The Emerging Human Pathogen Photorhabdus asymbiotica Is a Facultative Intracellular Bacterium and Induces Apoptosis of Macrophage-Like Cells▿

Photorhabdus species are gram-negative entomopathogenic bacteria of the family Enterobacteriaceae. Among the different members of the genus, one species, Photorhabdus asymbiotica, is a pathogen of both insects and humans. The pathogenicity mechanisms of this bacterium are unknown. Here we show that P. asymbiotica is a facultative intracellular pathogen that is able to replicate inside human macrophage-like cells. Furthermore, P. asymbiotica was shown for the first time in an intracellular location after insect infection. We also demonstrated that among Australian and American clinical isolates, only the Australian strains were able to invade nonphagocytic human cells. In cell culture infection experiments, Australian clinical isolates as well as cell-free bacterial culture supernatant induced strong apoptosis of a macrophage cell line at 6 h postinfection. American isolates also induced cellular death, but much later than that induced by Australian ones. Mammalian cultured cells analyzed for key features of apoptosis displayed apoptotic nuclear morphology, activation of the initiator caspases 8 and 9 and the executioner caspases 3 and 7, and poly(ADP-ribose) polymerase proteolysis, suggesting activation of both the intrinsic and extrinsic apoptotic pathways

IL-1 stimulates a diverging signaling pathway in EL4 6.1 thymoma cells. IL-2 release, but not IL-2 receptor expression, is sensitive to pertussis toxin.

Abstract We reassessed the involvement of Bordetella pertussis toxin (PTX)-sensitive proteins in the IL-1 signaling pathway on the responses induced by IL-1 on the murine thymoma cell line EL4 6.1. We demonstrate that the ADP-ribosyltransferase activity of PTX, and not its cell-anchoring B oligomer part, is responsible for the inhibition of IL-1-induced IL-2 release, since 1) the concentration of PTX (&amp;lt; or = 1 ng/ml) required to block the secretion is 100 to 1000 times lower than the concentration needed with the B oligomer; and 2) the mutated PT-9K/129G, devoid of ADP-ribosyltransferase activity, was inactive at 100 ng/ml. We found that partial ADP-ribosylation of the Gi2/Gi3 proteins before stimulation with IL-1 was sufficient to obtain full inhibition of IL-2 release. PTX did not however inhibit the appearance on the cell surface of the high affinity IL-2 receptors or the IL-2 release induced by PMA. In addition, we show that PTX prevented the expression of the IL-2 mRNA induced by IL-1, without affecting the binding of IL-2 specific nuclear factors to the T cell distal element of the IL-2 promoter. Furthermore, PTX also inhibited IL-1-induced proliferation of non-transformed thymocytes. In conclusion, our results demonstrate that IL-1-induced IL-2 release is sensitive to PTX-catalyzed ADP-ribosylation and that IL-1 activates a diverging pathway on EL4 6.1 cells.</jats:p

Chlamydial IFN-γ immune evasion is linked to host infection tropism

Chlamydiae are obligate intracellular pathogens that can exhibit a broad host range in infection tropism despite maintaining near genomic identity. Here, we have investigated the molecular basis for this unique host-pathogen relationship. We show that human and murine chlamydial infection tropism is linked to unique host and pathogen genes that have coevolved in response to host immunity. This intimate host-pathogen niche revolves around a restricted repertoire of host species-specific IFN-γ-mediated effector responses and chlamydial virulence factors capable of inhibiting these effector mechanisms. In human epithelial cells, IFN-γ induces indoleamine 2,3-dioxygenase expression that inhibits chlamydial growth by depleting host tryptophan pools. Human chlamydial strains, but not the mouse strain, avoid this response by the production of tryptophan synthase that rescues them from tryptophan starvation. Conversely, in murine epithelial cells IFN-γ induces expression of p47 GTPases, but not indoleamine 2,3-dioxygenase. One of these p47 GTPases (Iigp1) was shown by small interfering RNA silencing experiments to specifically inhibit human strains, but not the mouse strain. Like human strains and their host cells, the murine strain has coevolved with its murine host by producing a large toxin possessing YopT homology, possibly to circumvent host GTPases. Collectively, our findings show chlamydial host infection tropism is determined by IFN-γ-mediated immunity

Crystal structure of Yersinia enterocolitica type III secretion chaperone SycT

Pathogenic Yersinia species use a type III secretion (TTS) system to deliver a number of cytotoxic effector proteins directly into the mammalian host cell. To ensure effective translocation, several such effector proteins transiently bind to specific chaperones in the bacterial cytoplasm. Correspondingly, SycT is the chaperone of YopT, a cysteine protease that cleaves the membrane-anchor of Rho-GTPases in the host. We have analyzed the complex between YopT and SycT and determined the structure of SycT in three crystal forms. Biochemical studies indicate a stoichometric effector/chaperone ratio of 1:2 and the chaperone-binding site contains at least residues 52–103 of YopT. The crystal structures reveal a SycT homodimer with an overall fold similar to that of other TTS effector chaperones. In contrast to the canonical five-stranded anti-parallel β-sheet flanked by three α-helices, SycT lacks the dimerization α-helix and has an additional β-strand capable of undergoing a conformational change. The dimer interface consists of two β-strands and the connecting loops. Two hydrophobic patches involved in effector binding in other TTS effector chaperones are also found in SycT. The structural similarity of SycT to other chaperones and the spatial conservation of effector-binding sites support the idea that TTS effector chaperones form a single functional and structural group