Oligomeric Coiled-Coil Adhesin YadA Is a Double-Edged Sword

Yersinia adhesin A (YadA) is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Suprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt) Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs) upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadAV98D,N99A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions

Capnocytophaga canimorsus. interaction with the innate immune system

We show that Capnocytophaga canimorsus strain 5 (Cc5) is even more resistant to phagocytosis and killing by murine macrophages (J774.1) and human polymorphonuclear neutrophils (PMNs) than Yersinia enterocolitica, which is known as a model bacterium for resistance against phagocytosis due to its type 3 secretion system (Grosdent et al., 2002). We observed that Cc5 even becomes completely resistant to phagocytosis at high multiplicity of infection (moi of 50). In addition, we demonstrate that the Cc5 transposon mutant Y1C12, identified during a serum sensitivity screen, has an increased sensitivity to phagocytosis and killing by either murine macrophages or human PMNs even in the unopsonized state. This indicated that not an increased susceptibility for antibody binding or complement deposition led to an increased phagocytosis of the mutant, but that rather the outer surface was more readily recognized by the phagocytes. Furthermore, we demonstrate that Cc5 induces the formation of neutrophil extracellular traps upon infection of human PMNs in vitro and that Cc5 is trapped and killed within neutrophil extracellular traps, indicating sensitivity of Cc5 towards antimicrobial peptides present in PMN granules. Analysis of serum resistance in Cc5 revealed that serum resistance is probably linked to its lipopolysaccharide, which prevents deposition of the membrane attack complex on the bacterial surface. Moreover, we have observed that upon growth in the presence of cells, Cc5 releases or modifies factor(s) in the medium, which interfere with the killing ability of macrophages. Investigating the underlying mechanism, we could show that Cc5 does not affect phagosome maturation, but blocks the oxidative burst. This capacity was shown to depend on the release of the zinc metallopeptidase pitrilysin by Cc5. First analyses on the prevalence of the hypothetical virulence factors serum resistance and interference with the oxidative burst indicated that C. canimorsus strains might display strain variability. While 59% of the strains (50% of case strains, 61% of dog isolates) were able to block the killing ability of macrophages, 60% of the strains were highly serum resistant (100% of case strains, 54% of dog isolates). However, serum resistance could not be directly linked to a specific polysaccharide structure in C. canimorsus. November 2009, Salome Casutt-Meye

YadA renders <i>Y. enterocolitica</i> sensitive to NET-dependent killing.

<p>(<b>A</b>) <i>Y. enterocolitica</i> induces DNA release upon infection of PMNs. PMNs were infected for 120 min with <i>Y. enterocolitica</i> E40 (wt or ΔYadA) at an moi of 1. DNA release was quantified by Sytox staining. Untreated PMNs were used as negative control and NET formation was induced by PMA as positive control. Mean values from three or more experiments and standard deviation are shown including statistical significance in comparison to untreated PMNs with ** p<0.01 and * p<0.05 using one-way ANOVA. (<b>B</b>) % of <i>Y. enterocolitica</i> E40 (wt, ΔYadA and ΔYadA endowed with pSAM16 encoding YadA_Ψtb) killed by PMA-triggered NETs (120 min infection at a moi of 1). Phagocytosis was prevented by the addition of Cytochalasin D (CytD). Mean values from three or more experiments and standard deviation are shown. Statistical significance is shown in comparison to <i>Y. enterocolitica</i> wt with *** p<0.001 using one-way ANOVA. (<b>C</b>) Scanning electron micrograph (SEM) of untreated human PMNs and (<b>D</b>) of NETs formed by human PMNs treated with PMA. (<b>E</b>) SEM of <i>Y. enterocolitica</i> E40 wt bacteria (expressing YadA) trapped in NETs after 120 min infection at an moi of 1. (<b>F</b>) <i>Y. enterocolitica</i> E40 ΔYadA bacteria induce NET formation but are not trapped (same conditions as in A) (SEM). NET structure covers the whole bottom.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Collagen binding contributes to the sensitivity of <i>Y. enterocolitica</i> to NETs.

<p>(<b>A</b>) Mutation of the collagen binding motif has no effect on serum resistance. % survival of <i>Y. enterocolitica</i> E40 ΔYadA, E40 ΔYadA (pFR1) expressing YadA_ent, or E40 ΔYadA (pFR2) expressing YadA_entV98D,N99A, incubated for 1 hour in the presence of 10% normal human serum (NHS) or heat-inactivated human serum (HI NHS). (<b>B</b>) % Survival to NET-dependent killing of <i>Y. enterocolitica</i> E40 ΔYadA, E40 ΔYadA (pMA1) expressing YadA_ent, or E40 ΔYadA (pSAM23) expressing YadA_entV98D,N99A. (<b>C</b>) YadA_entV98D,N99A forms trimers. SDS PAGE analysis of total cells from <i>Y</i>. <i>enterocolitica</i> E40 ΔYadA, E40 ΔYadA (pFR1) expressing YadA_ent, or E40 ΔYadA (pFR2) expressing YadA_entV98D,N99A. The band corresponding to trimeric YadA is boxed.</p

NETs contain collagen.

<p>(<b>A</b>) Untreated PMNs. DNA (blue) and collagen (green) visualized by immunofluorescence. (<b>B</b>) Same staining after the PMNs have been infected with <i>Y. enterocolitica</i> E40 for 120 min at an moi of 1. Smears represent NETs. 60 x magnification, DNA (blue), collagen (green) and brightfield.</p