Capnocytophaga canimorsus, a commensal bacterium of the canine oral flora, has been repeatedly isolated since 1976 from severe human infections transmitted by dog bites. Here, we show that C. canimorsus exhibits robust growth when it is in direct contact with mammalian cells, including phagocytes. This property was found to be dependent on a surface-exposed sialidase allowing C. canimorsus to utilize internal aminosugars of glycan chains from host cell glycoproteins. Although sialidase probably evolved to sustain commensalism, by releasing carbohydrates from mucosal surfaces, it also contributed to bacterial persistence in a murine infection model: the wild type, but not the sialidase-deficient mutant, grew and persisted, both when infected singly or in competition. This study reveals an example of pathogenic bacteria feeding on mammalian cells, including phagocytes by deglycosylation of host glycans, and it illustrates how the adaptation of a commensal to its ecological niche in the host, here the dog's oral cavity, contributes to being a potential pathogen.

Capnocytophaga canimorsus, a commensal bacterium from dogs' mouths, can cause septicemia or meningitis in humans through bites or scratches. Here, we describe and characterize the inflammatory response of human and mouse macrophages on C. canimorsus infection. Macrophages infected with 10 different strains failed to release tumor necrosis factor (TNF)- alpha and interleukin (IL)-1 alpha . Macrophages infected with live and heat-killed (HK) C. canimorsus 5 (Cc5), a strain isolated from a patient with fatal septicemia, did not release IL-6, IL-8, interferon- gamma , macrophage inflammatory protein-1 beta , and nitric oxide (NO). This absence of a proinflammatory response was characterized by the inability of Toll-like receptor (TLR) 4 to respond to Cc5. Moreover, live but not HK Cc5 blocked the release of TNF- alpha and NO induced by HK Yersinia enterocolitica. In addition, live Cc5 down-regulated the expression of TLR4 and dephosphorylated p38 mitogen-activated protein kinase. These results highlight passive and active mechanisms of immune evasion by C. canimorsus, which may explain its capacity to escape from the host immune system.

C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-β-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including β-galactosidases, β-N-Acetylhexosaminidases and α-mannosidases.

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.

SummaryCapnocytophaga canimorsus are commensal Gramnegative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the FlavobacteriaBacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.