Escape from Immune Surveillance by Capnocytophaga canimorsus

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)-α and interleukin (IL)-1α. 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-γ, macrophage inflammatory protein-1β, 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-α 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 syste

XIAP-mediated Caspase Inhibition in Hodgkin's Lymphoma–derived B Cells

The malignant Hodgkin and Reed-Sternberg cells of Hodgkin's lymphoma (HL) and HL-derived B cell lines were previously shown to be resistant to different apoptotic stimuli. We show here that cytochrome c fails to stimulate caspases-9 and -3 activation in cytosolic extracts of HL-derived B cells, which is due to high level expression of X-linked inhibitor of apoptosis (XIAP). Coimmunoprecipitation studies revealed that XIAP, apoptosis protease-activating factor–1, and caspase-3 are complexed in HL-derived B cell lysates. Even after stimulation with exogenous cytochrome c and dATP, XIAP impairs the proteolytic processing and activation of caspase-3. In cytosolic extracts, inhibition of XIAP by the second mitochondria-derived activator of caspases (Smac)/DIABLO, or immunodepletion of XIAP restores cytochrome c–triggered processing and activation of caspase-3. Smac or a Smac-derived agonistic peptide also sensitized intact HL-derived B cells for the apoptotic action of staurosporine. Finally, Hodgkin and Reed-Sternberg cells of primary tumor HL tissues also constitutively and abundantly express XIAP. The results of this paper suggest that high level XIAP expression is a hallmark of HL, which may play a crucial role in resistance to apoptosis

Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus

Capnocytophaga canimorsus is a dog's and cat's oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus

Resistance of Capnocytophaga canimorsus to Killing by Human Complement and Polymorphonuclear Leukocytes▿

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

Aminosugars but not sialic acids sustain growth of <i>C. canimorsus.</i>

<p>Viable counts after challenge with 2×10⁶ wt <i>Cc5</i> (black) or <i>siaC</i> (grey) grown for 24 h with J774.1 in cRPMI (control) or in the same condition with the addition of Neu5Ac, Neu5Ac- CMP, 12.5 ng/ml enzyme SiaC_FL, SiaC_Y488C or NanH from <i>C. perfringens</i> (A) or with the addition of GalNAc, GlcNAc or LacNAc (B) or with the addition of mannose, galactose, glucose or sialyl-lactose (C). Mean values from 3 or more experiments and s.d. are shown including statistical difference between wt <i>Cc5</i> and <i>siaC</i> with * p<0.05, ** p<0.01 and *** p<0.001 for each pair of columns (2-tailed unpaired Student's t test). The grey dotted line indicates the bacterial number inoculated.</p

Surface localized sialidase is required for growth.

<p>(A) Sialidase activity of intact bacteria, measured with the substrate MUAN as the mean of triplicate measurements and s.d. of a representative experiment. (B) Viable counts after challenge with 2×10⁶<i>Cc5</i> (black), <i>siaC</i> (light grey) or <i>siaC</i> complemented with plasmids containing <i>siaC</i>_FL, <i>siaC</i>_Δ1–21 and <i>siaC</i>_Y488C after 24 h in presence of J774.1 with the grey dotted line indicating the bacterial number inoculated. Sialidase was detected by immunoblotting with a polyclonal antibody against SiaC in total cells (TC). (C) Outer membrane protein fractions (OMP), cell free supernatants (SN) of the J774.1 cultures shown in (B) including as control TC of <i>Cc5</i> were analyzed by immunoblotting for the presence of SiaC. (D) Surface localization of SiaC was tested by immunofluorescence on paraformaldehyde fixed but not permeabilized bacteria using anti-SiaC followed by anti- rabbit IgG conjugated to FITC.</p

<i>C. canimorsus</i> desialylates macrophage and epithelial cell surfaces.

<p>(A) The targets of the lectins used in this study are schematically represented (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000164#ppat.1000164-Varki2" target="_blank">[44]</a>). Surface carbohydrates of J774.1 macrophages (B) or HeLa epithelial cells (C) were analyzed by lectin binding after 2 h of infection with 4×10⁷ wt (<i>Cc5</i>) or <i>siaC</i> bacteria. Cells were fixed with paraformaldehyde and incubated for 1 h with lectin SNA, which recognizes terminal sialic acids (2- 6 or 2- 3) linked to Gal or to GalNAc or PNA that binds to the disaccharide Gal 1–3 GalNAc only after removal of terminal sialic acids. SiaC was added to cells alone or with <i>siaC</i> bacteria at 100 ng/ml. Biotinylated lectins were visualized by FITC conjugated streptavidin.</p

Identification of the Tn integration site and analysis of mRNA present in wt <i>C. canimorsus</i> 5.

<p>(A) Amino acid sequence of the <i>C. canimorsus</i> sialidase showing the signal peptide (italics) and the BNR/asp repeats (Ser/Thr-X-Asp-X-Gly-X-Thr-Trp/Phe) of bacterial sialidases (boxed). Domain predictions were analyzed by InterProScan <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000164#ppat.1000164-Quevillon1" target="_blank">[42]</a>. The residues conserved in sialidases at the C-terminus are underlined and the tyrosine 488 is bold <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000164#ppat.1000164-Roggentin1" target="_blank">[43]</a>. The Tn<i>4351</i> integration site in SiaC at amino acid 77 is indicated, boxed in grey and bold. (B) Genetic locus of the sialidase gene (<i>siaC</i>) including its upstream genes, <i>gntR</i>-like gene (CAPCA_MM1) and putative N-acyl-glucosamine epimerase encoding gene (CAPCA_MM2); and downstream coding sequence (CAPCA_MM3). (C) Reverse transcription performed on total RNA with specific primers (5129 or 5132) followed by PCR to identify transcripts present in wt <i>Cc5</i> (cDNA). PCR reactions were also performed using genomic DNA (gDNA) as template instead of cDNA as a positive control. As a control, reverse transcription was performed without reverse transcriptase in a parallel assay and used as template for the subsequent PCR reaction (-RT).</p