In this study, we investigated the activity of transcription factor NF-kappaB in macrophages infected with Yersinia enterocolitica. Although triggering initially a weak NF-kappaB signal, Y. enterocolitica inhibited NF-kappaB activation in murine J774A.1 and peritoneal macrophages within 60 to 90 min. Simultaneously, Y. enterocolitica prevented prolonged degradation of the inhibitory proteins IkappaB-alpha and IkappaB-beta observed by treatment with lipopolysaccharide (LPS) or nonvirulent, plasmid-cured yersiniae. Analysis of different Y. enterocolitica mutants revealed a striking correlation between the abilities of these strains to inhibit NF-kappaB and to suppress the tumor necrosis factor alpha (TNF-alpha) production as well as to trigger macrophage apoptosis. When NF-kappaB activation was prevented by the proteasome inhibitor MG-132, nonvirulent yersiniae as well as LPS became able to trigger J774A.1 cell apoptosis and inhibition of the TNF-alpha secretion. Y. enterocolitica also impaired the activity of NF-kappaB in epithelial HeLa cells. Although neither Y. enterocolitica nor TNF-alpha could induce HeLa cell apoptosis alone, TNF-alpha provoked apoptosis when activation of NF-kappaB was inhibited by Yersinia infection or by the proteasome inhibitor MG-132. Together, these data demonstrate that Y. enterocolitica suppresses cellular activation of NF-kappaB, which inhibits TNF-alpha release and triggers apoptosis in macrophages. Our results also suggest that Yersinia infection confers susceptibility to programmed cell death to other cell types, provided that the appropriate death signal is delivered.

A characteristic of the three human-pathogenic Yersinia spp. (the plague agent Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica) is the expression of the virulence (V)-antigen (LcrV). LcrV is a released protein which is involved in contact-induced secretion of yersinia antihost proteins and in evasion of the host's innate immune response. Here we report that recombinant LcrV signals in a CD14- and toll-like receptor 2 (TLR2)-dependent fashion leading to immunosuppression by interleukin 10 induction. The impact of this immunosuppressive effect for yersinia pathogenesis is underlined by the observation that TLR2-deficient mice are less susceptible to oral Y. enterocolitica infection than isogenic wild-type animals. In summary, these data demonstrate a new ligand specificity of TLR2, as LcrV is the first known secreted and nonlipidated virulence-associated protein of a Gram-negative bacterium using TLR2 for cell activation. We conclude that yersiniae might exploit host innate pattern recognition molecules and defense mechanisms to evade the host immune response.

Bartonella henselae causes vasculoproliferative disorders in humans. We identified a nonfimbrial adhesin of B. henselae designated as Bartonella adhesin A (BadA). BadA is a 340-kD outer membrane protein encoded by the 9.3-kb badA gene. It has a modular structure and contains domains homologous to the Yersinia enterocolitica nonfimbrial adhesin (Yersinia adhesin A). Expression of BadA was restored in a BadA-deficient transposon mutant by complementation in trans. BadA mediates the binding of B. henselae to extracellular matrix proteins and to endothelial cells, possibly via beta1 integrins, but prevents phagocytosis. Expression of BadA is crucial for activation of hypoxia-inducible factor 1 in host cells by B. henselae and secretion of proangiogenic cytokines (e.g., vascular endothelial growth factor). BadA is immunodominant in B. henselae-infected patients and rodents, indicating that it is expressed during Bartonella infections. Our results suggest that BadA, the largest characterized bacterial protein thus far, is a major pathogenicity factor of B. henselae with a potential role in the induction of vasculoproliferative disorders.

The balance between regulatory and inflammatory immune responses is critical to maintain intestinal homeostasis. Furthermore, the nature of the inflammatory response needs to be tailored to the tissue to provide proper protective immunity while preserving host integrity. TLR2 (Toll-like receptor 2) is a unique TLR in that it has been shown to promote regulatory and inflammatory T cell responses. Using Yersinia enterocolitica, we show that oral infection promotes T(H)17 immunity, whereas systemic infection promotes T(H)1 immunity. Furthermore, induction of T(H)17 immunity during oral infection is dependent on TLR1 and results from the combinatorial effect of TLR2/TLR1-induced IL-6 and IL-23 and the presence of TGF-β in the intestinal environment. Interestingly, TLR2/TLR1 was not involved in T(H)1 immune responses during systemic infection, whereas the TLR2/TLR6 receptor complex induced IL-10(+) regulatory T cell responses during both systemic and oral infections. Our results reveal that the route of infection is central in determining which pathways provide protective immunity. Furthermore, they also demonstrate that TLR2 has dual immune functions in the gut and identify TLR1 as a critical innate receptor for protective intestinal T(H)17 immunity.

Toll-like receptor 4 (TLR4), which signals through the adapter molecules myeloid differentiation factor 88 (MyD88) and toll/interleukin 1 receptor domain-containing adapter inducing IFN-β (TRIF), is required for protection against Gram-negative bacteria. TRIF is known to be important in TLR3-mediated antiviral signaling, but the role of TRIF signaling against Gram-negative enteropathogens is currently unknown. We show that TRIF signaling is indispensable for establishing innate protective immunity against Gram-negative Yersinia enterocolitica. Infection of wild-type mice rapidly induced both IFN-β and IFN-γ in the mesenteric lymph nodes. In contrast, TRIF-deficient mice were defective in these IFN responses and showed impaired phagocytosis in regional macrophages, resulting in greater bacterial dissemination and mortality. TRIF signaling may be universally important for protection against Gram-negative pathogens, as TRIF-deficient macrophages were also impaired in killing both Salmonella and Escherichia coli in vitro. The mechanism of TRIF-mediated protective immunity appears to be orchestrated by macrophage-induced IFN-β and NK cell production of IFN-γ. Sequential induction of IFN-β and IFN-γ leads to amplification of macrophage bactericidal activity sufficient to eliminate the invading pathogens at the intestinal interface. Our results demonstrate a previously unknown role of TRIF in host resistance to Gram-negative enteropathogens, which may lead to effective strategies for combating enteric infections.