H. influenzae of protease type and other virulence factors, we examined the distribution of gal protease types among defined strains of N. gonorrhoeae. We examined 889 independent isolates of N. gonorrhoeae Gal Gal Gal Gal GalNAc GalNAc GalNAc GalNAc GalNAc l Fab-Cys-Pro-Val-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Cys-Fc l C.ramosum S.pneumoniae H.influenzae 1 N.gonorrhoeae 2 N.gonorrhoeae 1 S.sanguis N.meningitidis 2 N.meningitidis 1 B.melaninogenicus H.influenzae 2 FG. 2. The hinge region of human gal, showing the locations of the peptide bonds cleaved by the gal proteases produced by N. gonorrhoeae, N. meningitidis, H. influenzae, S. pneumoniae, Streptococcus sanguis, Bacteroides melaninogenicus, and Clostridium ramosum.

3 VOL. 55, 1987 GONOCOCCAL gal PROTEASES 933 -SE RO0T YP ES A UXO0TY PE S (1A-) TOTAL AHiU PAHU Protol Pro Aret Met PA AM AU PAM PAU PAUM PM PH PAMi [ X [ i [8 TOTALS SE RO0T YP ES A U XOTY PE S (i-) TOTAL AHOU PAHU Protol Pro Arg Met PA AM AU PAM PAU PAUM PM PH PAH ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ H X TOTALS FG. 3. Auxotypes and serotypes of gonococcal isolates producing type 1 gal protease. Shaded squares at left show positive reactions with (upper panel) the six protein LA-specific monoclonal antibody reagents used to divide serogroup LA into serovars; numbers 1 to 6 represent monoclonal antibodies 4A12, 4G5, 2F12, 6D9, 5G9, and 5D1, respectively, and (lower panel) the six protein LB-specific monoclonal antibody reagents used to divide serogroup LB into serovars; numbers 1 to 6 represent monoclonal antibodies 3C8, 1F5, 2Li6, 2G2, 2D4, and 2H1, respectively (9). Abbreviations: AHU, requires arginine, hypoxanthine, and uracil; PAHU, AHU strain that also requires proline; Proto, prototrophic; Pro, requires proline; Arg, requires arginine; Met, requires methionine; PA, requires proline and arginine; AH, requires arginine and hypoxanthine; AU, requires arginine and uracil; PAH, requires proline, arginine, and hypoxanthine; PAU, requires proline, arginine, and uracil; PAUM, requires proline, arginine, uracil, and methionine; PM, requires proline and methionine; PH, requires proline and hypoxanthine; PAH, requires proline, arginine, and hypoxanthine. in this study. Each isolate was auxqtyped on defined medium and 7 (0.8%) produced no detectable enzyme. Correlation of (7) and classified serologically by coagglutination by using gal protease type with auxotype showed that all 192 AHU monoclonal antibodies (9). gal protease type was deter- and PAHU (requiring proline in addition to arginine, hypomined by analysis of gal cleavage products by SDS-PAGE xanthine, and uracil) isolates, regardless of serovar, geo- (14, 16) (Fig. 3 and 4). graphic distribution, or disease syndrome, produced type 1 Of 889 gonocnc-cal kisoltes, examined, 199 (22.4%) pro- protease. These included isolates from patients from several duced type 1 enzyme, 683 (76.8%) produced type 2 enzyme, cities in the United States and from Western Europe, Aus-

4 934 MULKS AND KNAPP NFECT. MMUN. _SE RO0T YP ES A U XOTY PE S (1A ) TOTAL AHU PAHU Protol Pro Arg.1 Met PA AM AU PAM PAU PAUM PM PH PAH El 13 1 i X. ~ lii ii i lislisl l li 17 TOTALS SE RO0T Y PES A U XOTY PE S (ib-) TOTAL AHU PAHU Protol Pro Arst Met.1 PA AM AU PAM PAU PAUM PM PH PAH liii ii iii lo E-A X: i ll 1 31 VA i i il h t:j i i.. i i i l li ii i l l TOTALS FG. 4. Auxotypes and serotypes of gonococcal isolates producing type 2 gal protease. For descriptions of monoclonal antibody reactions and abbreviations, see legend to Fig. 3. tralia, and Japan who had uncomplicated gonorrhea, pelvic protein TB (WT/l) serogroup produced type 2 gal proteinflammatory disease, and disseminated gonococcal infec- ase. tion. n contrast, of 13 other auxotypes examined, only 7 of To determine whether protease type 1 can be directly 690 (1.0%) produced type 1 enzyme. correlated with disseminated gonococcal infection, we ex- Correlation of gal protease type with serovar showed amined 24 strains of N. gonorrhoeae which had been isothat with few exceptions, type 1 gal protease-producing lated from separate, confirmed cases of disseminated gonostrains belonged to the two AHU-related serovars of the coccal infection. Of these isolates, 9 of 24 (37.5%) produced protein A serogroup (W), i.e., serovars A-1 and A-2. type 1 protease and 15 of 24 (62.5%) produced type 2 However, only 198 of 235 (84.3%) strains belonging to these enzyme; 8 of 24 (33.3%) were AHU strains and 16 of 24 two ser'ovars produced type 1 enzyme. Most isolates belong- (66.7%) belonged to other auxotypes. However, 18 of 24 ing to the non-ahu-related serovars of the protein A (25%) belonged to the A-1 or A-2 protein serovar, serogroup (serovars A-3 to A-18) and to all serovars of the whereas 6 of 24 (25%) belonged to various protein TB

5 VOL. 55, 1987 serovars. These data suggest that protein A, particularly serovars A-1 and A-2, is strongly correlated with disseminated gonococcal infection but that the AHU auxotype and gal protease type 1 are not directly correlated with disseminated gonococcal infection. DSCUSSON We studied three independent phenotypic variables, i.e., auxotype, serovar, and protease type, and found that there was an association of these phenotypes in AHU strains. All AHU strains examined produced gal protease type 1, and 188 of 192 (97.9%) belonged to the protein A-1 and A-2 serovars. n addition, all AHU strains and, with a single exception, all strains which produced type 1 protease were Dam' (i.e., methylated the adenine residue of GATC sequences; data not shown). However, in no case did we find a 100% two-way correlation between any pair of these traits, confirming that these are indeed independent variables. n our study on the evolution of gonococcal phenotypes in Copenhagen, Denmark (8), we also found linkage of these phenotypic traits in all AHU strains isolated between 1946 and 1979; we also found that all AHU strains examined contained the 2.6-megadalton gonococcal cryptic plasmid and did not contain the 24.5-megadalton gonococcal conjugal plasmid. We found that these strains show a distinct lack of diversity compared with strains of other auxotypes and serovars. These data suggest that AHU strains may belong to an extremely conserved clone. The means by which this linkage of independent phenotypic variables is conserved is as yet unclear, but it could be due to an extremely close physical linkage of the genes controlling these phenotypes, to an as yet undescribed biochemical linkage, to some characteristic which limits genetic variation in these strains, or to a positive selection pressure in vivo for strains with this particular constellation of phenotypic traits. n addition to the variability in gonococcal gal protease phenotypes, we observed considerable variability at the DNA level, as evidenced by restriction site maps of genes specifying gonococcal gal protease activity (4, 5, 10, 13, 20). We found that the iga genes from strains which produce type 1 gal protease have identical restriction maps. n contrast, the iga genes from strains producing type 2 enzymes show considerable restriction site polymorphism (13). Among 13 strains producing type 2 proteases, we found seven variations in the iga genes (iga-2a to iga-2g), all of which differed from the single restriction site pattern (iga-1) found in 12 strains producing type 1 protease (M. H. Mulks, D. A. Simpson, and R. J. Shoberg, Proceedings of the Fifth nternational Conference on Pathogenic Neisseriae, in press). Biochemical comparison of the two types of gonococcal gal proteases revealed several differences in these enzymes, including differences in molecular weight, stability, and susceptibility to enzyme inhibitors (D. A. Simpson, R. P. Hausinger, and M. H. Mulks, in preparation). We have also observed antigenic differences between the two types of enzymes (M. H. Mulks, unpublished data). Whether there are subtle differences among type 2 enzyme proteins from different strains, reflecting the genetic polymorphism in iga-2 genes, has not been determined. n summary, we found that (i) there were two types of gonococcal gal protease which could be distinguished by substrate specificity; (ii) the type of protease produced correlated with both auxotype and serovar, especially in AHU isolates; (iii) there was no direct correlation evident GONOCOCCAL gal PROTEASES 935 between gal protease type and disseminated gonococcal infection; and (iv) there were also variations in the gene encoding gal protease activity which may account for the two phenotypes described, but which were not always responsible for obvious phenotypic differences. t is not known whether the type of gal protease produced makes any contribution to gonococcal virulence, but the linkage of gal protease type with known virulence markers and the potential role of these enzymes in the pathophysiology of N. gonorrhoeae are under further investigation. ACKNOWLEDGMENTS This work was supported by Public Health Service grant A (to M.H.M.) and Program Project Grant A from the National nstitute of Allergy and nfectious Diseases and by a Michigan State University All-University Research nitiation grant (to M.H.M.). LTERATURE CTED 1. Bohnhoff, M., J. A. Morello, and S. A. Lerner Auxotypes, penicillin susceptibility, and serogroups of Neisseria gonorrhoeae from disseminated and uncomplicated infections. J. nfect. Dis. 154: Bricker, J., M. H. Mulks, A. G. Plaut, E. R. Moxon, and A. Wright gal proteases of Haemophilus influenzae: cloning and characterization in E. coli K-12. Proc. Natl. Acad. Sci. USA 80: Cannon, J. G., T. M. Buchanan, and P. F. Sparling Confirmation of association of protein 1 serotype of Neisseria gonorrhoeae with ability to cause disseminated infection. nfect. mmun. 40: Fishman, Y., J. Bricker, J. V. Gilbert, A. G. Plaut, and A. Wright Cloning of the type 1 immunoglobulin protease from Neisseria gonorrhoeae and secretion of the enzyme from Escherichia coli, p n G. Schoolnik (ed.), The pathogenic neisseriae. American Society for Microbiology, Washington, D.C. 5. Halter, R., J. Pohlner, and T. Meyer ga protease of Neisseria gonorrhoeae: isolation and characterization of the gene and its extracellular product. EMBO J. 3: Kilian, M., B. Thomsen, T. E. Petersen, and H. S. Bleeg Occurrence and nature of bacterial ga proteases. Ann. N.Y. Acad. Sci. 409: Knapp, J. S., and K. K. Holmes Disseminated gonococcal infection caused by Neisseria gonorrhoeae with unique nutritional requirements. J. nfect. Dis. 132: Knapp, J. S., M. H. Mulks,. Lind, H. B. Short, and V. L. Clark Evolution of gonococcal populations in Copenhagen, Denmark, , p n G. Schoolnik (ed.), The pathogenic neisseriae. American Society for Microbiology, Washington, D.C. 9. Knapp, J. S., M. R. Tam, R. C. Nowinski, K. K. Holmes, and E. G. Sandstrom Serological classification of Neisseria gonorrhoeae using monoclonal antibodies directed against gonococcal outer membrane protein. J. nfect. Dis. 150: Koomey, J. M., R. E. Gill, and S. Falkow Genetic and biochemical analysis of gonococcal gal protease: cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity. Proc. Natl. Acad. Sci. USA 79: Kornfeld, S. J., and A. G. Plaut Secretory immunity and the bacterial ga proteases. Rev. nfect. Dis. 3: Mulks, M. H Microbial ga proteases, p n. A. Holder (ed.), Microbial enzymes and virulence. CRC Press nc., Boca Raton, Fla. 13. Mulks, M. H., and J. S. Knapp mmunoglobulin Al protease types of Neisseria gonorrhoeae, p n G. Schoolnik (ed.), The pathogenic neisseriae. American Society for Microbiology, Washington, D.C. 14. Mulks, M. H., S. J. Kornfeld, B. Frangione, and A. G. Plaut Relationship between the specificity of ga proteases and

6 936 MULKS AND KNAPP serotypes in Haemophilus influenzae. J. nfect. Dis. 146: Mulks, M. H., and A. G. Plaut ga protease production as a characteristic distinguishing pathogenic from harmless Neisseriaceae. N. Engl. J. Med. 299: Mulks, M. H., A. G. Plaut, H. A. Feldman, and B. Frangione ga proteases of two distinct specificities are released by Neisseria meningitidis. J. Exp. Dis. 152: Mulks, M. H., A. G. Plaut, and M. Lamm Gonococcal ga protease reduces inhibition of bacterial attachment by human secretory ga, p n S. Normark and D. Danielsson (ed.), Genetics and immunobiology of pathogenic NFECT. MMUN. neisseria. University of Umea, Umea, Sweden. 18. Plaut, A. G The gal proteases of pathogenic bacteria. Annu. Rev. Microbiol. 37: Plaut, A. G., J. V. Gilbert, and R. Wistar, Jr Loss of antibody activity in human immunoglobulin A exposed to extracellular immunoglobulin A proteases of Neisseria gonorrhoeae and Streptococcus sanguis. nfect. mmun. 17: Rahr, S., R. Halter, H. Muller, J. Pohlner, and T. F. Meyer Genetic analysis of neisserial immunoglobulin A proteases, p n G. Schoolnik (ed.), The pathogenic neisseriae. American Society for Microbiology, Washington, D.C.