BACKGROUND
clostridium perfringens is a medically important clostridial pathogen and an etiological agent, causing several diseases in humans and animals; the former includes gas gangrene, food poisoning, necrotizing enterocolitis of infants and enteritis necroticans  <cit> .
it is an obligate anaerobic rod-shaped bacterium commonly found in the gastrointestinal tracts of both animals and humans and widely distributed in soil and sewage.
the ability of clostridium perfringens to cause disease is associated with the production of a variety of extracellular toxins .
on the basis of differential production of toxins, the strains of c. perfringens can be divided into five types a through e  <cit> .
type a strains cause gas gangrene, the most destructive of all diseases, which is characterized by rapid destruction of tissue with production of gas.
the incidence of disease ranged from 1% or less of wounded personnel during world war ii to 10% of wounded personnel during world war i. hundreds of thousands of soldiers died of gas gangrene as a result of battlefield injuries, and c. perfringens was widely recognised as being the most important causal organism of the disease.
moreover, c. perfringens and its toxins have been listed as potential biological and toxin warfare  agents and warrants attention towards developing strategies pertaining to detection and protection.
interest in vaccine against gas gangrene has been intermittent with most effort during the world wars i and ii and devoted to the therapeutic use of antisera.
such antisera raised against toxoids of all of the five species of clostridia associated with gas gangrene were shown to have benefits if the serum was given soon after trauma  <cit> .
active immunization against the disease has received little attention until a few years back  <cit> .
many of the earlier studies used formaldehyde toxoids but due to inherent problems associated with these preparations, the subsequent studies employed genetic approaches.
immunization with isolated c-domain  of alpha toxin has shown both, high level circulating antibodies and protection of mice against as high as  <dig> ld <dig> dose of the toxin  <cit> .
apart from description of antibody responses to well known c. perfringens alpha toxin, responses to non-toxin antigens have been little explored.
a number of clinical studies in other pathogenic bacteria including c. difficile have highlighted the importance of non-toxin protein antigens in disease expression  <cit> .
for instance, c. difficile surface layer protein  has been shown to contain antigenic epitopes and play role in colonization of the bacterium to gastrointestinal tissues  <cit> .
complete genome sequences for three of its widely studied strains; c. perfringens strain  <dig>  c. perfringens atcc 13124t , and c. perfringens sm <dig>  have been recently determined and compared  <cit> .
several striking findings have emerged from the complete genome sequencing data of this clostridial pathogen.
comparisons of the three genomes have revealed considerable genomic diversity with > <dig> unique "genomic islands" identified and using pcr based analysis it was also demonstrated that the large genomic islands were widely variable across a large collection of c. perfringens strains  <cit> .
proteome maps of sequenced organism are important research tools for the authentication of hypothetical proteins, the identification of components of different cellular proteome fractions and for yielding information concerning the occurrence and abundance of proteins.
such proteome maps in the public domain have been generated for many pathogens and are of great value in identifying new virulence factors and the antigens of potential diagnostic and/or curative value against infections with pathogens.
despite a sudden spurt of activity towards proteomic characterization of bacterial pathogens, for reasons unknown, clostridia have largely been ignored.
clostridium difficile is the only clostridial species for which analysis of proteome has been carried out to some extent  <cit> .
to invade, multiply and colonize tissues of the host, a pathogen must be able to evade the host immune system, and obtain nutrients essential for growth.
the factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis.
growth of microorganisms in vitro, under conditions which mimic certain aspects of the host environment, such as temperature  <cit> , ph  <cit> , nutrient conditions, and interaction with host derived cells  <cit> , can provide valuable information on microbial pathogenesis.
proteome analysis is one of the best tools for understanding the basic biology of microorganisms including pathogenesis, physiology, and mechanisms of avoiding host immune system.
in this study we report identification of major surface and cell envelope proteins from clostridium perfringens atcc <dig> and those differentially expressed in cells grown on cooked meat medium  in comparison with cells grown in reference state tpyg  medium.
cooked meat medium  <cit>  provides substrate in the form of muscle tissue, for the myonecrotic cells of c. perfringens which produces phospholipase c as one of its major virulence factor.
most cases of gas gangrene are associated with an initial focus of infection which develops into a rapidly spreading disease with active invasion of surrounding connective or muscle tissue.
RESULTS
protein identification
a total of  <dig> dominant protein spots in three gels  were marked and analyzed after in gel digestion with trypsin using mldi-tof-ms and/or esi-ms/ms .
this included  <dig> surface associated proteins,  <dig> cell envelope proteins, and  <dig> cmm specific differentially expressed proteins.
the gels were analyzed quantitatively to determine the relative abundance of spots and also the fold difference of expression in cmm specific proteins.
since our protein identification was based on ion search at ncbi nonredundant database in the taxonomic group of bacteria  or firmicutes , chances of false positive hits are substantially reduced.
we estimated the mw and pi values of the protein spots on the 2-de gels and compared them with theoretical mw and pi values of corresponding proteins from c. perfringens atcc <dig>  most of the experimental values matched well with theoretical values, indicating unambiguous identification .
any discrepancies between experimental and theoretical masses might have been caused by post-translational proteolytic processing and modification.
the differences between the two pi values might be attributed to the cleavage of alkaline regions and phosphorylation of multiple residues.
cmm induced changes in total cellular protein profile
figures 1a and 1b show a portion of 2-de gels of total cellular protein from c. perfringens atcc <dig> cells, grown on tpyg and cmm, respectively.
the analytical and biological replicates  of the corresponding 2-de gels are shown in additional file  <dig> and  <dig>  growth on cmm resulted in over expression of several proteins of which  <dig> most prominent ones have been identified.
to identify the up-regulated proteins, the spots  were excised from the gel, digested with trypsin and subjected to ms/ms analysis as detailed in methods.
riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited  <dig>  to  <dig>  fold increase in the expression level in cells grown on cmm .
other over expressed proteins include butyryl-coa dehydrogenase, udp-glucose 4-epimerase, and electron transfer flavoprotein, showing almost qualitative change in expression  in cmm grown c. perfringens cells.
ornithine carbamoyltransferase   was the most abundant of the over-expressed proteins and has also been identified in the surface protein fraction of this bacterium  .
similarly, cystathionine beta-lyase  showing  <dig> -fold difference of expression in cmm-grown cells of c. perfringens was also observed as a dominant cell surface protein  of the bacterium.
curiously, almost all the proteins over-expressed in cmm grown cells were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism.
cell surface and envelope proteins
a total of  <dig> surface-localized proteins and  <dig> cell envelope proteins were identified by proteomic analysis of c. perfringens atcc <dig> .
for six of the surface proteins the identification was based on ms/ms analysis of the trypsin digested protein, in addition to sequencing of one or more peptides; the independent datasets resulted in same protein match in database search .
the identified homologs exhibited high amino acid sequence identity  with corresponding proteins from c. perfringens atcc <dig>  as revealed by blastp results.
the 2-de gel pattern and the identification data of the envelope proteins suggest that rubredoxin and atp synthase f <dig>  alpha and beta subunit existed as multiple electropherotypes .
rubredoxin/rubrerythrin  were the most abundant cell envelope associated proteins which is known to exist as multiple homologs in the c. perfringens atcc <dig> genome showing different pi values.
except for the spot mp <dig>  all the identified proteins were assigned to the cog functional category of energy production and conversion.
triosephosphate isomerase, phosphoglycerate kinase, glutamate synthase , cell wall-associated serine proteinase, and sucrose-6-phosphate dehydrogenase were the major components in the surface protein fraction of the c. perfringens strain .
charge variants of aminopeptidase, cystathionine beta-lyase, and translation elongation factor p were some other surface proteins identified.
when searched against cog database, most of the dominant surface proteins were predicted to be involved in amino acid transport and metabolism , carbohydrate transport and metabolism , and translation, ribosomal structure and biogenesis .
immunogenic proteins
choloylglycine hydrolase family protein , glutamate synthase , sucrose-6-phosphate dehydrogenase , and ornithine carbamoyltransferase  were found to be immunogenic cell surface proteins as revealed by the western blot analysis of 2-de separated surface proteins probed with mouse anti-cpwc serum .
we could not identify a few other immunogenic surface proteins visible on western blot.
c. perfringens atcc <dig> cells were grown on cmm and tpyg till late exponential phase and equal amount of whole cell lysate was separated on one dimensional sds-page.
western blot was generated using polyclonal serum from mice surviving gas gangrene infection ; highlighting proteins recognized by antibodies from c. perfringens infected mice.
remarkable differences were observed in the profile of immunogenic proteins, especially in the regions corresponding to molecular masses of 40– <dig> kda and 58– <dig> kda.
sequence analysis of identified proteins
based on blast search results, all the proteins identified in the present investigation appeared to be highly conserved  among c. perfringens strains and were not strain specific  .
most of the proteins  were also conserved among other clostridial members showing >70% amino acid sequence identity.
sucrose-6-phosphate dehydrogenase, threonine dehydratase, and n-acetylmuramoyl-l-alanine amidase exhibited 50–60% sequence identity while choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein shared only <50% identity with their closest homologs in bacterial domain.
all the identified proteins were analyzed using various bioinformatics software programs, such as signalp, secretomep, psort, lipop, tmhmm, and prosite for predicting protein secretion and localization.
for instance, n-acetylmuramoyl-l-alanine amidase and cell wall-associated serine proteinase obtained from cell surface fraction of strain atcc <dig> were predicted by signalp to be secreted in the classical sec pathway, which is characterized by the presence of a signal peptide  <cit>  .
both these proteins containing the signal peptides possessed cleavage site for signal peptidase  <dig> .
interestingly, cell wall-associated serine proteinase was also predicted; to harbor two transmembrane helices , suggesting an extracytoplasmic but cell-associated location; contain an lpxtg motif  for cell wall anchorage; and a cell wall associated localization .
psort algorithm predicted most of the proteins  to have cytoplasmic localization.
choloylglycine hydrolase family protein, cell wall-associated serine proteinase, rubredoxin/rubrerythrin, and rhomboid family protein were predicted by secretomep to be secreted by non-classical sec pathway characterized by the lack of typical export signals  <cit> .
discussion
cooked meat medium was developed by robertson  <cit>  in  <dig> for use in the cultivation of certain anaerobes isolated from wounds.
the present formulation for cmm is a modification of robertson's original formula.
cooked meat medium is still widely used for the cultivation and maintenance of clostridia and the medium is recommended for use in the enumeration and identification of clostridium perfringens from food  <cit> .
cooked meat medium provides a favorable environment for the growth of c. perfringens, since the muscle protein in the heart tissue granules is a source of amino acids and other nutrients.
the muscle tissue also provides reducing substances, particularly glutathione, which permits the growth of strict anaerobes  <cit> .
the combination of 2-de and ms has clearly identified major proteins over-expressed in cells of c. perfringens atcc <dig> when grown on cmm.
we have identified eleven prominent proteins showing over expression cmm grown whole cell proteome of c. perfringens atc <dig> cells .
for a bacterial protein to be considered as a candidate vaccine antigen, it should preferably be conserved , secreted or surface localized, and immunogenic .
ornithine carbamoyltransferase  was an abundant protein up-regulated in cmm-grown cells.
it was also identified as an immunogenic surface protein of this bacterium  .
in another study, ornithine carbamoyltransferase has been isolated as putative adhesin from surface molecule preparation of staphylococcus epidermidis  <cit> .
cotc is a bonafied cell wall protein of streptococcus agalactiae  <cit> , s. pyogenes  <cit> , s. sanguis  <cit> , and s. suis  <cit> .
taken together, this makes cotc a putative vaccine candidate against c. perfringens infection.
similarly, cystathionine beta-lyase  that was over-expressed in cmm-grown cells of c. perfringens, has been previously shown as a dominant cell surface protein of the bacterium, indicating a possible role of this protein in pathogenesis and a potential as putative vaccine candidate.
electron transfer flavoprotein, over-expressed in cmm grown cells has been recognized in earlier studies as cross reactive protein of c. tetani when probed with mouse anti c. perfringens  polyclonal serum  <cit>  and also as an extracellular protein in bacillus anthracis  <cit>  and mycobacterium tuberculosis  <cit> .
antibodies from animals surviving gas gangrene infection recognized proteins from both tpyg and cmm grown cells of c. perfringens atcc <dig> , though remarkable differences were observed in the profile of immunogenic proteins, especially in the regions corresponding to molecular masses of 40– <dig> kda and 58– <dig> kda.
curiously, six proteins in the molecular mass range of 40– <dig> kda have also been shown to be over-expressed in c. perfringens atcc <dig> cells when grown on cmm, using 2-de profiling of whole cell proteins.
these proteins varied in their observed pi values from  <dig>  –  <dig>  and are likely to migrate closely on a one dimensional sds-page.
the results indicate that with reference to tpyg grown cells, some additional proteins expressed in vivo  are also expressed when c. perfringens atcc <dig> cells are grown on cmm.
based on the results obtained in the present investigation, it will be highly speculative to suggest that cmm provides host simulated conditions for c. perfringens.
in a pre-gangrenous infection, c. perfringens cells encounter live muscle and immune cells that will be responding and fighting to kill the bacterium.
by comparison, cooked meat media  is processed, granulated and boiled muscle tissue.
further work using proteome from cells obtained from infected host and those from cmm and tpyg grown cells may provide further clue in this direction.
most of the cell envelope and up-regulated proteins existed as multiple isoelectropherotypes and often differences in their observed and theoretical pi values were more pronounced, compared to those observed for molecular masses .
we cannot exclude a possibility that there are major post translational events in these proteins resulting in pi value differences.
nevertheless, earlier observations have indicated that different isoelectropherotypes of polypeptides in 2-de gels do not always arise from true post translational modifications, but also from the 2-de procedure itself  <cit> .
the outer surface of bacteria is of great importance to the understanding of bacterial pathogenesis.
elements of the surface are implicated in bacterial defense mechanisms and virulence related functions e.g.
adhesion, invasion, direct injury, and induction of septic shock.
there is no information available with respect to surface proteins of this medically important bacterium.
in the present study, several of the surface proteins and those over-expressed in cmm grown cells were largely assigned putative function in amino acid transport and metabolism , suggesting that this organism is adapted to protein rich environment of host tissue.
together, these identified and predicted proteins could be useful targets for the development of improved vaccines against gangrenous infections.
two of the surface proteins of c. perfringens, ornithine carbamoyltransferase and phosphoglycerate kinase have also been identified as immunogenic proteins in the outer surface protein preparation of s. agalactiae and s. pyogenes  <cit> .
curiously, sera directed against the two proteins were shown to protect neonatal animals from s. agalactieae infection in a passive immunization experiment.
in earlier studies, phosphoglycerate kinase was reported on the surface of s. pneumoniae, was antigenic in humans, and elicited protective immune responses in mouse model  <cit>  .
also in schistosoma mansoni, phosphoglycerate kinase has been identified as a protective antigen  <cit> .
another surface protein, ef-g, identified in this study was found to be immuno-reactive against sera from broiler chicken immune to necrotic entritis  <cit> .
the protein was secreted into the culture supernatant and unique to virulent c. perfringens strain cp <dig> causing necrotic entritis.
notably, ef-g is regulated by the virr-virs virulence regulon of c. perfringens  <cit> .
moreover, ef-g has been demonstrated as an immunogenic protein and was identified in both cell surface and extracellular fraction of b. anthracis  <cit> .
further, choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein can be excellent surface protein markers for specific detection of c. perfringens from environment and food as they share very low percent amino acid sequence identity with there nearest homologs  and are conserved among the c. perfringens strains .
some of the surface proteins from c. perfringens atcc <dig> showed metabolic functions that would typically place them in the cytoplasm.
moreover, except for n-acetylmuramoyl-l-alanine amidase and cell wall-associated serine proteinase, these proteins have no n-terminal signal peptide and do not possess the canonical gram-positive anchor motif lpxtg .
several surface-associated cytoplasmic proteins reported in this study were also detected on the bacterial surface in previous proteomic analysis .
for example, phosphoglycerate kinase was reported on the surface of s. pneumoniae  <cit> , s. agalactiae  <cit> , s. pyogenes  <cit> , and s. oralis  and also as secreted protein in b. anthracis  <cit> .
increasing number of reports have shown presence of proteins on the surface of gram positive bacteria or secreted into the medium that one would otherwise expect to be cytoplasmic  <cit> .
in a previous study, the culture supernatant of c. perfringens at the late exponential growth phase was shown to contain intracellular proteins that had no putative signal sequences, such as ribokinase, β-hydroxybutyryl-coenzyme a dehydrogenase, fructosebisphosphate aldolase, and elongation factor g  <cit> .
in other studies also, a significant number of cytoplasmic proteins have been identified as cell-wall associated proteins/immunogens  <cit> .
in spite of a growing list of cytoplasmic proteins identified on the bacterial surface, the mechanism of their surface localization and attachment to the bacterial envelope remain unclear.
internal signal sequences, posttranslational acylation, or an association with a secreted protein are hypothesized as possible means  <cit> .
the mapping of proteins to the bacterial surface may well suggest alternate functions for proteins with other well established roles.
for instance, the glycolytic enzyme α-enolase has been shown as plasmin-binding protein on the outside of the bacterial cells  <cit> .
for most of the cell envelope proteins identified here, a surface localization cannot be ruled out as not all of the proteins from the cell surface fraction could be identified.
the translation elongation factor tu  has been shown to be surface associated protein in s. pyogenes  <cit>  and other gram-positive bacteria  <cit> .
little is known about the possible functions of surface-associated elongation factors on the bacterial surface.
nevertheless, elongation factor of lactococcus johnsonii is shown to be involved in attachment of this pathogen to human intestinal cells and mucins  <cit> , while the same protein in mycobacterium pneumoniae binds fibronectin, which mediates the attachment of pathogen to host cells  <cit> .
it has also been reported as immunogenic spore protein of bacillus anthracis  <cit>  and a virulence determinant in coxiella burnetii  <cit> .
CONCLUSIONS
eleven prominent proteins showing over expression on cmm grown cells using whole cell proteome of c. perfringens atc <dig> have been identified by 2-de ms approach.
in addition the predominant cell surface and cell envelope  proteins were also identified and a few were found to be common with those observed as over-expressed in cmm grown cells.
cystathionine beta-lyase and ornithine carbamoyltransferase identified in this study can be putative vaccine candidates as they are over-expressed in cmm grown cells, are surface localized, the latter is immunogenic, and their homologs in other pathogenic bacteria have been shown to be immunogenic/virulence factor.
in addition phosphoglycerate kinase, n-acetylmuramoyl-l-alanine amidase, and translation elongation factor tu and ef-g can also be putative vaccine candidates as they are abundant on the cell surface fraction and their homologs in other gram positive pathogenic bacteria have been shown to be immunogenic/virulence determinants.
we propose choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein as potential surface protein markers for specific detection of c. perfringens from environment and food.

 clostridium perfringens is a medically important clostridial pathogen and an etiological agent, causing several diseases in humans and animals; the former includes gas gangrene, food poisoning, necrotizing enterocolitis of infants and enteritis necroticans. it is an obligate anaerobic rod-shaped bacterium commonly found in the gastrointestinal tracts of both animals and humans and widely distributed in soil and sewage. the ability of clostridium perfringens to cause disease is associated with the production of a variety of extracellular toxins. on the basis of differential production of toxins, the strains of c. perfringens can be divided into five types a through e. type a strains cause gas gangrene, the most destructive of all diseases, which is characterized by rapid destruction of tissue with production of gas. the incidence of disease ranged from 1% or less of wounded personnel during world war ii to 10% of wounded personnel during world war i. hundreds of thousands of soldiers died of gas gangrene as a result of battlefield injuries, and c. perfringens was widely recognised as being the most important causal organism of the disease. moreover, c. perfringens and its toxins have been listed as potential biological and toxin warfare agents and warrants attention towards developing strategies pertaining to detection and protection. interest in vaccine against gas gangrene has been intermittent with most effort during the world wars i and ii and devoted to the therapeutic use of antisera. such antisera raised against toxoids of all of the five species of clostridia associated with gas gangrene were shown to have benefits if the serum was given soon after trauma. active immunization against the disease has received little attention until a few years back. many of the earlier studies used formaldehyde toxoids but due to inherent problems associated with these preparations, the subsequent studies employed genetic approaches. immunization with isolated c-domain of alpha toxin has shown both, high level circulating antibodies and protection of mice against as high as ld dose of the toxin. apart from description of antibody responses to well known c. perfringens alpha toxin, responses to non-toxin antigens have been little explored. a number of clinical studies in other pathogenic bacteria including c. difficile have highlighted the importance of non-toxin protein antigens in disease expression. for instance, c. difficile surface layer protein has been shown to contain antigenic epitopes and play role in colonization of the bacterium to gastrointestinal tissues. complete genome sequences for three of its widely studied strains; c. perfringens strain c. perfringens atcc 13124t, and c. perfringens sm have been recently determined and compared. several striking findings have emerged from the complete genome sequencing data of this clostridial pathogen. comparisons of the three genomes have revealed considerable genomic diversity with > unique "genomic islands" identified and using pcr based analysis it was also demonstrated that the large genomic islands were widely variable across a large collection of c. perfringens strains. proteome maps of sequenced organism are important research tools for the authentication of hypothetical proteins, the identification of components of different cellular proteome fractions and for yielding information concerning the occurrence and abundance of proteins. such proteome maps in the public domain have been generated for many pathogens and are of great value in identifying new virulence factors and the antigens of potential diagnostic and/or curative value against infections with pathogens. despite a sudden spurt of activity towards proteomic characterization of bacterial pathogens, for reasons unknown, clostridia have largely been ignored. clostridium difficile is the only clostridial species for which analysis of proteome has been carried out to some extent. to invade, multiply and colonize tissues of the host, a pathogen must be able to evade the host immune system, and obtain nutrients essential for growth. the factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. growth of microorganisms in vitro, under conditions which mimic certain aspects of the host environment, such as temperature, ph, nutrient conditions, and interaction with host derived cells, can provide valuable information on microbial pathogenesis. proteome analysis is one of the best tools for understanding the basic biology of microorganisms including pathogenesis, physiology, and mechanisms of avoiding host immune system. in this study we report identification of major surface and cell envelope proteins from clostridium perfringens atcc and those differentially expressed in cells grown on cooked meat medium in comparison with cells grown in reference state tpyg medium. cooked meat medium provides substrate in the form of muscle tissue, for the myonecrotic cells of c. perfringens which produces phospholipase c as one of its major virulence factor. most cases of gas gangrene are associated with an initial focus of infection which develops into a rapidly spreading disease with active invasion of surrounding connective or muscle tissue.  protein identification a total of dominant protein spots in three gels were marked and analyzed after in gel digestion with trypsin using mldi-tof-ms and/or esi-ms/ms. this included surface associated proteins, cell envelope proteins, and cmm specific differentially expressed proteins. the gels were analyzed quantitatively to determine the relative abundance of spots and also the fold difference of expression in cmm specific proteins. since our protein identification was based on ion search at ncbi nonredundant database in the taxonomic group of bacteria or firmicutes, chances of false positive hits are substantially reduced. we estimated the mw and pi values of the protein spots on the 2-de gels and compared them with theoretical mw and pi values of corresponding proteins from c. perfringens atcc most of the experimental values matched well with theoretical values, indicating unambiguous identification. any discrepancies between experimental and theoretical masses might have been caused by post-translational proteolytic processing and modification. the differences between the two pi values might be attributed to the cleavage of alkaline regions and phosphorylation of multiple residues. cmm induced changes in total cellular protein profile figures 1a and 1b show a portion of 2-de gels of total cellular protein from c. perfringens atcc cells, grown on tpyg and cmm, respectively. the analytical and biological replicates of the corresponding 2-de gels are shown in additional file and growth on cmm resulted in over expression of several proteins of which most prominent ones have been identified. to identify the up-regulated proteins, the spots were excised from the gel, digested with trypsin and subjected to ms/ms analysis as detailed in methods. riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited to fold increase in the expression level in cells grown on cmm. other over expressed proteins include butyryl-coa dehydrogenase, udp-glucose 4-epimerase, and electron transfer flavoprotein, showing almost qualitative change in expression in cmm grown c. perfringens cells. ornithine carbamoyltransferase was the most abundant of the over-expressed proteins and has also been identified in the surface protein fraction of this bacterium. similarly, cystathionine beta-lyase showing -fold difference of expression in cmm-grown cells of c. perfringens was also observed as a dominant cell surface protein of the bacterium. curiously, almost all the proteins over-expressed in cmm grown cells were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism. cell surface and envelope proteins a total of surface-localized proteins and cell envelope proteins were identified by proteomic analysis of c. perfringens atcc. for six of the surface proteins the identification was based on ms/ms analysis of the trypsin digested protein, in addition to sequencing of one or more peptides; the independent datasets resulted in same protein match in database search. the identified homologs exhibited high amino acid sequence identity with corresponding proteins from c. perfringens atcc as revealed by blastp results. the 2-de gel pattern and the identification data of the envelope proteins suggest that rubredoxin and atp synthase f alpha and beta subunit existed as multiple electropherotypes. rubredoxin/rubrerythrin were the most abundant cell envelope associated proteins which is known to exist as multiple homologs in the c. perfringens atcc genome showing different pi values. except for the spot mp all the identified proteins were assigned to the cog functional category of energy production and conversion. triosephosphate isomerase, phosphoglycerate kinase, glutamate synthase, cell wall-associated serine proteinase, and sucrose-6-phosphate dehydrogenase were the major components in the surface protein fraction of the c. perfringens strain. charge variants of aminopeptidase, cystathionine beta-lyase, and translation elongation factor p were some other surface proteins identified. when searched against cog database, most of the dominant surface proteins were predicted to be involved in amino acid transport and metabolism, carbohydrate transport and metabolism, and translation, ribosomal structure and biogenesis. immunogenic proteins choloylglycine hydrolase family protein, glutamate synthase, sucrose-6-phosphate dehydrogenase, and ornithine carbamoyltransferase were found to be immunogenic cell surface proteins as revealed by the western blot analysis of 2-de separated surface proteins probed with mouse anti-cpwc serum. we could not identify a few other immunogenic surface proteins visible on western blot. c. perfringens atcc cells were grown on cmm and tpyg till late exponential phase and equal amount of whole cell lysate was separated on one dimensional sds-page. western blot was generated using polyclonal serum from mice surviving gas gangrene infection; highlighting proteins recognized by antibodies from c. perfringens infected mice. remarkable differences were observed in the profile of immunogenic proteins, especially in the regions corresponding to molecular masses of 40– kda and 58– kda. sequence analysis of identified proteins based on blast search results, all the proteins identified in the present investigation appeared to be highly conserved among c. perfringens strains and were not strain specific. most of the proteins were also conserved among other clostridial members showing >70% amino acid sequence identity. sucrose-6-phosphate dehydrogenase, threonine dehydratase, and n-acetylmuramoyl-l-alanine amidase exhibited 50–60% sequence identity while choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein shared only <50% identity with their closest homologs in bacterial domain. all the identified proteins were analyzed using various bioinformatics software programs, such as signalp, secretomep, psort, lipop, tmhmm, and prosite for predicting protein secretion and localization. for instance, n-acetylmuramoyl-l-alanine amidase and cell wall-associated serine proteinase obtained from cell surface fraction of strain atcc were predicted by signalp to be secreted in the classical sec pathway, which is characterized by the presence of a signal peptide. both these proteins containing the signal peptides possessed cleavage site for signal peptidase. interestingly, cell wall-associated serine proteinase was also predicted; to harbor two transmembrane helices, suggesting an extracytoplasmic but cell-associated location; contain an lpxtg motif for cell wall anchorage; and a cell wall associated localization. psort algorithm predicted most of the proteins to have cytoplasmic localization. choloylglycine hydrolase family protein, cell wall-associated serine proteinase, rubredoxin/rubrerythrin, and rhomboid family protein were predicted by secretomep to be secreted by non-classical sec pathway characterized by the lack of typical export signals. discussion cooked meat medium was developed by robertson in for use in the cultivation of certain anaerobes isolated from wounds. the present formulation for cmm is a modification of robertson's original formula. cooked meat medium is still widely used for the cultivation and maintenance of clostridia and the medium is recommended for use in the enumeration and identification of clostridium perfringens from food. cooked meat medium provides a favorable environment for the growth of c. perfringens, since the muscle protein in the heart tissue granules is a source of amino acids and other nutrients. the muscle tissue also provides reducing substances, particularly glutathione, which permits the growth of strict anaerobes. the combination of 2-de and ms has clearly identified major proteins over-expressed in cells of c. perfringens atcc when grown on cmm. we have identified eleven prominent proteins showing over expression cmm grown whole cell proteome of c. perfringens atc cells. for a bacterial protein to be considered as a candidate vaccine antigen, it should preferably be conserved, secreted or surface localized, and immunogenic. ornithine carbamoyltransferase was an abundant protein up-regulated in cmm-grown cells. it was also identified as an immunogenic surface protein of this bacterium. in another study, ornithine carbamoyltransferase has been isolated as putative adhesin from surface molecule preparation of staphylococcus epidermidis. cotc is a bonafied cell wall protein of streptococcus agalactiae, s. pyogenes, s. sanguis, and s. suis. taken together, this makes cotc a putative vaccine candidate against c. perfringens infection. similarly, cystathionine beta-lyase that was over-expressed in cmm-grown cells of c. perfringens, has been previously shown as a dominant cell surface protein of the bacterium, indicating a possible role of this protein in pathogenesis and a potential as putative vaccine candidate. electron transfer flavoprotein, over-expressed in cmm grown cells has been recognized in earlier studies as cross reactive protein of c. tetani when probed with mouse anti c. perfringens polyclonal serum and also as an extracellular protein in bacillus anthracis and mycobacterium tuberculosis. antibodies from animals surviving gas gangrene infection recognized proteins from both tpyg and cmm grown cells of c. perfringens atcc, though remarkable differences were observed in the profile of immunogenic proteins, especially in the regions corresponding to molecular masses of 40– kda and 58– kda. curiously, six proteins in the molecular mass range of 40– kda have also been shown to be over-expressed in c. perfringens atcc cells when grown on cmm, using 2-de profiling of whole cell proteins. these proteins varied in their observed pi values from – and are likely to migrate closely on a one dimensional sds-page. the results indicate that with reference to tpyg grown cells, some additional proteins expressed in vivo are also expressed when c. perfringens atcc cells are grown on cmm. based on the results obtained in the present investigation, it will be highly speculative to suggest that cmm provides host simulated conditions for c. perfringens. in a pre-gangrenous infection, c. perfringens cells encounter live muscle and immune cells that will be responding and fighting to kill the bacterium. by comparison, cooked meat media is processed, granulated and boiled muscle tissue. further work using proteome from cells obtained from infected host and those from cmm and tpyg grown cells may provide further clue in this direction. most of the cell envelope and up-regulated proteins existed as multiple isoelectropherotypes and often differences in their observed and theoretical pi values were more pronounced, compared to those observed for molecular masses. we cannot exclude a possibility that there are major post translational events in these proteins resulting in pi value differences. nevertheless, earlier observations have indicated that different isoelectropherotypes of polypeptides in 2-de gels do not always arise from true post translational modifications, but also from the 2-de procedure itself. the outer surface of bacteria is of great importance to the understanding of bacterial pathogenesis. elements of the surface are implicated in bacterial defense mechanisms and virulence related functions e.g. adhesion, invasion, direct injury, and induction of septic shock. there is no information available with respect to surface proteins of this medically important bacterium. in the present study, several of the surface proteins and those over-expressed in cmm grown cells were largely assigned putative function in amino acid transport and metabolism, suggesting that this organism is adapted to protein rich environment of host tissue. together, these identified and predicted proteins could be useful targets for the development of improved vaccines against gangrenous infections. two of the surface proteins of c. perfringens, ornithine carbamoyltransferase and phosphoglycerate kinase have also been identified as immunogenic proteins in the outer surface protein preparation of s. agalactiae and s. pyogenes. curiously, sera directed against the two proteins were shown to protect neonatal animals from s. agalactieae infection in a passive immunization experiment. in earlier studies, phosphoglycerate kinase was reported on the surface of s. pneumoniae, was antigenic in humans, and elicited protective immune responses in mouse model. also in schistosoma mansoni, phosphoglycerate kinase has been identified as a protective antigen. another surface protein, ef-g, identified in this study was found to be immuno-reactive against sera from broiler chicken immune to necrotic entritis. the protein was secreted into the culture supernatant and unique to virulent c. perfringens strain cp causing necrotic entritis. notably, ef-g is regulated by the virr-virs virulence regulon of c. perfringens. moreover, ef-g has been demonstrated as an immunogenic protein and was identified in both cell surface and extracellular fraction of b. anthracis. further, choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein can be excellent surface protein markers for specific detection of c. perfringens from environment and food as they share very low percent amino acid sequence identity with there nearest homologs and are conserved among the c. perfringens strains. some of the surface proteins from c. perfringens atcc showed metabolic functions that would typically place them in the cytoplasm. moreover, except for n-acetylmuramoyl-l-alanine amidase and cell wall-associated serine proteinase, these proteins have no n-terminal signal peptide and do not possess the canonical gram-positive anchor motif lpxtg. several surface-associated cytoplasmic proteins reported in this study were also detected on the bacterial surface in previous proteomic analysis. for example, phosphoglycerate kinase was reported on the surface of s. pneumoniae, s. agalactiae, s. pyogenes, and s. oralis and also as secreted protein in b. anthracis. increasing number of reports have shown presence of proteins on the surface of gram positive bacteria or secreted into the medium that one would otherwise expect to be cytoplasmic. in a previous study, the culture supernatant of c. perfringens at the late exponential growth phase was shown to contain intracellular proteins that had no putative signal sequences, such as ribokinase, β-hydroxybutyryl-coenzyme a dehydrogenase, fructosebisphosphate aldolase, and elongation factor g. in other studies also, a significant number of cytoplasmic proteins have been identified as cell-wall associated proteins/immunogens. in spite of a growing list of cytoplasmic proteins identified on the bacterial surface, the mechanism of their surface localization and attachment to the bacterial envelope remain unclear. internal signal sequences, posttranslational acylation, or an association with a secreted protein are hypothesized as possible means. the mapping of proteins to the bacterial surface may well suggest alternate functions for proteins with other well established roles. for instance, the glycolytic enzyme α-enolase has been shown as plasmin-binding protein on the outside of the bacterial cells. for most of the cell envelope proteins identified here, a surface localization cannot be ruled out as not all of the proteins from the cell surface fraction could be identified. the translation elongation factor tu has been shown to be surface associated protein in s. pyogenes and other gram-positive bacteria. little is known about the possible functions of surface-associated elongation factors on the bacterial surface. nevertheless, elongation factor of lactococcus johnsonii is shown to be involved in attachment of this pathogen to human intestinal cells and mucins, while the same protein in mycobacterium pneumoniae binds fibronectin, which mediates the attachment of pathogen to host cells. it has also been reported as immunogenic spore protein of bacillus anthracis and a virulence determinant in coxiella burnetii.  eleven prominent proteins showing over expression on cmm grown cells using whole cell proteome of c. perfringens atc have been identified by 2-de ms approach. in addition the predominant cell surface and cell envelope proteins were also identified and a few were found to be common with those observed as over-expressed in cmm grown cells. cystathionine beta-lyase and ornithine carbamoyltransferase identified in this study can be putative vaccine candidates as they are over-expressed in cmm grown cells, are surface localized, the latter is immunogenic, and their homologs in other pathogenic bacteria have been shown to be immunogenic/virulence factor. in addition phosphoglycerate kinase, n-acetylmuramoyl-l-alanine amidase, and translation elongation factor tu and ef-g can also be putative vaccine candidates as they are abundant on the cell surface fraction and their homologs in other gram positive pathogenic bacteria have been shown to be immunogenic/virulence determinants. we propose choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein as potential surface protein markers for specific detection of c. perfringens from environment and food. 
most of the proteins over-expressed in cmm were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism. many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized. the factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. based upon bioinformatic analysis; choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein were predicted as surface protein markers for specific detection of c. perfringens from the environment and food. riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited to fold increase in the expression level in cells growing on cmm. ornithine carbamoyltransferase and cystathionine beta-lyase were over-expressed in cells grown on cooked meat medium and also identified in the surface protein fraction and the former was immunogenic; making them potential vaccine candidates. clostridium perfringens is a medically important clostridial pathogen causing diseases in man and animals. using 2-de mass spectrometry strategy, we identified major surface and cell envelope proteins from clostridium perfringens atcc and those differentially expressed in cells grown on cooked meat medium in comparison with cells grown in reference state. to invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth.
 clostridium perfringens is a medically important clostridial pathogen causing diseases in man and animals. to invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth. the factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized.  using 2-de mass spectrometry strategy, we identified major surface and cell envelope proteins from clostridium perfringens atcc and those differentially expressed in cells grown on cooked meat medium in comparison with cells grown in reference state. riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited to fold increase in the expression level in cells growing on cmm.  ornithine carbamoyltransferase and cystathionine beta-lyase were over-expressed in cells grown on cooked meat medium and also identified in the surface protein fraction and the former was immunogenic; making them potential vaccine candidates. based upon bioinformatic analysis; choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein were predicted as surface protein markers for specific detection of c. perfringens from the environment and food. most of the proteins over-expressed in cmm were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism. 