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Identification of Methionine-processed HPr in the Equine Pathogen Streptococcus equi
SYSTEMATIC AND _ht..:....tp_://w_w_w_.ur_ba_nf_is_ch_er_.de-.:./jo_u_rn_als_/s_am_ _ _ _ _ _ _ _ _ _ _ _ APPLIED MICROBIOLOGY System. Appl. Microbiol. 23, 330-332 (2000)
© Urban & Fischer Verlag
Identification of Methionine-processed HPr in the Equine Pathogen Streptococcus equi lAIN C. SUTCLIFFE, JOANNE TRIGG, and DEAN HARRINGTON School of Sciences, University of Sunderland, UK Received July 5, 2000
Summary Using preparative electrophoresis, a low molecular weight protein has been partially purified from a cell extract of the equine pathogen Streptococcus equi susp. equi. N-terminal sequence analysis and Western blotting revealed the protein to be HPr, a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Interestingly, the only form of the HPr protein detected in S. equi was one with the amino-terminal methionine removed, a modification that has previously been associated with surface localization of streptococcal HPr proteins. Key words: HPr - Strangles - Streptococcus
Streptococcus equi subsp. equi is the causative agent of strangles, a highly contagious and debilitating respiratory disease of the horse (CHANTER, 1997). Although a disseminated and normally fatal form of the disease manifests itself in 1-5% of cases during an outbreak, the majority of horses mount an effective immune response and recover from acute disease. During our investigations of the virulence determinants of S. equi we have analysed low molecular weight antigens in cell extracts that crossreact in Western blotting experiments with a convalescent serum from a horse that had recovered from an episode of strangles. During these experiments we have partially purified and characterized by N-terminal sequence analysis a protein identified here as HPr, a central component of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system (VADEBONCOEUR, 1995). Briefly, cells harvested from a 500 mL Todd-Hewitt broth culture of S. equi strain 4047 were harvested by centrifugation and washed twice with phosphate-buffered saline. Cell extracts were obtained by boiling the cell pellet for 10 minutes in 1.2 mL sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer followed by removal of cell debris by centrifugation. The extract was then fractionated using a Biorad MiniPrep preparative electrophoresis system: the sample (700 J.lL) was electrophoresed through a 7.5 cm 15% tube gel and the fractionated proteins were collected by continuous elution with water. A total of 100 fractions (250 J.lL each) were obtained and every fifth fraction was analysed by Western blotting (HAMILTON et aI., 2000) using a 111000 dilution of horse convalescent 0723-2020/00/23/03-330 $ 15.00/0
serum as the primary antibody and mouse anti-horse IgG (1130,000; Sigma Chemical Co., Dorset, UK) as secondary antibody. Fractions 40-45, containing proteins in the 14-20 kDa range, were pooled and concentrated using Nanosep centrifugal concentrators (3000 kDa molecular weight cut-off; Flowgen Ltd, UK). The pooled proteins were subjected to SDS-PAGE and then blotted onto Immobilon-PSQ transfer membrane (Millipore, UK). Reversible protein staining (Biosafe Coomassie reagent, Biorad laboratories, UK) revealed two major protein bands in the 15-16 kDa range and samples of each were excised from the blot and subjected to N-terminal protein sequencing. The lower molecular weight band gave no distinguishable sequence as it appeared to be a mixture of at least three proteins. However, the upper ca. 16 kDa band yielded the unambiguous 15 amino acid N-terminal sequence ASKDFHlVAETGIHA. Database searches using the BLAST search tool (ALTSCHUL et aI., 1997; http://www.ncbi.nim.nih.gov/BLAST) revealed that this sequence is 100% identical to amino acids 2-16 of the HPr proteins of Streptococcus salivarius (GAGNON et aI., 1993), Streptococcus mutans (BOYD et aI., 1994) and Streptococcus bovis (ASANUMA, N. and HINo, T.: GenBank accession number AB027569, 1999). The sequence is also 100% identical to amino acids 1-15 of methionAbbreviations: HPr - a phosphotransfer protein of PTS, PTS phosphoenolpyruvate:sugar phosphotransferase system, SDSPAGE - sodium dodecyl sulphate polyacrylamide gel electrophoresis
Methionine-Processes HPr of Streptococcus equi
1
2
<1 10
Fig. 1. Western blot analysis of S. equi using polyclonal rabbit antiserum to HPr from S. salivarius. Lane 1. Cell extract of S. equi strain 4047. Lane 2. Pooled preparative electrophoresis fractions 40-45 containing proteins of ca. 16 kDa. Positions of the molecular weight standards (kDa) are indicated on the right.
ine-processed HPrs (see below) from Streptococcus pyogenes (GERLACH et aI., 1992), Streptococcus sanguis (JENKINSON, 1989) and Streptococcus suis (DUBREIL et aI., 1996). The high degree of homology reflects the fact that the N-termini of HPr proteins are highly conserved as the active site histidine involved in phosphorelay is located at His-15 (VADEBONCOEUR, 1995). Although streptococcal HPr proteins have molecular weights of ca. 9 kDa, several have been observed to migrate at 12-16 kDa on SDS-PAGE (JENKINSON, 1989; GERLACH et aI., 1992; SUTCLIFFE et aI., 1993). To confirm the presence of HPr in the samples analysed, the original cell extract and pooled fractions 40-45 were electrophoresed trough a 15% SDS-PAGE gel, Western blotted and probed with a 111000 dilution of a polyclonal rabbit serum against S. salivarius HPr. A band of ca. 16 kDa which showed strong cross-reaction with the antiHPr was observed in both samples (Figure 1). Cross-reaction with the same antibody was also observed in cell extracts of two other strains of S. equi subsp. equi and in four strains of Streptococcus equi subsp. zooepidemicus (data not shown). These findings constitute the first identification of HPr in S. equi. A putative protein sequence 92 % identical to 80/87 amino acids of the S. salivarius HPr can be identified in the unedited test assembly of the S. equi strain 4047 genome (http://www.sanger.ac.uk/ Projects/S_equil). At present the predicted N-terminus is missing from this putative protein but sequence identical to 5 amino acids (TGIHA) in the sequence determined herein is present. Further examination of the S. equi genome sequence suggests that a complete PTS system (Enzyme I; HPr; Ell membrane-associated proteins) is present in this organism (data not shown). Methionine (Met) processing of HPr has been identified previously as a cause of HPr polymorphism in S.
331
salivarius and other oral streptococci (VADEBONCOEUR et aI., 1991; VADEBONCOEUR, 1995). Electrophoretic resolution of HPr-1 (Met-removed) and HPr-2 (Met-intact) is achieved only by using non-denaturing Tris-buffered PAGE as the two forms cannot be resolved by SDSPAGE. As we failed to identify an N-terminal Met in the protein obtained following SDS-PAGE, it can be concluded that the S. equi HPr protein is primarily in the HPr-1 Met-processed form . The absence of the N-terminal Met from HPr-1 has previously been linked to the surface localization of this HPr from in several streptococci (JENKINSON, 1989; SUTCLIFFE et aI., 1993; DUBREIL et aI., 1996) and the Met-processed HPr of S. pyogenes was originally identified as a mitogenic extracellular protein (GERLACH et aI., 1992). Although it remains possible that some Met-processed HPr may be associated with the cell surface in S. equi subsp. equi, the primary role of HPr in the PTS demands a cytoplasmic location for this phosphocarrier protein. The predominant presence of Met-processed HPr in cell extracts of this organism suggests that the cytoplasmic HPr of S. equi must also be in the HPr-1 form and consequently that the removal of the N-terminal Met does not dictate its localization. The significance of amino-terminal Met processing of streptococcal HPr proteins remains elusive and, in the absence of a signal sequence directing protein export, it remains to be determined how HPr may be translocated to the cell surface. In this context, it is notable that some streptococcal glycolytic enzymes are also surface localized by as yet undetermined mechanisms (PANCHOLI and FISCHETTI, 1998). Acknowledgement
We thank Dr. NEIL CHANTER (Animal Health Trust, Newmarket, UK) for supplying strains of S. equi and the convalescent equine serum; Mr. JOHN GILROY (Department of Biological Sciences, University of Durham, UK) for carrying out the protein sequencing and Dr. CHRISTIAN VADEBONCOEUR (Universite Laval, Quebec, Canada) for kindly suppying the polyclonal rabbit anti-HPr antiserum EL-63.
References ALTSCHUL, S. E, MADDEN, T. L., SCHAFFER, A. A., ZHANG, j., ZHANG, Z., MILLER, W. and LIPMAN, D. J.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programmes. Nucleic Acids Res. 25, 3389-3402 (1997). BOYD, D. A., CVITKOVITCH, D. G. and HAMILTON, I. R.: Sequence and expression of the genes for HPr (ptsH) and Enzyme I (pts) of the phosphoenolpyruvate-dependent phosphotransferase transport system from Streptococcus mutans. Infect. Immun. 62, 1156-1165 (1994). CHANTER, N.: Streptococci and enterococci as animal pathogens. J. App!. Microbio!. Symp. Supp!. 83, 100S-109S (1997). DUBREIL, J. D., JACQUES, M., BROCHU, D., FRENETTE, M. and VADEBONCOEUR, c.: Surface location of HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus suis. Microbiology 142, 837-843 (1996). GAGNON, G., VADEBONCOEUR, C. and FRENETTE, M.: Phosphotransferase system of Streptococcus salivarius: characterisa-
332
I. C. SUTCLIFFE, J. TRIGG and D. HARRINGTON
tion of the ptsH gene and its product. Gene 136, 27-34 (1993). GERLACH, D., ALOUT, H., MORAvEK, L., PAVLIK, M. and KOHLER, W.: The characterisation of two new low molecular weight proteins (LMPs) from Streptococcus pyogenes. Zbl. Bakt. 277,1-9 (1992). HAMILTON, A., HARRINGTON, D. and SUTCLIFFE, I. c.: Characterisation of acid phosphatase activities in the equine pathogen Streptococcus equi. Syst. Appl. Microbiol., in press (2000). JENKINSON, H.: Properties of a phosphocarrier protein (HPr) extracted from intact cells of Streptococcus sanguis. J. Gen. Microbiol. 135,3183-3197 (1989). PANCHOLI, V. and FISCHETTI, V. A.: a-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J. BioI. Chern. 273, 14503-14525 (1998). SUTCLIFFE, I. c., HOGG, S. D. and RUSSELL, R. R. B.: Identification of Streptococcus mutans antigen D as the HPr compo-
nent of the sugar-phosphotransferase transport system. FEMS Microbiol. Lett. 107,67-70 (1993). VADEBONCOEUR, c.: HPr: Heteromorphous Protein. Res. MicrobioI. 146,525-530 (1995). VADEBONCOEUR, c., KONISHI, Y., DUMAS, E, GAUTHIER, L. and FRENETTE, M.: HPr polymorphism in oral streptococci is caused by partial removal of the N-terminal methionine. Biochimie 73,1427-1430 (1991).
Corresponding author: lAIN SUTCLIFFE, Fleming Building, School of Sciences, The University of Sunderland, Sunderland SR2 3SD, UK Telephone: +44-191-515-2995; Fax: +44-191-515-3747; E-mail: [email protected]
© Urban & Fischer Verlag
Identification of Methionine-processed HPr in the Equine Pathogen Streptococcus equi lAIN C. SUTCLIFFE, JOANNE TRIGG, and DEAN HARRINGTON School of Sciences, University of Sunderland, UK Received July 5, 2000
Summary Using preparative electrophoresis, a low molecular weight protein has been partially purified from a cell extract of the equine pathogen Streptococcus equi susp. equi. N-terminal sequence analysis and Western blotting revealed the protein to be HPr, a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Interestingly, the only form of the HPr protein detected in S. equi was one with the amino-terminal methionine removed, a modification that has previously been associated with surface localization of streptococcal HPr proteins. Key words: HPr - Strangles - Streptococcus
Streptococcus equi subsp. equi is the causative agent of strangles, a highly contagious and debilitating respiratory disease of the horse (CHANTER, 1997). Although a disseminated and normally fatal form of the disease manifests itself in 1-5% of cases during an outbreak, the majority of horses mount an effective immune response and recover from acute disease. During our investigations of the virulence determinants of S. equi we have analysed low molecular weight antigens in cell extracts that crossreact in Western blotting experiments with a convalescent serum from a horse that had recovered from an episode of strangles. During these experiments we have partially purified and characterized by N-terminal sequence analysis a protein identified here as HPr, a central component of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system (VADEBONCOEUR, 1995). Briefly, cells harvested from a 500 mL Todd-Hewitt broth culture of S. equi strain 4047 were harvested by centrifugation and washed twice with phosphate-buffered saline. Cell extracts were obtained by boiling the cell pellet for 10 minutes in 1.2 mL sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer followed by removal of cell debris by centrifugation. The extract was then fractionated using a Biorad MiniPrep preparative electrophoresis system: the sample (700 J.lL) was electrophoresed through a 7.5 cm 15% tube gel and the fractionated proteins were collected by continuous elution with water. A total of 100 fractions (250 J.lL each) were obtained and every fifth fraction was analysed by Western blotting (HAMILTON et aI., 2000) using a 111000 dilution of horse convalescent 0723-2020/00/23/03-330 $ 15.00/0
serum as the primary antibody and mouse anti-horse IgG (1130,000; Sigma Chemical Co., Dorset, UK) as secondary antibody. Fractions 40-45, containing proteins in the 14-20 kDa range, were pooled and concentrated using Nanosep centrifugal concentrators (3000 kDa molecular weight cut-off; Flowgen Ltd, UK). The pooled proteins were subjected to SDS-PAGE and then blotted onto Immobilon-PSQ transfer membrane (Millipore, UK). Reversible protein staining (Biosafe Coomassie reagent, Biorad laboratories, UK) revealed two major protein bands in the 15-16 kDa range and samples of each were excised from the blot and subjected to N-terminal protein sequencing. The lower molecular weight band gave no distinguishable sequence as it appeared to be a mixture of at least three proteins. However, the upper ca. 16 kDa band yielded the unambiguous 15 amino acid N-terminal sequence ASKDFHlVAETGIHA. Database searches using the BLAST search tool (ALTSCHUL et aI., 1997; http://www.ncbi.nim.nih.gov/BLAST) revealed that this sequence is 100% identical to amino acids 2-16 of the HPr proteins of Streptococcus salivarius (GAGNON et aI., 1993), Streptococcus mutans (BOYD et aI., 1994) and Streptococcus bovis (ASANUMA, N. and HINo, T.: GenBank accession number AB027569, 1999). The sequence is also 100% identical to amino acids 1-15 of methionAbbreviations: HPr - a phosphotransfer protein of PTS, PTS phosphoenolpyruvate:sugar phosphotransferase system, SDSPAGE - sodium dodecyl sulphate polyacrylamide gel electrophoresis
Methionine-Processes HPr of Streptococcus equi
1
2
<1 10
Fig. 1. Western blot analysis of S. equi using polyclonal rabbit antiserum to HPr from S. salivarius. Lane 1. Cell extract of S. equi strain 4047. Lane 2. Pooled preparative electrophoresis fractions 40-45 containing proteins of ca. 16 kDa. Positions of the molecular weight standards (kDa) are indicated on the right.
ine-processed HPrs (see below) from Streptococcus pyogenes (GERLACH et aI., 1992), Streptococcus sanguis (JENKINSON, 1989) and Streptococcus suis (DUBREIL et aI., 1996). The high degree of homology reflects the fact that the N-termini of HPr proteins are highly conserved as the active site histidine involved in phosphorelay is located at His-15 (VADEBONCOEUR, 1995). Although streptococcal HPr proteins have molecular weights of ca. 9 kDa, several have been observed to migrate at 12-16 kDa on SDS-PAGE (JENKINSON, 1989; GERLACH et aI., 1992; SUTCLIFFE et aI., 1993). To confirm the presence of HPr in the samples analysed, the original cell extract and pooled fractions 40-45 were electrophoresed trough a 15% SDS-PAGE gel, Western blotted and probed with a 111000 dilution of a polyclonal rabbit serum against S. salivarius HPr. A band of ca. 16 kDa which showed strong cross-reaction with the antiHPr was observed in both samples (Figure 1). Cross-reaction with the same antibody was also observed in cell extracts of two other strains of S. equi subsp. equi and in four strains of Streptococcus equi subsp. zooepidemicus (data not shown). These findings constitute the first identification of HPr in S. equi. A putative protein sequence 92 % identical to 80/87 amino acids of the S. salivarius HPr can be identified in the unedited test assembly of the S. equi strain 4047 genome (http://www.sanger.ac.uk/ Projects/S_equil). At present the predicted N-terminus is missing from this putative protein but sequence identical to 5 amino acids (TGIHA) in the sequence determined herein is present. Further examination of the S. equi genome sequence suggests that a complete PTS system (Enzyme I; HPr; Ell membrane-associated proteins) is present in this organism (data not shown). Methionine (Met) processing of HPr has been identified previously as a cause of HPr polymorphism in S.
331
salivarius and other oral streptococci (VADEBONCOEUR et aI., 1991; VADEBONCOEUR, 1995). Electrophoretic resolution of HPr-1 (Met-removed) and HPr-2 (Met-intact) is achieved only by using non-denaturing Tris-buffered PAGE as the two forms cannot be resolved by SDSPAGE. As we failed to identify an N-terminal Met in the protein obtained following SDS-PAGE, it can be concluded that the S. equi HPr protein is primarily in the HPr-1 Met-processed form . The absence of the N-terminal Met from HPr-1 has previously been linked to the surface localization of this HPr from in several streptococci (JENKINSON, 1989; SUTCLIFFE et aI., 1993; DUBREIL et aI., 1996) and the Met-processed HPr of S. pyogenes was originally identified as a mitogenic extracellular protein (GERLACH et aI., 1992). Although it remains possible that some Met-processed HPr may be associated with the cell surface in S. equi subsp. equi, the primary role of HPr in the PTS demands a cytoplasmic location for this phosphocarrier protein. The predominant presence of Met-processed HPr in cell extracts of this organism suggests that the cytoplasmic HPr of S. equi must also be in the HPr-1 form and consequently that the removal of the N-terminal Met does not dictate its localization. The significance of amino-terminal Met processing of streptococcal HPr proteins remains elusive and, in the absence of a signal sequence directing protein export, it remains to be determined how HPr may be translocated to the cell surface. In this context, it is notable that some streptococcal glycolytic enzymes are also surface localized by as yet undetermined mechanisms (PANCHOLI and FISCHETTI, 1998). Acknowledgement
We thank Dr. NEIL CHANTER (Animal Health Trust, Newmarket, UK) for supplying strains of S. equi and the convalescent equine serum; Mr. JOHN GILROY (Department of Biological Sciences, University of Durham, UK) for carrying out the protein sequencing and Dr. CHRISTIAN VADEBONCOEUR (Universite Laval, Quebec, Canada) for kindly suppying the polyclonal rabbit anti-HPr antiserum EL-63.
References ALTSCHUL, S. E, MADDEN, T. L., SCHAFFER, A. A., ZHANG, j., ZHANG, Z., MILLER, W. and LIPMAN, D. J.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programmes. Nucleic Acids Res. 25, 3389-3402 (1997). BOYD, D. A., CVITKOVITCH, D. G. and HAMILTON, I. R.: Sequence and expression of the genes for HPr (ptsH) and Enzyme I (pts) of the phosphoenolpyruvate-dependent phosphotransferase transport system from Streptococcus mutans. Infect. Immun. 62, 1156-1165 (1994). CHANTER, N.: Streptococci and enterococci as animal pathogens. J. App!. Microbio!. Symp. Supp!. 83, 100S-109S (1997). DUBREIL, J. D., JACQUES, M., BROCHU, D., FRENETTE, M. and VADEBONCOEUR, c.: Surface location of HPr, a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus suis. Microbiology 142, 837-843 (1996). GAGNON, G., VADEBONCOEUR, C. and FRENETTE, M.: Phosphotransferase system of Streptococcus salivarius: characterisa-
332
I. C. SUTCLIFFE, J. TRIGG and D. HARRINGTON
tion of the ptsH gene and its product. Gene 136, 27-34 (1993). GERLACH, D., ALOUT, H., MORAvEK, L., PAVLIK, M. and KOHLER, W.: The characterisation of two new low molecular weight proteins (LMPs) from Streptococcus pyogenes. Zbl. Bakt. 277,1-9 (1992). HAMILTON, A., HARRINGTON, D. and SUTCLIFFE, I. c.: Characterisation of acid phosphatase activities in the equine pathogen Streptococcus equi. Syst. Appl. Microbiol., in press (2000). JENKINSON, H.: Properties of a phosphocarrier protein (HPr) extracted from intact cells of Streptococcus sanguis. J. Gen. Microbiol. 135,3183-3197 (1989). PANCHOLI, V. and FISCHETTI, V. A.: a-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J. BioI. Chern. 273, 14503-14525 (1998). SUTCLIFFE, I. c., HOGG, S. D. and RUSSELL, R. R. B.: Identification of Streptococcus mutans antigen D as the HPr compo-
nent of the sugar-phosphotransferase transport system. FEMS Microbiol. Lett. 107,67-70 (1993). VADEBONCOEUR, c.: HPr: Heteromorphous Protein. Res. MicrobioI. 146,525-530 (1995). VADEBONCOEUR, c., KONISHI, Y., DUMAS, E, GAUTHIER, L. and FRENETTE, M.: HPr polymorphism in oral streptococci is caused by partial removal of the N-terminal methionine. Biochimie 73,1427-1430 (1991).
Corresponding author: lAIN SUTCLIFFE, Fleming Building, School of Sciences, The University of Sunderland, Sunderland SR2 3SD, UK Telephone: +44-191-515-2995; Fax: +44-191-515-3747; E-mail: [email protected]