Clostridium difficile is the leading cause of infectious diarrhea in hospitals worldwide, because of its virulence, spore-forming ability and persistence1,2. C. difficile-associated diseases (CDAD) are induced by antibiotic treatment or disruption of the normal gastrointestinal flora3,4. Recently, morbidity and mortality resulting from CDAD have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns1,2,5,6. Since 2002, epidemic toxinotype III NAP1/027 strains1,2, which produce high levels of the major virulence factors, toxin A and toxin B, have emerged. These toxins have 63% amino acid sequence similarity7 and are members of the large clostridial glucosylating toxin family, which are monoglucosyltransferases that are proinflammatory, cytotoxic and enterotoxic in the human colon8–10. Inside host cells, both toxins catalyze the transfer of glucose onto the Rho family of GTPases, leading to cell death8, 11. However, the role of these toxins in the context of a C. difficile infection is unknown. Here we describe the construction of isogenic tcdA and tcdB mutants of a virulent C. difficile strain and their use in the hamster disease model to show that toxin B is a key virulence determinant. Previous studies showed that purified toxin A alone can induce most of the pathology observed following infection of hamsters with C. difficile8,9, 12 and that toxin B is not toxic in animals unless it is co-administered with toxin A, suggesting that the toxins act synergistically12. Our work provides evidence that toxin B, not toxin A, is essential for virulence, which represents a major paradigm shift. Furthermore, it is clear that the importance of these toxins in the context of infection cannot be predicted exclusively from studies using purified toxins, reinforcing the importance of using the natural infection process to dissect the role of toxins in disease.
Despite the widely held belief that gastric acid serves as a barrier to bacterial pathogens, there are almost no experimental data to support this hypothesis. We have developed a mouse model to quantify the effectiveness of gastric acid in mediating resistance to infection with ingested bacteria. Mice that were constitutively hypochlorhydric due to a mutation in a gastric H ؉ /K ؉ -ATPase (proton pump) gene were infected with Yersinia enterocolitica, Salmonella enterica serovar Typhimurium, Citrobacter rodentium, or Clostridium perfringens cells or spores. Significantly greater numbers of Yersinia, Salmonella, and Citrobacter cells (P < 0.006) and Clostridium spores (P ؍ 0.02) survived in hypochlorhydric mice, resulting in reduced median infectious doses. Experiments involving intraperitoneal infection or infection of mice treated with antacids indicated that the increased sensitivity of hypochlorhydric mice to infection was entirely due to the absence of stomach acid. Apart from establishing the role of gastric acid in nonspecific immunity to ingested bacterial pathogens, our model provides an excellent system with which to investigate the effects of hypochlorhydria on susceptibility to infection and to evaluate the in vivo susceptibility to gastric acid of orally administered therapies, such as vaccines and probiotics.
The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alphatoxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence.Clostridium perfringens type A is the causative agent of human gas gangrene, or clostridial myonecrosis, and human food poisoning (25,27). It produces many secreted hydrolytic enzymes and toxins, including alpha-toxin and perfringolysin O. C. perfringens strains can also encode up to three sialidases, but the three sialidase genes, nanH, nanI, and nanJ, are not present in all of the strains that have been completely sequenced. Strain ATCC 13124 encodes all three sialidases (18), while strain 13 encodes both of the large sialidases, NanI and NanJ, but not the smaller NanH enzyme (32). The food poisoning isolate SM101 encodes NanH but not NanI or NanJ (18).Sialidases have been implicated in the virulence of several bacterial pathogens. They have been shown to enhance the pathogenesis of disease through synergistic effects with other bacterial factors. For example, Vibrio cholerae sialidase enhances the activity of cholera toxin (10), Pseudomonas aeruginosa sialidase increases the binding of this organism to the cells of susceptible patients (6), and the two sialidases of Streptococcus pneumoniae contribute to the progression of infection in several animal models (16,23,37). More recently, a surfaceexposed sialidase was shown to be required for persistence of the canine pathogen Capnophagia canimorsus (15). Alpha-toxin is an essential virulence factor in gas gangrene (2), and perfringolysin O, although not essential, has been found to have a synergistic role with alpha-toxin, enhancing the disease process (3). Synergy between alpha-toxin and the NanI sialidase was also observed in experiment...
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