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

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. Forty-eight 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-γ) and interleukin 2 (IL-2), suggesting a Th1 type immune response.Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals; the former include gas gangrene, food poisoning, necrotizing enterocolitis of infants, and enteritis necroticans (28, 37, 45). C. perfringens is an obligately 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 C. perfringens to cause disease is associated with the production of a variety of extracellular toxins (13 different toxins have been reported so far). On the basis of differential production of toxins, the strains of C. perfringens can be divided into five types, A through E (35), of which type A and type C strains are implicated in human diseases while other types are of veterinary importance. Type A strains cause gas gangrene, the most destructive of all clostridial diseases, which is characterized by rapid destruction of tissue with production of gas (4, 42). 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 (27). Hundreds of thousands of soldiers died of gas gangrene as a result of battlefield injuries, and C. perfringens was widely recognized as the most important causal organism of the disease. Besides gas gangrene, type A strains also cause gastrointestinal diseases in humans (food poisoning, antibiotic-associated diarrhea, sporadic diarrhea, sudden infant death syndrome) and animals (diarrhea in foals and pigs, etc.). C. perfringens type C strains cause necrotic enteritis in humans and animals, in addition to enterotoxemia in sheep. Moreover, C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; therefore, efforts to develop strategies for detection and protection are warranted.Interest in a vaccine against gas gangrene has been intermittent; most efforts were made during World Wars I and II and were devoted to the therapeutic use of antisera. Such antisera, raised against toxoids of all five species of clostridia associated with gas gangrene, were shown to have benefits if they were given soon after trauma (20). Active immunization against the disease has received little attention until a few years ago (32, 43, 44). A number of clinical studies of other pathogenic bacteria, including Clostridium difficile, have highlighted the importance of nontoxin protein antigens in disease expression, especially in colonization by the bacterium (9, 12, 16, 26).Despite a sudden spurt of activity in the proteomic characterization of bacterial pathogens, for reasons unknown, clostridia have been largely ignored. Clostridium difficile is the only clostridial species whose proteome has been analyzed to some extent (34). Proteomic methodology has been used to elucidate proteins regulated by the VirR/VirS system in Clostridium perfringens (40).To invade, multiply in, and colonize host tissues, 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 for the understanding of microbial pathogenesis. The exoproteins of Gram-positive bacteria are likely to contain some of these key factors. The term “secretome” refers to and takes into account both the protein secretion systems and the secreted proteins; in monoderm bacteria (Gram-positive cell envelope architecture), these proteins can also be found in the membrane and/or cell wall. The proteins found in the extracellular milieu of Gram-positive bacteria are hence extracellular proteins, or exoproteins, which form the exoproteome; these exoproteins are not necessarily secreted by known secretion systems (15). This terminology is used for the subproteome described in this study.Further, the correct identification of C. perfringens pathovars is critical for epidemiological studies and for the development of effective preventive measures, including vaccination. It is not well understood why C. perfringens produces illnesses that differ so greatly in severity. Specific illnesses may reflect the particular type of tissue that high numbers of the organism are able to invade. Clearly, the presence of certain toxins is one explanation, but it falls short in many instances. For example, strains that cause food poisoning may differ from those that cause gas gangrene only by the presence of an enterotoxin gene in the former, yet food-poisoning strains have never been found to cause gas gangrene. Elucidation of the exoproteins of this bacterium is likely to reveal factors responsible for the host specificity of the different C. perfringens pathovars.The present investigation was carried out with the following objectives: (i) identification of dominant exoproteins from the C. perfringens type A strain ATCC 13124 (a gas gangrene isolate and the species type strain) and comparison with those of type C strains, (ii) elucidation of immunogenic components of the exoproteome, (iii) in silico analysis of the identified proteins with respect to localization, predicted function, and likely potential as surface markers for detection and as vaccine candidates, and (iv) validation of selected candidates with respect to vaccine potential in an experimental mouse model of gas gangrene. To the best of our knowledge, ours is the first proteomic elucidation of the C. perfringens exoproteome. The identification of extracellular proteins of C. perfringens type A and type C strains in the present investigation advances our understanding of the pathogenesis of the disease and opens significant opportunities for the identification of diagnostic markers and the development of vaccines against gas gangrene in humans.