SHORT COMMUNICATIONS Research in Veterinary Science 1992, 53, 244-246
Testing memngeal strains of Streptococcus suis to detect M protein genes J. D. MOGOLLON, C. PIJOAN, Department of Clinical and Populations Sciences, M. P. MURTAUGH, Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St Paul, Minnesota 55108, P. P. CLEARY, Department of Microbiology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455, J. E. COLLINS, Department of Veterinary Diagnostic Investigation, College of Veterinary Medicine, University of Minnesota, St Paul Minnesota 55108, USA
Previous reports have suggested that the surface proteins found in meningeal strains of Streptococcus suis might be similar to the M protein of group A streptococci. Fifty-five strains of S suis, including human and swine meningeal and pneumonic isolates, were tested for M protein genes by DNA probes representing the constant domain of the 3' end of the group A, M protein gene. None of the S suis strains examined was positive, indicating that these organisms either lack M protein genes or harbour different genes, not expressing the constant domains of protein M from group A.
MENINGITIS due to Streptococcus suis type 2 in weaned pigs has emerged as an important problem in the North American pig industry during recent years (Hoffman and Henderson 1985, Sanford 1989). S suis type 2 has also been isolated from the tonsils of a large proportion of healthy adults in affected herds, suggesting that many strains are avirulent (CliftonHadley et al 1985). Unfortunately, available diagnostic techniques do not differentiate isolates in terms of virulence factors, which for S suis have not been defined. The emergence of S suis as a major pathogen could be due to the evolution by some strains of antiphagocytic surface determinants similar to the M protein (Arend s and Zanen 1984, Vecht et al 1989). Virulence in S pyogenes (group A) has been attributed to the antipliagocytic properties of the M protein. This protein is arranged as a coiled-coil dimer with the carboxy terminus attached to the peptidoglycan of the cell wall (Fischetti et al 1988). The amino-terminal region is distal to the cell surface and is exposed to selective immunological pressure (Kehoe et al 1987). M protein or M-like proteins have also been described in group B (Maxted 1948), C (Woolcock 1974), E (Daynes and Armstrong 1973) and G (Maxted and Potter 1967, Simpson et al 1987) and have been associated with antiphagocytic properties. To determine by DNA hybridisation if S suis strains possess M protein genes, two reference strains of S pyogenes (CS130 and CS190) and five human strains
isolated from cases of human meningitis in Hong Kong, were obtained from the Center for Reference and Research on Streptococci, University of Minnesota. Twenty strains of S suis from several outbreaks of meningitis were provided by the veterinary diagnostic laboratory of South Dakota State University. Ten S suis strains were from a Minnesota herd experiencing an outbreak of meningitis and 20 strains of S suis isolated from pneumonia in pigs were collected from the veterinary diagnostic laboratory of the University of Minnesota. Identification was based on standard biochemical tests and serotyping (Hommez et al 1986). Two hybridisation probes were used. One was derived from a polymerase chain reaction (PCR) amplification. It represented sequences about 450 base pairs (bp) from the C repeats of type M 1 protein (emm 1 gene) (A. Sukorov and P. P. Cleary, unpublished data). The other probe was plasmid pP134, which contains a 1.2 kb fragment (Simpson et al 1987) of the conserved domains from type 12 M protein (emm 12 gene) of group A streptococci. The PCR product and plasmid pP134 were labelled by random priming in the presence of digoxigenin-dUTP (Feinberg and Vogelstein 1983). DNA from S suis was extracted (Mogollon et al 1990) and 3 ~tg were spotted onto nylon membranes (Schleicher and Schuell Nytran) using a Bio-Dot apparatus (Bio-Rad) and immobilised by baking at 80°C for one hour. Hybridisation was carried out under low stringency conditions. Briefly, membranes were prehybridised at 49°C for two hours in a solution containing 5 × saline sodium citrate (ssc), 0-1 per cent sarkosyl, 0-02 per cent sodium dodecyl sulphate (SDS) and 1 per cent of tlie blocking powder (milk proteins) supplied by the manu-facturer. Hybridisation was then performed by incorporating 50 ng of the digoxigeninPCR or pP134 probe into the same solution at 49°C overnight. After hybridisation, the membranes were washed twice with 2 × ssc, and 0-1 per cent SDS at room temperature followed twice with 0.2 × ssc and 0.1 per cent SDS at 49°C for 15 minutes each. Bound
244
Proteins in meningeal Streptococcus suis
f
B
C
t
2
2
5
3 4
4
5 6 7'
A
CS130
245
5
CSI90
FIG 1 : Dot blot hybridisation of Streptococcus strains isolated from man. Samples and strain designations are as follows: row 1, Spyogenes CS130; row 2, S suis 84-551 ; row 3, S suis 87-552; row 4, S suis 87-553; row 5, S suis 87-554; row 6, S suis 87-555; and row 7, S pyogenes CS190. Hybridisation and development conditions were as described in the text
probes were detected by sequential incubations with antidigoxigenin antibody conjugated to alkaline phosphatase and developed in a solution of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate toluidinium salts. Blots were allowed to develop overnight. The digoxigenin-labelled probes specifically identified reference strains of Spyogenes known to harbour genes coding for M protein. However, these same probes did not detect M protein genes in any strain of S suis examined despite the use of low stringency conditions (Figs 1 and 2). Very faint background reactions were observed on the membranes of negative strains. Previous hybridisation studies (Scott et al 1986) showed that the 3' end of the M protein genes of S pyogenes are highly conserved among different serotypes. Since the hybridisation probes were derived from the 3' conserved region, S suis clearly lacks sequences for commonly conserved regions of Spyogenes M protein genes. S suis might have a distinct class of genes that code for surface proteins different from the M class of proteins. Vecht et al (1989, 1990) demonstrated the presence
6
FIG 2: Dot blot hybridisation of Streptococcus suis isolated from cases of meningitis in swine. All spots are independently isolated samples of S suis except for the spots at 1A and 5C, which are S pyogenes CS130 and CS190. Hybridisation and development conditions were as described in the text
of a protein of high molecular weight on the surface of S suis strains; these proteins were antigenic and were putatively M-like but they were not characterised biochemically to substantiate that they were truly Mlike. The present results suggest that both virulent and avirulent strains of S suis lack the genes encoding conserved domains of all M and M-like proteins previously described in the streptococci. Consequently the nature and function of the proteins found by Vecht et al (1989, 1990) remain under question. It is now apparent that the surface proteins of Gram-positive cocci play an important part in pathogenicity (Forester et al 1983, Fischetti et al 1988). Therefore, the possibility that surface proteins may contribute to or provide markers for meningeal strains of S suis merits further study.
Acknowledgements This work was supported in part by a grant from the National Pork Producers Council and by a us Department of Agriculture formula grant.
246
J. D. Mogollon, C. Pijoan, M. P. Murtaugh, P. P. Cleary, J. E. Collins
References ARENDS, J. P. & ZANEN, H. C. (1984) A subtype of Streptococcus suis type 2 isolated from cases of meningitis in man and pigs. Proceedings of the 9th Lancefield International Symposium on Streptococci Disease. pp343-344 CLIFTON-HADLEY, F. A., ALEXANDER, T. J. L. & ENRIGHT, M. R. (1985) Diagnosis of Streptococcus suis type 2 infections in pigs. Pig Veterinary Society Proceedings 14, 27-34 DAYNES, R. A. & ARMSTRONG, C. H. (1973) An antiphagocytic factor associated with group E streptococci. Infection andlmmunity 7, 298-304 FEINBERG, A. P. & VOGELSTEIN, B. (1983) A technique for radiolabening DNArestriction endonuclease fragnaents to high specific activity. Analytical Biochemistry 132, 6-13 FISCHETTI, V., JONES, K. F., HOLLINGSHEAD, S. K. & SCOTT, J. R. (1988) Structure, function and genetics of streptococcal M protein. Review of Infectious Diseases 10, $356-$359 FORESTER, H. HUNTER, N. & KNOX, K. W. (1983) Characteristics of a high molecular weight extracellular protein of Streptococcus mutans. Journal of General Microbiology 129, 27792788 HOFFMAN, L., & HENDERSON, L. (1985) The significance of Streptococcus suis in swine disease: clinical, pathologic and bacteriologic data from a two year study. Proceedings of the American Association of Veterinary Laboratory Diagnosticians Meeting. pp201-210 HOMMEZ, J., DEVRIESE, L. A., HENRICHSEN, J. & CASTRYCK, F. (1986) Identification and characterization of Streptococcus suis. Veterinary Microbiology 11, 349-355 KEHOE, M. A., MILLER, L., POIRIER, T. P., BEACHEY, E. H., LEE, M. & HARRINGTON, D. (t987) Genetics of type 5 M protein of Streptococcuspyogenes. Streptococcal genetics. American Society for Microbiology. pp112-t16 MAXTED, W. R. (1948) Occurrence of the M substance of type 28
group A in Streptococci of Lancefield group B, C and G. Journal of General Microbiology 3, 1-6 MAXTED, W. R. & POTTER, E. V. (1967) The presence of type 12 M protein antigen in group G Streptococci. Journal of General Microbiology 49, 119-125 MOGOLLON, J. D., PIJOAN, C., MURTAUGH, M. P., COLLINS, J., KAPLAN, E. & CLEARY, P. P. (1990) Characterization of prototype and clinically defined strains of Streptococcus suis by genomic fingerprinting. Journal of Clinical Microbiology 28, 2462-2466 SANFORD, S. E. (1989) Streptococcus suis: a strategic update. Proceedings of the the American Association of Swine Practitioners pp130-132 SCOTT, J. R., HOLLINGSHEAD, S. K. & FISCHETTI, V. A. (1986) Homologous regions within M protein genes in group A Streptococci of different serotypes. Infection and Immunity 52, 609612 SIMPSON, W. J., ROBBINS, J. C. & CLEARY, P. P. (1987) Evidence for group A-related M protein genes in human but not animal-associated group G streptococcal pathogens. Microbial Pathogenesis 3, 339-350 VECHT, U., ARENDS, J. P., VAN DER MOLEN, E. J. & VAN LEEGOED, L. (1989) Differences in virulence between two strains of Streptococcus suis type 2 after experimentally induced infection of new born germ-free pigs. American Journal of Veterinary Research 50, 1037-1043 VECHT, U., WISSELINK, H. J., VAN DIJK, & SMITH, H. E. (1990) Differences in virulence of StrepWcoccus suis type 2 strains. Proceedings of the 1lth Congress of the International Pig Veterinary Society p173 WOOLOCK, J. B. (1974) Purification and antigenicity of an M-like protein of Streptococcus equi. Infection and Immunity 10, 116-122
Received May 1, 1991 Accepted September 10, 1991
Testing memngeal strains of Streptococcus suis to detect M protein genes J. D. MOGOLLON, C. PIJOAN, Department of Clinical and Populations Sciences, M. P. MURTAUGH, Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St Paul, Minnesota 55108, P. P. CLEARY, Department of Microbiology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455, J. E. COLLINS, Department of Veterinary Diagnostic Investigation, College of Veterinary Medicine, University of Minnesota, St Paul Minnesota 55108, USA
Previous reports have suggested that the surface proteins found in meningeal strains of Streptococcus suis might be similar to the M protein of group A streptococci. Fifty-five strains of S suis, including human and swine meningeal and pneumonic isolates, were tested for M protein genes by DNA probes representing the constant domain of the 3' end of the group A, M protein gene. None of the S suis strains examined was positive, indicating that these organisms either lack M protein genes or harbour different genes, not expressing the constant domains of protein M from group A.
MENINGITIS due to Streptococcus suis type 2 in weaned pigs has emerged as an important problem in the North American pig industry during recent years (Hoffman and Henderson 1985, Sanford 1989). S suis type 2 has also been isolated from the tonsils of a large proportion of healthy adults in affected herds, suggesting that many strains are avirulent (CliftonHadley et al 1985). Unfortunately, available diagnostic techniques do not differentiate isolates in terms of virulence factors, which for S suis have not been defined. The emergence of S suis as a major pathogen could be due to the evolution by some strains of antiphagocytic surface determinants similar to the M protein (Arend s and Zanen 1984, Vecht et al 1989). Virulence in S pyogenes (group A) has been attributed to the antipliagocytic properties of the M protein. This protein is arranged as a coiled-coil dimer with the carboxy terminus attached to the peptidoglycan of the cell wall (Fischetti et al 1988). The amino-terminal region is distal to the cell surface and is exposed to selective immunological pressure (Kehoe et al 1987). M protein or M-like proteins have also been described in group B (Maxted 1948), C (Woolcock 1974), E (Daynes and Armstrong 1973) and G (Maxted and Potter 1967, Simpson et al 1987) and have been associated with antiphagocytic properties. To determine by DNA hybridisation if S suis strains possess M protein genes, two reference strains of S pyogenes (CS130 and CS190) and five human strains
isolated from cases of human meningitis in Hong Kong, were obtained from the Center for Reference and Research on Streptococci, University of Minnesota. Twenty strains of S suis from several outbreaks of meningitis were provided by the veterinary diagnostic laboratory of South Dakota State University. Ten S suis strains were from a Minnesota herd experiencing an outbreak of meningitis and 20 strains of S suis isolated from pneumonia in pigs were collected from the veterinary diagnostic laboratory of the University of Minnesota. Identification was based on standard biochemical tests and serotyping (Hommez et al 1986). Two hybridisation probes were used. One was derived from a polymerase chain reaction (PCR) amplification. It represented sequences about 450 base pairs (bp) from the C repeats of type M 1 protein (emm 1 gene) (A. Sukorov and P. P. Cleary, unpublished data). The other probe was plasmid pP134, which contains a 1.2 kb fragment (Simpson et al 1987) of the conserved domains from type 12 M protein (emm 12 gene) of group A streptococci. The PCR product and plasmid pP134 were labelled by random priming in the presence of digoxigenin-dUTP (Feinberg and Vogelstein 1983). DNA from S suis was extracted (Mogollon et al 1990) and 3 ~tg were spotted onto nylon membranes (Schleicher and Schuell Nytran) using a Bio-Dot apparatus (Bio-Rad) and immobilised by baking at 80°C for one hour. Hybridisation was carried out under low stringency conditions. Briefly, membranes were prehybridised at 49°C for two hours in a solution containing 5 × saline sodium citrate (ssc), 0-1 per cent sarkosyl, 0-02 per cent sodium dodecyl sulphate (SDS) and 1 per cent of tlie blocking powder (milk proteins) supplied by the manu-facturer. Hybridisation was then performed by incorporating 50 ng of the digoxigeninPCR or pP134 probe into the same solution at 49°C overnight. After hybridisation, the membranes were washed twice with 2 × ssc, and 0-1 per cent SDS at room temperature followed twice with 0.2 × ssc and 0.1 per cent SDS at 49°C for 15 minutes each. Bound
244
Proteins in meningeal Streptococcus suis
f
B
C
t
2
2
5
3 4
4
5 6 7'
A
CS130
245
5
CSI90
FIG 1 : Dot blot hybridisation of Streptococcus strains isolated from man. Samples and strain designations are as follows: row 1, Spyogenes CS130; row 2, S suis 84-551 ; row 3, S suis 87-552; row 4, S suis 87-553; row 5, S suis 87-554; row 6, S suis 87-555; and row 7, S pyogenes CS190. Hybridisation and development conditions were as described in the text
probes were detected by sequential incubations with antidigoxigenin antibody conjugated to alkaline phosphatase and developed in a solution of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate toluidinium salts. Blots were allowed to develop overnight. The digoxigenin-labelled probes specifically identified reference strains of Spyogenes known to harbour genes coding for M protein. However, these same probes did not detect M protein genes in any strain of S suis examined despite the use of low stringency conditions (Figs 1 and 2). Very faint background reactions were observed on the membranes of negative strains. Previous hybridisation studies (Scott et al 1986) showed that the 3' end of the M protein genes of S pyogenes are highly conserved among different serotypes. Since the hybridisation probes were derived from the 3' conserved region, S suis clearly lacks sequences for commonly conserved regions of Spyogenes M protein genes. S suis might have a distinct class of genes that code for surface proteins different from the M class of proteins. Vecht et al (1989, 1990) demonstrated the presence
6
FIG 2: Dot blot hybridisation of Streptococcus suis isolated from cases of meningitis in swine. All spots are independently isolated samples of S suis except for the spots at 1A and 5C, which are S pyogenes CS130 and CS190. Hybridisation and development conditions were as described in the text
of a protein of high molecular weight on the surface of S suis strains; these proteins were antigenic and were putatively M-like but they were not characterised biochemically to substantiate that they were truly Mlike. The present results suggest that both virulent and avirulent strains of S suis lack the genes encoding conserved domains of all M and M-like proteins previously described in the streptococci. Consequently the nature and function of the proteins found by Vecht et al (1989, 1990) remain under question. It is now apparent that the surface proteins of Gram-positive cocci play an important part in pathogenicity (Forester et al 1983, Fischetti et al 1988). Therefore, the possibility that surface proteins may contribute to or provide markers for meningeal strains of S suis merits further study.
Acknowledgements This work was supported in part by a grant from the National Pork Producers Council and by a us Department of Agriculture formula grant.
246
J. D. Mogollon, C. Pijoan, M. P. Murtaugh, P. P. Cleary, J. E. Collins
References ARENDS, J. P. & ZANEN, H. C. (1984) A subtype of Streptococcus suis type 2 isolated from cases of meningitis in man and pigs. Proceedings of the 9th Lancefield International Symposium on Streptococci Disease. pp343-344 CLIFTON-HADLEY, F. A., ALEXANDER, T. J. L. & ENRIGHT, M. R. (1985) Diagnosis of Streptococcus suis type 2 infections in pigs. Pig Veterinary Society Proceedings 14, 27-34 DAYNES, R. A. & ARMSTRONG, C. H. (1973) An antiphagocytic factor associated with group E streptococci. Infection andlmmunity 7, 298-304 FEINBERG, A. P. & VOGELSTEIN, B. (1983) A technique for radiolabening DNArestriction endonuclease fragnaents to high specific activity. Analytical Biochemistry 132, 6-13 FISCHETTI, V., JONES, K. F., HOLLINGSHEAD, S. K. & SCOTT, J. R. (1988) Structure, function and genetics of streptococcal M protein. Review of Infectious Diseases 10, $356-$359 FORESTER, H. HUNTER, N. & KNOX, K. W. (1983) Characteristics of a high molecular weight extracellular protein of Streptococcus mutans. Journal of General Microbiology 129, 27792788 HOFFMAN, L., & HENDERSON, L. (1985) The significance of Streptococcus suis in swine disease: clinical, pathologic and bacteriologic data from a two year study. Proceedings of the American Association of Veterinary Laboratory Diagnosticians Meeting. pp201-210 HOMMEZ, J., DEVRIESE, L. A., HENRICHSEN, J. & CASTRYCK, F. (1986) Identification and characterization of Streptococcus suis. Veterinary Microbiology 11, 349-355 KEHOE, M. A., MILLER, L., POIRIER, T. P., BEACHEY, E. H., LEE, M. & HARRINGTON, D. (t987) Genetics of type 5 M protein of Streptococcuspyogenes. Streptococcal genetics. American Society for Microbiology. pp112-t16 MAXTED, W. R. (1948) Occurrence of the M substance of type 28
group A in Streptococci of Lancefield group B, C and G. Journal of General Microbiology 3, 1-6 MAXTED, W. R. & POTTER, E. V. (1967) The presence of type 12 M protein antigen in group G Streptococci. Journal of General Microbiology 49, 119-125 MOGOLLON, J. D., PIJOAN, C., MURTAUGH, M. P., COLLINS, J., KAPLAN, E. & CLEARY, P. P. (1990) Characterization of prototype and clinically defined strains of Streptococcus suis by genomic fingerprinting. Journal of Clinical Microbiology 28, 2462-2466 SANFORD, S. E. (1989) Streptococcus suis: a strategic update. Proceedings of the the American Association of Swine Practitioners pp130-132 SCOTT, J. R., HOLLINGSHEAD, S. K. & FISCHETTI, V. A. (1986) Homologous regions within M protein genes in group A Streptococci of different serotypes. Infection and Immunity 52, 609612 SIMPSON, W. J., ROBBINS, J. C. & CLEARY, P. P. (1987) Evidence for group A-related M protein genes in human but not animal-associated group G streptococcal pathogens. Microbial Pathogenesis 3, 339-350 VECHT, U., ARENDS, J. P., VAN DER MOLEN, E. J. & VAN LEEGOED, L. (1989) Differences in virulence between two strains of Streptococcus suis type 2 after experimentally induced infection of new born germ-free pigs. American Journal of Veterinary Research 50, 1037-1043 VECHT, U., WISSELINK, H. J., VAN DIJK, & SMITH, H. E. (1990) Differences in virulence of StrepWcoccus suis type 2 strains. Proceedings of the 1lth Congress of the International Pig Veterinary Society p173 WOOLOCK, J. B. (1974) Purification and antigenicity of an M-like protein of Streptococcus equi. Infection and Immunity 10, 116-122
Received May 1, 1991 Accepted September 10, 1991
