Comparative Proteomic Analysis of Extracellular Proteins of Clostridium perfringens Type A and Type C Strains

Abstract

Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Fortyeight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-␥) and interleukin 2 (IL-2), suggesting a Th1 type immune response.

Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility

“…The C. perfringens SagA protein has a signal sequence compatible for secretion via the Sec pathway (not shown). The SagA homolog in C. perfringens strain ATCC 13124 was identified as the most abundant secreted protein, even more so than alpha toxin and collagenase, two wellstudied secreted toxins (24). This is consistent with our observation that even maximum expression from the P bgaL promoter on a multicopy plasmid did not fully complement the sagA mutants.…”

“…This is consistent with our observation that even maximum expression from the P bgaL promoter on a multicopy plasmid did not fully complement the sagA mutants. The C. perfringens SagA protein displayed three different isoforms in two-dimensional gel electrophoresis, which suggests posttranslational modification of the protein (24).…”

The MprB Extracytoplasmic Domain Negatively Regulates Activation of the Mycobacterium tuberculosis MprAB Two-Component System

“…M. tuberculosis MprB and its orthologs all possessed two transmembrane domains flanking a relatively large ECD of ϳ100 amino acids, a cytoplasmic HAMP (present in histidine kinases, adenylate cyclases, methyl-accepting proteins, and phosphatases) domain, and sensor kinase DHp (dimerization and histidine phosphotransfer) and CA (catalytic and ATP-binding) domains (data not shown). The DHp domain is necessary for SK function, since it contains residues conferring SK-RR specificity (43)(44)(45)(46)(47)(48), SK dimerization and autophosphorylation activity (49)(50)(51), and kinase and phosphatase activity (52). We rationalized that the DHp domain of MprB orthologs may therefore be the most conserved.…”

“…While DnaK was originally thought to be exclusively cytoplasmic, several studies have now reported the localization of DnaK outside the cell, including in Mycobacterium spp. (67)(68)(69)(70) and other Gram-positive bacteria (51,(71)(72)(73)(74). DnaK is a member of the well-conserved Hsp70 family of proteins found in all kingdoms of life.…”

Comparative analysis of extractable proteins from Clostridium perfringens type A and type C strains showing varying degree of virulence

“…Active immunization using inactive toxin moiety was demonstrated to yield positive results in animal model [7,8]. Importance of non-toxin protein antigens in disease expression, especially in colonization by the pathogenic bacteria, including Clostridium difficile, has been recently recognized [9,10]. Preparedness for bioterrorism or state sponsored warfare using biological agents requires development of effective pre-exposure prophylactic regimen as post-exposure therapies are plagued with antibiotic resistant strains.…”