Evaluation of Human Oral Organisms and Pathogenic Streptococcus for Production of IgA Protease

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1 TE JOURNAL OF INFETIOUS DISEASES VOL. 131, SUPPLEMENT MAY by the University of hicago. All rights reserved. Evaluation of uman Oral Organisms and Pathogenic Streptococcus for Production of IgA Protease Robert J. Genco, Andrew G. Plaut, and Robert. Moellering, Jr. From the Department of Oral Biology, School of Dentistry, State University of New York at Buffalo, Buffalo, New York; and the Department of Medicine, Tufts-New England Medical enter ospital, and the Department of Medicine, Massachusetts General ospital-arvard Medical School, Boston, Massachusetts IgA protease is a proteolytic enzyme found in whole human saliva and in dental plaque that cleaves both secretory and myeloma IgA of human origin to yield intact Fabo and Fco fragments. To determine which bacteria are capable of producing this enzyme, we have examined a variety of strains normally found in the human oral cavity and a number of streptococci of known Lancefield group serotype. Streptococci of groups A, B,, D, F, G,, M, and N, Streptococcus mutans, Streptococcus sanguis, Streptococcus mitior, Streptococcus salivarius, Streptococcus faecalis, Veillonella, Lactobacillus, Actinomyces, Propionibacterium, Bacteroides, and Fusobacterium were grown in liquid medium, and fluids were examined for IgA protease activity. Only S. sanguis and clinically isolated group streptococci elaborated IgA protease under the culture conditions used. Negative strains could not be stimulated to produce the enzyme when cultured in the presence of secretory IgA. Among the natural oral bacteria, capacity to produce IgA protease is restricted to certain species of Streptococcus, notably those of the group serotype. Since secretory immunity is mediated by the IgA class of antibody, the presence of this enzyme at mucosal surfaces could modify the secretory immune function. Immunoglobulins are enzymatically cleaved at the susceptible hinge region of the heavy polypeptide chain to yield Fab and Fe fragments. uman immunoglobulin A (IgA) is cleaved by several proteolytic enzymes yielding a typical Fabo fragment, but the Fe region is further degraded to peptides of low molecular weight. Recently, an enzyme designated IgA protease has been identified in culture filtrates of certain alimentary tract bacteria, notably This study was supported by grants no. AM AI 16607,DE 02814, and DE 00167from the National Institutes of ealth, by contract no from the U.S. Army Medical Research and Development ommand, and by research career development award no. AI to Dr. Plaut. We thank Drs. harles Ellis, R. Todd Evans, Paul Mashimo, Sigmund Socransky, Burton Rosan, Robert Fitzgerald, Ronald Gibbons, and Benjamin ammond for providing bacterial strains and Dr. Barbara Watson for grouping them. We also thank Ms. Joanne Gilbert, Ms. arole Sadowski, and Mr. Richard all for their help. Please address requests for reprints to Dr. Robert Genco, Department of Oral Biology, School of Dentistry, State University of New York at Buffalo, 4510 Main Street, Buffalo, "New York Streptococcus sanguis [1, 2], and this enzyme yields an intact Fca fragment when it hydrolyzes human serum and secretory IgA. IgA protease is an EDTA-sensitive neutral endopeptidase with unique specificity for human secretory IgA and serum IgA of subclass IgAI [3]; the enzyme does not cleave proteins of subclass IgA2 or other human immunoglobulins. Since this unusual specificity may be of significance in the overall function of secretory IgA antibody at mucosal surfaces and since S. sanguis is a numerically important human oral microorganism, we have undertaken this study to examine a wide range of bacteria for production of IgA protease. Materials and Methods Detection of IgA protease activity. Enzymatic activity was identified by incubation of purified human IgA myeloma proteins at a concentration of mg/ml in Tris-I buffer (0.05 M, p 8.1) with an equal volume of enzyme at 37 prepared as outlined below. IgA S17

2 S18 Genco, Plaut, and Moellering of subclass IgAI was purified from the serum of patients with multiple myeloma, and secretory IgA was purified from human colostrum [4]. Proteolysis was detected by immunoelectrophoresis of digests developed with unabsorbed rabbit antiserum known to contain specificity for light and heavy chains. Electrophoretic separation and identification of Fabo and Fea fragments was facilitated by selection as substrate of an IgA myeloma protein of slow (cathodal) electrophoretic mobility. ighly active enzyme preparations produce essentially complete 19A cleavage in 30 min. The criteria for the identification of Fea include its reaction with a(heavy)-chain-specific antisera, its failure to precipitate with light chain antisera, its faster electrophoretic mobility with respect to the uncleaved IgA, and its reaction of immunologic nonidentity with Faba [1]. Bacterial strains. Some strains of S. sanguis, Streptococcus salivarius, Streptococcus faecalis, Lactobacillus, Actinomyces, and Propionibacterium were obtained from the American Type ulture ollection (A T; Rockville, Md.). The AT strain numbers are given in table 1. Additional strains of S. sanguis were obtained from Dr. B. Rosan (University of Pennsylvania School of Dental ealth, Philadelphia, Pa.); S. mutans from Dr. R. Fitzgerald (Veterans' Administration ospital, Miami, Fla.); Streptococcus mitior and S. sanguis from Dr. R. Gibbons and Actinomyces from Dr. S. Socransky (both of Forsyth Dental enter, Boston, Mass.); and Veillonella from Dr. R. T. Evans and Fusobacterium from Dr. P. Mashimo (both of the State University of New York at Buffalo, School of Dentistry, Buffalo, N.Y.). Purity of strains was monitored by examination of colonies cultured on an appropriate solid medium. The streptococci identified as groups A, B,, D, F, G,, M, and N were human clinical isolates from a variety of sources. The organisms were grouped by Dr. B. Watson (Massachusetts General ospital, Boston, Mass.) and were frozen in horse blood for storage. Before being tested for IgA protease, these streptococci were grown on brucella agar with 5% horse blood (ST o., North Andover, Mass.). Representative colonies were picked and subcultured in Todd-ewitt broth. For evaluation of enzyme production, all or- Table 1. Production of IgA protease by representative human oral organisms. Proteolytic enzyme Organism production Gram-positive cocci Streptococcus sanguis Strains no. AT 10556, AT 9811, M-ll, 72 x 35, 72 x 41, 6512,410,62 x 23,62 x 49, 79PR3 Strains no. 66 x 49, 6211 Strain no. AT Strains no. AT 10558, 71 x 26, B-4, hallis, Wickey, M-5, 71 x 50, 903 Streptococcus mutans Strains no. E-49a, FA-l b, AT c, omz-176 d, LM-7 e Streptococcus salivarius, Strain no. AT Streptococcus mitior Strains no. S-3, 26 Streptococcus faecalis Strains no. AT 4200, 828, 4083, 6055, Gram-negative cocci Veillonella, strain V-1 Veillonella alcalescens (5DROG) Gram-positive rods Lactobacillus casei Strains no. AT 11578,7469, 11582,4646 Lactobacillus fermenti, strain no. AT Lactobacillus salivarius, strain no. AT Actinomyces viscosus Strains no. T-14, T-6 Actinomyces israelii Strains no. AT 12836, 12597, Actinomyces naeslundii, strain no. I Actinomyces bovis, strain no. AT Propionibacterium acnes Strains no , 6919 Gram-negative rods Bacteroides melaninogenicus Strains no. BE, K 110 Fusobacterium fusiformis Strains no. 1, 8 Fusobacterium nucleatum Strains no. 3, 6, 7 Fusobacterium polymorphum, Strain no. 5 Weak* Weak* NOTE. The superscripts a-d refer to the Bratthall serotypes [5J, and E refers to the Lancefield group. AT = American Type ulture ollection (Rockville, Md.). * ulture filtrates contained a weakly active enzyme yielding minimal Faba but no Fco fragment from serum and neither from secretory IgA. This enzyme has not been further characterized.

3 IgA Protease of Streptococcus 819 ganisms were grown in an appropriate liquid medium [2] under an oxygen tension that yielded abundant growth in hr at 37. ulture supernatants were freed of bacteria by centrifugation and brought to 60% saturation with (N41 S04 at 24 to precipitate IgA protease. The precipitates were dissolved again in 10% of the original volume with Tris-I buffer (0.05 M, p 8.1). After dialysis against the same buffer, the solutions were tested for IgA protease activity as described above. Results As shown in table 1, IgA protease activity was found in the culture fluids of approximately half of the strains of S. sanguis tested. A representative immunoelectrophoresis demonstrating the cleavage of IgA is shown in figure 1. Other organisms commonly found in human dental plaque did not elaborate the enzyme; these included S. mutans, S. mitior, S. salivarius, S. faecalis, Veillonella, Lactobacillus, Actinomyces, Propionibacterium acnes, Bacteroides, and Fusobacterium. ulture fluids demonstrating no enzyme activity were reex- Figure 1. Immunoelectrophoretic analysis of the cleavage of human colostral secretory IgA (siga) by IgA protease (p'ase). A, the top well contains siga. (The duplication of the precipitin band is an experimental artifact.) The bottom well shows the same siga treated by IgA protease from Streptococcus sanguis (AT no ) and shows typical Fabo and Fca fragments. The antiserum in the trough is unabsorbed rabbit antiserum to human IgA. B, the top well shows siga treated with IgA protease from S. sanguis (AT no. 9811), and the bottom well shows the same siga treated with enzyme from group Streptococcus (Or..) isolated from a patient with bacterial endocarditis. There is no apparent difference in the fragments produced by the two enzyme preparations. The antiserum is the same as in A. amined for activity after incubation for several days with IgA substrate and remained negative. The results obtained with clinically isolated streptococci of known Lancefield groups are summarized in table 2. Among 16 group strains tested, 12 showed definite production of IgA protease. The other groups tested (A, B,, 'D, F, G, M, and N) were negative. ulture filtrates of both group M organisms caused a cathodal displacement (shift) of the IgA myeloma paraprotein substrate but yielded no fragments. Since this effect was also seen with human serum transferrin treated by these preparations, we have concluded that this effect is not specific for immunoglobulins and may be caused by another enzyme, perhaps a sialidase, elaborated by group M organisms. To exclude the possibility that organisms not producing IgA protease were elaborating an inhibitor to the enzyme, we undertook the following experiment. Representative culture filtrates of negative strains were mixed with filtrates showing IgA protease activity, and this mixture was tested again for enzyme activity. In no case were inhibitory culture fluids identified.

4 820 Genco, Plaut, and Moellering Table 2. IgA protease production by streptococci isolated from various clinical sources. ulture no Source Gallbladder Femur erebrospinal fluid External ear canal Bile erebrospinal fluid Throat Lancefield group A A B B D (Streptococcus bovis) D (S. bovis) D (Enterococcus) D (Enterococcus) F F G G M M N N * ulture filtrates of group M organisms contained a carbohydrase-type enzyme as described in the text. Despite the fact that strains positive for IgA protease continued to produce the enzyme after repeated subculture in media free of IgA substrate, we attempted to stimulate enzyme production in negative strains by growing them in liquid media supplemented with 10% (vol/vol) sterile human parotid fluid containing secretory IgA. Among three negative strains stimulated in this way, no IgA protease activity could be detected. As shown in figure 1A and B, the fragments derived from colostral IgA cleaved by IgA protease of S. sanguis (AT no ) and that of a representative group Streptococcus were indistinguishable by immunoelectrophoresis. Proteolytic enzyme production arbohydrase-type* arbohydrase-type* Discussion The IgA protease activity of whole human saliva appears to be derived mainly from bacterial sources rather than from the major salivary glands since parotid fluid collected from the orifice of the parotid duct has been found to be inactive, whereas extracts or cultures of dental plaque have been found to contain IgA protease [2]. Dental plaque is composedmainly of bacteria, and upon screening of the major genera and species of bacteria found in supragingival plaque, IgA protease was detected only in culture fluids of S. sanguis. Therefore, we are led to conclude that most of the IgA protease in mixed human saliva is produced by S. sanguis.

5 IgA Protease of Streptococcus S21 S. sanguis is known to be an early colonizer of the tooth surface, but it is not known whether IgA protease influences subsequent colonization of plaque by other species. Other organisms found in plaque, including those that are transient and present in low numbers, or those that are found in unique locations such as in the depths of periodontal pockets were not tested for IgA protease activity. The finding that IgA protease production is a feature common to S. sanguis and group Streptococcus provides further evidence for the close relationship between these organisms. White and Niven [6] characterized strains of S. sanguis obtained from patients with subacute bacterial endocarditis as a-hemolytic organisms that formed dextran from sucrose, hydrolyzed arginine, fermented lactose, trehalose, and salicin, but did not ferment glycerol, mannitol, or sorbitol. Streptococci with these properties are commonly found in the mouth and on the teeth of man [7]. Porterfield [8] and Dodd [9] have shown that some strains of S. sanguis carry the group antigen. More recently, oleman and Williams [10] reviewed the data on the presence of the antigen on S. sanguis and pointed out discrepancies with use of group antisera directed to the Perriyer strain (AT strain no ) as compared to antisera directed to the hallis strain (NT strain no. 7868; National Type ulture ollection, Washington, D..). They found, however, that 14 of 35 strains of S. sanguis gave positive results when tested with group antisera. Our findings of IgA protease production by group streptococci and by most strains of S. sanguis examined lends further support to the concept of a close relationship between these organisms. The finding of an IgA protease of unique specificity for IgA but not for IgG, IgM, or 19B in human saliva and feces points to a role for this enzyme in modifying the biologic activity of secretory IgA antibodies bathing the gastrointestinal tract and associated structures. It has recently been shown [11, 12] that salivary IgA antibodies directed to the cariogenic bacterium S. mutans can markedly inhibit colonization of the teeth by this organism. These experiments suggest that salivary antibodies may exert a major influence on the colonization of mucous membranes and associated structures by bacteria. It is clear that colonization of mucosal or dental surfaces by bacteria is an important step for the full expression of virulence by bacterial pathogens responsible for diseases such as streptococcal pharyngitis, bacterial diarrheas, dental caries, and periodontitis. Thus, local conditions that modify the effects of secretory IgA antibodies on colonization could influence the initiation or progression of these mucosal and dental diseases. Although no direct evidence is available, it is conceivable that IgA protease produced by S. sanguis, a ubiquitous oral organism, can cleave secretory IgA antibodies and reduce the ability of these antibodies to prevent colonization of the alimentary canal by transient organisms, some of which may be pathogenic. References 1. Mehta, S. K., Plaut, A. G., alvanico, N. J., Tomasi, T. B., Jr. uman immunoglobulin A. Production of an Fe fragment by an enteric microbial proteolytic enzyme. J. ImmunoI. 111: , Plaut, A. G., Genco, R. J., Tomasi, T. B., Jr. Isolation of an enzyme from Streptococcus sanguis which specifically cleaves IgA. J. ImmunoI. 113: , 3. Plaut, A. G., Wistar, R., apra, J. D. Differential susceptibility of human IgA immunoglobulins to streptococcal IgA protease. J. lin. Invest. 54: , 4. Tomasi, T. B., Grey,. M. Structure and function of immunoglobulins A. Progr. Allergy 16:81-213, Bratthall, D. Demonstration of five serological groups of streptococcal strains resembling Streptococcus mutans. OdontoI. Rev. 21: , White, J.., Niven,. F., Jr. Streptococcus S.B.E.: a Streptococcus associated with subacute bacterial endocarditis. J. BacterioI. 51: , arlsson, J. A numerical taxonomic study of human oral streptococci. OdontoI. Rev. 19: , Porterfield, R. S. lassification of streptococci of subacute bacterial endocarditis. J. Gen. MicrobioI. 4:92-101, Dodd, R. L. Serologic relationship between Streptococ-. cus group and Streptococcus sanguis. Proc. Soc. Exp. BioI. Med. 70: , olman, G., Williams, R. E. O. Taxonomy of some human viridans streptococci. In L. W. Wannamaker and J. M. Matsen [ed.]. Streptococci and streptococcal diseases. Academic Press, New York and London, 1972, p Taubman, M. A., Smith, D. J. Effects of local immunization with Streptococcus mutans on induction of salivary immunoglobulin A antibody and experimental dental caries in rats. Infec. Immun. 9: , 12. Emmings, F. G., Evans, R. T., Genco, R. J. Induction of salivary IgA antibodies to S. mutans in Macaca irus [abstract]. J. Dent. Res. 53:184,