At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)-lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.

Vascular disrupting agents (VDAs) such as DMXAA (5,6-dimethylxanthenone-4-acetic acid) represent a novel approach for cancer treatment. DMXAA has potent anti-tumor activity in mice and, despite significant pre-clinical promise, failed human clinical trials. The anti-tumor activity of DMXAA has been linked to its ability to induce type I interferons in macrophages although the molecular mechanisms involved are poorly understood. Here we identify STING as a direct receptor for DMXAA leading to TBK1 and IRF3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic-dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anti-cancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.

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The Gram-negative bacteria Yersinia pestis , causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-κB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-κB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition.

SummaryThe Yersinia pestis virulence factor YopJ is a potent inhibitor of the NF-kB and MAPK signalling pathways, however, its molecular mechanism and relevance to pathogenesis are the subject of much debate. In this report, we characterize the effects of this type III effector protein on bone fide signalling events downstream of Toll-like receptors (TLRs), critical sensors in innate immunity. YopJ inhibited TLR-mediated NF-kB and MAP kinase activation, as suggested by previous studies. In addition, induction of the TLRmediated interferon response was blocked by YopJ, indicating that YopJ also inhibits IRF3 signalling. Examination of the NF-kB signalling pathway in detail suggested that YopJ acts at the level of TAK1 (MAP3K7) activation. Further studies revealed a YopJdependent decrease in the ubiquitination of TRAF3 and TRAF6. These data support the hypothesis that YopJ is a deubiquitinating protease that acts on TRAF proteins to prevent or remove the K63-polymerized ubiquitin conjugates required for signal transduction. Our data do not directly address the alternative hypothesis that YopJ is an acetyltransferase that acts on the activation loop of IKK and MKK proteins, but support the conclusion that the critical function of YopJ is to deubiquinate TRAF proteins.