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Incubation of Streptococcus uberis with extracellular matrix proteins enhances adherence to and internalization into bovine mammary epithelial cells
FEMS Microbiology Letters 178 (1999) 81^85
Incubation of Streptococcus uberis with extracellular matrix proteins enhances adherence to and internalization into bovine mammary epithelial cells Raul A. Almeida, Douglas A. Luther, Stephen P. Oliver * Department of Animal Science, Institute of Agriculture, The University of Tennessee, Knoxville, TN 37996, USA Received 28 May 1999; accepted 28 June 1999
Abstract Two strains of Streptococcus uberis (UT 888 and UT 366) isolated from cows with clinical mastitis were co-cultured with bovine mammary epithelial cells (MAC-T) with and without laminin, fibrinogen, fibronectin or collagen. Incubation of S. uberis with extracellular matrix proteins (ECMPs) increased adherence to and internalization into MAC-T cells. Both strains of S. uberis exhibited greater adherence when co-cultured in the presence of collagen than with any other ECMP. However, adherence was always higher when strains were co-cultured with ECMP than in medium alone. S. uberis UT 888 adhered better to MAC-T cells than S. uberis UT 366. The influence of ECMPs on bacterial internalization into MAC-T cells was similar to adherence, however, differences among ECMPs were less noticeable. S. uberis UT 888 had a higher internalization index than S. uberis UT 366. It is possible that ECMPs induce or up-regulate proteins that selectively adhere to ECMPs which could serve as a bridge between the eukaryotic cell and the bacterial pathogen that leads to internalization of the ECMP-bound pathogen into the mammary epithelial cell. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Extracellular matrix protein; Adherence; Internalization; Streptococcus uberis
1. Introduction Streptococcus uberis is classi¢ed as an environmental pathogen with emerging importance as a mastitiscausing organism in dairy cattle. The incidence of S. uberis infections is markedly elevated during the non-lactating period and during early lactation and conventional control measures are not e¡ective in
* Corresponding author. Tel.: +1 (423) 974-7260; Fax: +1 (423) 974-6465; E-mail: [email protected]
controlling this pathogen [1]. Previous reports from our laboratory showed that S. uberis adhered to and internalized into bovine mammary epithelial cells [2^ 3]. Expression of `de novo' protein factors was required for internalization of S. uberis into bovine mammary epithelial cells [2,4]. More recently, we showed that cultivation of S. uberis with extracellular matrix proteins (ECMPs) induced expression of surface-associated proteins [5]. The purpose of the present study was to determine if pre-incubation of S. uberis with ECMPs in£uenced adherence to and internalization into bovine mammary epithelial cells.
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 3 6 - 5
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R.A. Almeida et al. / FEMS Microbiology Letters 178 (1999) 81^85
2. Materials and methods 2.1. Bacterial strains and growth conditions S. uberis strains UT 888 and UT 366, both isolated from dairy cows with clinical mastitis, were used in this study. Isolates were characterized extensively and identi¢ed to the species level and classi¢ed according to their DNA ¢ngerprint pattern as described previously [6,7]. Streptococcus pyogenes M6, kindly provided by Dr V. Fischetti (Rockefeller University, New York, USA), was used as a positive control. For adherence and internalization assays, bacterial suspensions kept at 370³C were thawed in a 37³C water bath, streaked onto blood agar plates and incubated for 16 h at 37³C. Bacteria were then inoculated into Todd-Hewitt broth (Difco, Detroit, MI, USA) for 2 h at 37³C in an orbital shaker. Bacterial suspensions were diluted in cell growth medium (CGM) as described previously [5] to a concentration of 107 colony forming units (CFU) ml31 . 2.2. Mammary epithelial cells A bovine mammary epithelial cell line (MAC-T) kindly provided by J.D. Turner, McGill University, Canada, was used [8]. MAC-T cells were grown in 24-well cell culture plates at 37³C in 5% CO2 :95% air (v/v) in CGM. 2.3. Internalization and adherence assays Internalization and adherence assays were performed following procedures described previously [3,5] with slight modi¢cations. Brie£y, S. uberis strains UT 888 and UT 366 were co-cultured with MAC-T cells in DMEM with and without addition of laminin (10 Wg ml31 ; Sigma, St. Louis, MO, USA), ¢brinogen (10 Wg ml31 ; Sigma), ¢bronectin (10 Wg ml31 ; Sigma) or collagen (25 Wg ml31 ; Sigma) for 2 h at 37³C in 5% CO2 :95% air (v/v). After incubation, monolayers were washed three times with PBS and CGM containing penicillin (5 Wg ml31 ; Sigma) and gentamicin (100 Wg ml31 ; Sigma) was added and incubated for 2 h at 37³C. After medium containing antibiotics was removed, MAC-T cell monolayers were washed three times with PBS,
treated with 0.25% trypsin and further lysed with Triton X-100 (Amersham, Arlington Heights, IL, USA) at a ¢nal concentration of 0.025% (v/v) in sterile distilled water. MAC-T cell lysates were 10fold serially diluted, plated in triplicate on blood agar plates and incubated overnight at 37³C. CFU per ml were determined by standard colony counting techniques. Cell-associated bacteria were measured in triplicate parallel wells following a treatment similar to that described previously but omitting the antibiotic treatment. Numbers of adherent bacteria were calculated by subtracting the number of internalized bacteria from the number of cell-associated bacteria.
3. Results and discussion Incubation of S. uberis with laminin, collagen, ¢bronectin and ¢brinogen resulted in increased adherence to and internalization into bovine mammary epithelial cells as compared with S. uberis incubated in DMEM only (Fig. 1). Both strains of S. uberis exhibited greater adherence when co-cultured in the presence of collagen than with any other ECMP (Fig. 1). The number of adherent bacteria was always higher when strains were co-cultured with ECMP than in DMEM alone. S. uberis UT 888 showed a greater adherence to MAC-T cells than S. uberis UT 366, but lower than S. pyogenes M6 (Fig. 1). The e¡ect of incubation with ECMPs on bacterial internalization into mammary epithelial cells showed a similar pattern as with adherence (Fig. 2). However, di¡erences among ECMPs were less noticeable with internalization than with adherence. S. uberis UT 888 had a higher internalization index than S. uberis UT 366, probably corresponding to a higher number of adherent bacteria. Although bacterial pathogens that are pro¢cient at internalizing host cells have evolved a number of mechanisms for intracellular invasion, some common themes have emerged. Among these are expression of speci¢c surface bacterial proteins required for internalization [9] and expression of surface bacterial proteins upon exposure to ECMPs and serum proteins [10]. With group A streptococci, it was reported that serum proteins, ¢brinogen or laminin acted as an
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Fig. 1. E¡ect of incubation of ECMPs on adherence of S. uberis to bovine mammary epithelial cells. S. uberis UT 366 (A), S. uberis UT 888 (B) and S. pyogenes M6 (C) were incubated in DMEM with or without (CNTRL) ¢bronectin (FN, 10 Wg ml31 ), ¢brinogen (FG, 10 Wg ml31 ), laminin (LM, 10 Wg ml31 ) or collagen (COLL, 25 Wg ml31 ) and co-cultured with bovine mammary epithelial cells. Data represent the means of six independent observations and S.E.M.
agonist of internalization and this e¡ect was dependent on expression of M1 [11]. Previous results from our laboratory indicated that cultivation of S. uberis with collagen and laminin induced expression of surface-associated proteins [5]. Results of the present study showed that incubation of S. uberis with ECMPs, and particularly with collagen and laminin, resulted in an increased ability of S. uberis to adhere to and internalize into bovine mammary epithelial cells. Taken together, these results suggests that ECMPs induce
or are involved in up-regulation of proteins that selectively adhere to ECMPs which could serve as a bridge between the host cell and the pathogen. It is likely that the interaction between ECMPs and cell surface receptor(s) transduces signal(s) that trigger an actin-mediated `zipper phagocytosis' process, leading to internalization of the ECMP-bound pathogen into the mammary epithelial cell [9]. Previous reports showed that internalization of S. uberis into bovine mammary epithelial cells was an actinmediated process and that S. uberis cells exploited
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Fig. 2. E¡ect of incubation of ECMPs on internalization of S. uberis into bovine mammary epithelial cells. S. uberis UT 366 (A), S. uberis UT 888 (B) and S. pyogenes M6 (C) incubated in DMEM with and without (CNTRL) ¢bronectin (FN, 10 Wg ml31 ), ¢brinogen (FG, 10 Wg ml31 ), laminin (LM, 10 Wg ml31 ), collagen (COLL, 25 Wg ml31 ) or DMEM (CNTRL) and co-cultured with bovine mammary epithelial cells. Data represent the means of six independent observations and S.E.M.
host cell signal transduction to generate their own `internalization signal' into the target cell ([2,9], Almeida and Oliver, unpublished observations). Considering results of the present study and previous reports from our laboratory [4,5], we conclude that ECMPs exert a positive regulation and expression of virulence factors in S. uberis that enhances colonization of mammary epithelial cells and persistence within mammary epithelial cells. Future research should address the e¡ectiveness of antibodies di-
rected against these putative virulence factors in preventing bacterial adherence and internalization.
Acknowledgements This work was supported by the Tennessee Agricultural Experiment Station, The University of Tennessee, College of Veterinary Medicine, Center of Excellence Research Program in Livestock Disease
FEMSLE 8926 11-8-99
R.A. Almeida et al. / FEMS Microbiology Letters 178 (1999) 81^85
and Human Health and USDA Animal Health Research Funds.
References [1] Todhunter, D.A., Smith, K.L. and Hogan, J.S. (1994) Environmental streptococcal mastitis. In: Proc. Natl. Mastitis Counc., pp. 92^95. Arlington, VA. [2] Matthews, K.R., Almeida, R.A. and Oliver, S.P. (1994) Bovine mammary epithelial cell invasion by Streptococcus uberis. Infect. Immun. 62, 5641^5646. [3] Almeida, R.A., Luther, D.A., Kumar, S.J., Calvinho, L.F., Bronze, M.S. and Oliver, S.P. (1996) Adherence of Streptococcus uberis to bovine mammary epithelial cells and to extracellular matrix proteins. J. Vet. Med. B43, 385^392. [4] Matthews, K.R., Luther, D.A. and Oliver, S.P. (1994) Protein expression by Streptococcus uberis in the presence of bovine mammary epithelial cells. J. Dairy Sci. 77, 3490. [5] Gilbert, F.B., Luther, D.A. and Oliver, S.P. (1997) Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells. FEMS Microbiol. Lett. 156, 161^164.
85
[6] Jayarao, B.M., Oliver, S.P., Matthews, K.R. and King, S.H. (1991) Comparative evaluation of Vitek Gram-positive identi¢cation system and API Rapid Strep system for identi¢cation of Streptococcus species of bovine origin. Vet. Microbiol. 26, 301^308. [7] Jayarao, B.M. and Oliver, S.P. (1994) Polymerase chain reaction-based DNA ¢ngerprint for identi¢cation of Streptococcus and Enterococcus species isolated from bovine milk. J. Food Prot. 57, 240^245. [8] Huynh, H.T., Robitaille, G. and Turner, J.D. (1991) Establishment of bovine mammary epithelial cells (MAC-T): An in Vitro model for bovine lactation. Exp. Cell Res. 197, 191^199. [9] Finlay, B.B. and Falkow, S. (1997) Common themes in microbial pathogenicity revisited. Mol. Biol. Rev. 61, 136^169. [10] Jaudon, J., Burstein, E., Hanski, E. and Sela, S. (1997) Proteins M6 and F1 are required for e¤cient invasion of group A streptococci into cultured epithelial cells. In: Streptococcus and the Host (Horaud, T., et al., Eds.), pp. 511^515. Plenum Press, New York. [11] Cue, D., Dombek, P.E., Lam, H. and Cleary, P.P. (1998) Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells. Infect. Immun. 66, 4593^4601.
FEMSLE 8926 11-8-99
Incubation of Streptococcus uberis with extracellular matrix proteins enhances adherence to and internalization into bovine mammary epithelial cells Raul A. Almeida, Douglas A. Luther, Stephen P. Oliver * Department of Animal Science, Institute of Agriculture, The University of Tennessee, Knoxville, TN 37996, USA Received 28 May 1999; accepted 28 June 1999
Abstract Two strains of Streptococcus uberis (UT 888 and UT 366) isolated from cows with clinical mastitis were co-cultured with bovine mammary epithelial cells (MAC-T) with and without laminin, fibrinogen, fibronectin or collagen. Incubation of S. uberis with extracellular matrix proteins (ECMPs) increased adherence to and internalization into MAC-T cells. Both strains of S. uberis exhibited greater adherence when co-cultured in the presence of collagen than with any other ECMP. However, adherence was always higher when strains were co-cultured with ECMP than in medium alone. S. uberis UT 888 adhered better to MAC-T cells than S. uberis UT 366. The influence of ECMPs on bacterial internalization into MAC-T cells was similar to adherence, however, differences among ECMPs were less noticeable. S. uberis UT 888 had a higher internalization index than S. uberis UT 366. It is possible that ECMPs induce or up-regulate proteins that selectively adhere to ECMPs which could serve as a bridge between the eukaryotic cell and the bacterial pathogen that leads to internalization of the ECMP-bound pathogen into the mammary epithelial cell. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Extracellular matrix protein; Adherence; Internalization; Streptococcus uberis
1. Introduction Streptococcus uberis is classi¢ed as an environmental pathogen with emerging importance as a mastitiscausing organism in dairy cattle. The incidence of S. uberis infections is markedly elevated during the non-lactating period and during early lactation and conventional control measures are not e¡ective in
* Corresponding author. Tel.: +1 (423) 974-7260; Fax: +1 (423) 974-6465; E-mail: [email protected]
controlling this pathogen [1]. Previous reports from our laboratory showed that S. uberis adhered to and internalized into bovine mammary epithelial cells [2^ 3]. Expression of `de novo' protein factors was required for internalization of S. uberis into bovine mammary epithelial cells [2,4]. More recently, we showed that cultivation of S. uberis with extracellular matrix proteins (ECMPs) induced expression of surface-associated proteins [5]. The purpose of the present study was to determine if pre-incubation of S. uberis with ECMPs in£uenced adherence to and internalization into bovine mammary epithelial cells.
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 3 6 - 5
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R.A. Almeida et al. / FEMS Microbiology Letters 178 (1999) 81^85
2. Materials and methods 2.1. Bacterial strains and growth conditions S. uberis strains UT 888 and UT 366, both isolated from dairy cows with clinical mastitis, were used in this study. Isolates were characterized extensively and identi¢ed to the species level and classi¢ed according to their DNA ¢ngerprint pattern as described previously [6,7]. Streptococcus pyogenes M6, kindly provided by Dr V. Fischetti (Rockefeller University, New York, USA), was used as a positive control. For adherence and internalization assays, bacterial suspensions kept at 370³C were thawed in a 37³C water bath, streaked onto blood agar plates and incubated for 16 h at 37³C. Bacteria were then inoculated into Todd-Hewitt broth (Difco, Detroit, MI, USA) for 2 h at 37³C in an orbital shaker. Bacterial suspensions were diluted in cell growth medium (CGM) as described previously [5] to a concentration of 107 colony forming units (CFU) ml31 . 2.2. Mammary epithelial cells A bovine mammary epithelial cell line (MAC-T) kindly provided by J.D. Turner, McGill University, Canada, was used [8]. MAC-T cells were grown in 24-well cell culture plates at 37³C in 5% CO2 :95% air (v/v) in CGM. 2.3. Internalization and adherence assays Internalization and adherence assays were performed following procedures described previously [3,5] with slight modi¢cations. Brie£y, S. uberis strains UT 888 and UT 366 were co-cultured with MAC-T cells in DMEM with and without addition of laminin (10 Wg ml31 ; Sigma, St. Louis, MO, USA), ¢brinogen (10 Wg ml31 ; Sigma), ¢bronectin (10 Wg ml31 ; Sigma) or collagen (25 Wg ml31 ; Sigma) for 2 h at 37³C in 5% CO2 :95% air (v/v). After incubation, monolayers were washed three times with PBS and CGM containing penicillin (5 Wg ml31 ; Sigma) and gentamicin (100 Wg ml31 ; Sigma) was added and incubated for 2 h at 37³C. After medium containing antibiotics was removed, MAC-T cell monolayers were washed three times with PBS,
treated with 0.25% trypsin and further lysed with Triton X-100 (Amersham, Arlington Heights, IL, USA) at a ¢nal concentration of 0.025% (v/v) in sterile distilled water. MAC-T cell lysates were 10fold serially diluted, plated in triplicate on blood agar plates and incubated overnight at 37³C. CFU per ml were determined by standard colony counting techniques. Cell-associated bacteria were measured in triplicate parallel wells following a treatment similar to that described previously but omitting the antibiotic treatment. Numbers of adherent bacteria were calculated by subtracting the number of internalized bacteria from the number of cell-associated bacteria.
3. Results and discussion Incubation of S. uberis with laminin, collagen, ¢bronectin and ¢brinogen resulted in increased adherence to and internalization into bovine mammary epithelial cells as compared with S. uberis incubated in DMEM only (Fig. 1). Both strains of S. uberis exhibited greater adherence when co-cultured in the presence of collagen than with any other ECMP (Fig. 1). The number of adherent bacteria was always higher when strains were co-cultured with ECMP than in DMEM alone. S. uberis UT 888 showed a greater adherence to MAC-T cells than S. uberis UT 366, but lower than S. pyogenes M6 (Fig. 1). The e¡ect of incubation with ECMPs on bacterial internalization into mammary epithelial cells showed a similar pattern as with adherence (Fig. 2). However, di¡erences among ECMPs were less noticeable with internalization than with adherence. S. uberis UT 888 had a higher internalization index than S. uberis UT 366, probably corresponding to a higher number of adherent bacteria. Although bacterial pathogens that are pro¢cient at internalizing host cells have evolved a number of mechanisms for intracellular invasion, some common themes have emerged. Among these are expression of speci¢c surface bacterial proteins required for internalization [9] and expression of surface bacterial proteins upon exposure to ECMPs and serum proteins [10]. With group A streptococci, it was reported that serum proteins, ¢brinogen or laminin acted as an
FEMSLE 8926 11-8-99
R.A. Almeida et al. / FEMS Microbiology Letters 178 (1999) 81^85
83
Fig. 1. E¡ect of incubation of ECMPs on adherence of S. uberis to bovine mammary epithelial cells. S. uberis UT 366 (A), S. uberis UT 888 (B) and S. pyogenes M6 (C) were incubated in DMEM with or without (CNTRL) ¢bronectin (FN, 10 Wg ml31 ), ¢brinogen (FG, 10 Wg ml31 ), laminin (LM, 10 Wg ml31 ) or collagen (COLL, 25 Wg ml31 ) and co-cultured with bovine mammary epithelial cells. Data represent the means of six independent observations and S.E.M.
agonist of internalization and this e¡ect was dependent on expression of M1 [11]. Previous results from our laboratory indicated that cultivation of S. uberis with collagen and laminin induced expression of surface-associated proteins [5]. Results of the present study showed that incubation of S. uberis with ECMPs, and particularly with collagen and laminin, resulted in an increased ability of S. uberis to adhere to and internalize into bovine mammary epithelial cells. Taken together, these results suggests that ECMPs induce
or are involved in up-regulation of proteins that selectively adhere to ECMPs which could serve as a bridge between the host cell and the pathogen. It is likely that the interaction between ECMPs and cell surface receptor(s) transduces signal(s) that trigger an actin-mediated `zipper phagocytosis' process, leading to internalization of the ECMP-bound pathogen into the mammary epithelial cell [9]. Previous reports showed that internalization of S. uberis into bovine mammary epithelial cells was an actinmediated process and that S. uberis cells exploited
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Fig. 2. E¡ect of incubation of ECMPs on internalization of S. uberis into bovine mammary epithelial cells. S. uberis UT 366 (A), S. uberis UT 888 (B) and S. pyogenes M6 (C) incubated in DMEM with and without (CNTRL) ¢bronectin (FN, 10 Wg ml31 ), ¢brinogen (FG, 10 Wg ml31 ), laminin (LM, 10 Wg ml31 ), collagen (COLL, 25 Wg ml31 ) or DMEM (CNTRL) and co-cultured with bovine mammary epithelial cells. Data represent the means of six independent observations and S.E.M.
host cell signal transduction to generate their own `internalization signal' into the target cell ([2,9], Almeida and Oliver, unpublished observations). Considering results of the present study and previous reports from our laboratory [4,5], we conclude that ECMPs exert a positive regulation and expression of virulence factors in S. uberis that enhances colonization of mammary epithelial cells and persistence within mammary epithelial cells. Future research should address the e¡ectiveness of antibodies di-
rected against these putative virulence factors in preventing bacterial adherence and internalization.
Acknowledgements This work was supported by the Tennessee Agricultural Experiment Station, The University of Tennessee, College of Veterinary Medicine, Center of Excellence Research Program in Livestock Disease
FEMSLE 8926 11-8-99
R.A. Almeida et al. / FEMS Microbiology Letters 178 (1999) 81^85
and Human Health and USDA Animal Health Research Funds.
References [1] Todhunter, D.A., Smith, K.L. and Hogan, J.S. (1994) Environmental streptococcal mastitis. In: Proc. Natl. Mastitis Counc., pp. 92^95. Arlington, VA. [2] Matthews, K.R., Almeida, R.A. and Oliver, S.P. (1994) Bovine mammary epithelial cell invasion by Streptococcus uberis. Infect. Immun. 62, 5641^5646. [3] Almeida, R.A., Luther, D.A., Kumar, S.J., Calvinho, L.F., Bronze, M.S. and Oliver, S.P. (1996) Adherence of Streptococcus uberis to bovine mammary epithelial cells and to extracellular matrix proteins. J. Vet. Med. B43, 385^392. [4] Matthews, K.R., Luther, D.A. and Oliver, S.P. (1994) Protein expression by Streptococcus uberis in the presence of bovine mammary epithelial cells. J. Dairy Sci. 77, 3490. [5] Gilbert, F.B., Luther, D.A. and Oliver, S.P. (1997) Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells. FEMS Microbiol. Lett. 156, 161^164.
85
[6] Jayarao, B.M., Oliver, S.P., Matthews, K.R. and King, S.H. (1991) Comparative evaluation of Vitek Gram-positive identi¢cation system and API Rapid Strep system for identi¢cation of Streptococcus species of bovine origin. Vet. Microbiol. 26, 301^308. [7] Jayarao, B.M. and Oliver, S.P. (1994) Polymerase chain reaction-based DNA ¢ngerprint for identi¢cation of Streptococcus and Enterococcus species isolated from bovine milk. J. Food Prot. 57, 240^245. [8] Huynh, H.T., Robitaille, G. and Turner, J.D. (1991) Establishment of bovine mammary epithelial cells (MAC-T): An in Vitro model for bovine lactation. Exp. Cell Res. 197, 191^199. [9] Finlay, B.B. and Falkow, S. (1997) Common themes in microbial pathogenicity revisited. Mol. Biol. Rev. 61, 136^169. [10] Jaudon, J., Burstein, E., Hanski, E. and Sela, S. (1997) Proteins M6 and F1 are required for e¤cient invasion of group A streptococci into cultured epithelial cells. In: Streptococcus and the Host (Horaud, T., et al., Eds.), pp. 511^515. Plenum Press, New York. [11] Cue, D., Dombek, P.E., Lam, H. and Cleary, P.P. (1998) Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells. Infect. Immun. 66, 4593^4601.
FEMSLE 8926 11-8-99