Resistance ofCapnocytophaga canimorsusto Killing by Human Complement and Polymorphonuclear Leukocytes

Abstract

Capnocytophaga canimorsus is a bacterium of the canine oral flora known since 1976 to cause rare but severe septicemia and peripheral gangrene in patients that have been in contact with a dog. It was recently shown that these bacteria do not elicit an inflammatory response (H. Shin, M. Mally, M. Kuhn, C. Paroz, and G. R. Cornelis, J. Infect. Dis. 195:375-386, 2007). Here, we analyze their sensitivity to the innate immune system. Bacteria from the archetype strain Cc5 were highly resistant to killing by complement. There was little membrane attack complex (MAC) deposition in spite of C3b deposition. Cc5 bacteria were as resistant to phagocytosis by human polymorphonuclear leukocytes (PMNs) as Yersinia enterocolitica MRS40, endowed with an antiphagocytic type III secretion system. We isolated Y1C12, a transposon mutant that is hypersensitive to killing by complement via the antibody-dependent classical pathway. The mutation inactivated a putative glycosyltransferase gene, suggesting that the Y1C12 mutant was affected at the level of a capsular polysaccharide or lipopolysaccharide (LPS) structure. Cc5 appeared to have several polysaccharidic structures, one being altered in Y1C12. The structure missing in Y1C12 could be purified by classical LPS purification procedures and labeled by tritiated palmitate, indicating that it is more likely to be an LPS structure than a capsule. Y1C12 bacteria were also more sensitive to phagocytosis by PMNs than wild-type bacteria. In conclusion, a polysaccharide structure, likely an LPS, protects C. canimorsus from deposition of the complement MAC and from efficient phagocytosis by PMNs.

NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus

“…Compositional analysis by GLC-MS of both core oligosaccharide fractions resulted in identical sugar residues being present in comparable proportions. As expected, the core oligosaccharide fraction of the mutant did not contain contaminating amylopectin (21) and sugars from the O-chain (9). Thus, the Y1C12 mutant was found to be representative for further structural analysis of the complete rough-type C. canimorsus LPS (3).…”

“…canimorsus 5 (Cc5) (3) and its complement-sensitive Y1C12 mutant (9) were grown on heart infusion agar 5% sheep blood supplemented with gentamicin and, in the case of Y1C12, with erythromycin. The Cc5-based Y1C12 mutant has a transposon insertion within a predicted glycosyltranferase-encoding gene, probably the N-acetylfucosamine transferase WbuB, necessary for the formation of the O-antigen (9). Bacteria were harvested by scraping off colonies from the agar surface of 500 plates, washed, and resuspended in PBS.…”

Molecular Characterization of Capnocytophaga canimorsus and Other Canine Capnocytophaga spp. and Assessment by PCR of Their Frequencies in Dogs

“…It was shown that human macrophages incubated with C. canimorsus failed to produce tumor necrosis factor alpha, interleukin-1␣ (IL-1␣), IL-6, Il-8, and gamma interferon; that TLR-4 could not be activated by the reference strain; and that even tumor necrosis factor alpha release by another pathogen, Yersinia enterocolitica, was impaired by live C. canimorsus (21). The resistance to phagocytosis of C. canimorsus by macrophages and the blocking of the ability of macrophages to kill other bacteria also were demonstrated by the same group of investigators (17), as well as its resistance to killing by complement and polymorphonuclear leukocytes (22). However, C. canimorsus sepsis is characterized by extensive signs of inflammation, and alternative virulence mechanisms should be present.…”

Capnocytophaga canimorsus Sepsis Following a Minor Dog Bite to the Finger: Case Report

“…8e10 Capnocytophaga canimorsus prevents immune responses by the host because it inhibits the release of tumor necrosis factor and other proinflammatory proteins by infected macrophages. 11 Moreover, C. canimorsus can evade the human immune system because it is resistant to killing by serum complement and polymorphonuclear leucocytes, which is likely due to the composition of its lipopolysaccharide structure. 11 It has specific virulence factors that enable it to resist phagocytosis by macrophages and to persist intracellularly within leucocytes, resulting in extensive bacteraemia.…”