B-211. The Emerging Human Pathogen Photorhabdus asymbiotica Induces Apoptosis through Caspase 3 Pathway in THP1 cells

S. C. P. Costa, G. Courties, J. Dornand, M. Brehelin, R. Zumbihl;
Univ. de Montpellier II, Montpellier, FRANCE.

Photorhabdus are members of the Enterobacteriaceae family and are symbiotically associated with the Heterorhabditidae nematodes. Photorhabdus species are highly pathogenic for a broad spectrum of insects. Recently, Photorhabdus have been isolated from clinical specimens in United States and in Australia. This new clinical entity was named P. asymbiotica. To date, 14 cases were documented and infections caused by P. asymbiotica were characterized by locally invasive soft tissue and disseminated bacteraemia. There is no defined route of infection. In the present study, we were interested in the understanding of P. asymbiotica action mechanism and differentiated THP1 cells were the model used for host-bacteria interaction assays. Phagocytosis is on of the immune defense reaction that bacteria must avoid in order to survive. Therefore, we realized a gentamycin killing assay and for the first time we demonstrate that, although there was low initial intracellular rate, the clinical isolate can survive within the macrophage. Furthermore, ELISA analysis reveled that TNF-αλπηα was secreted by THP1 cells only when P. asymbiotica were inactive by heat. THP1 cells after contact with live P asymbiotica became non adherent. When observed microscopically, THP1 cells showed apoptotic features, namely apoptotic bodies and chromatin condensation, that were absent from cells treated with inactive bacteria. Moreover, TUNEL staining of THP1 cells revealed an elevated cell apoptosis, reaching a percentage of 70%. When cells were treated with z-VAD-fmk (caspase general inhibitor), only 20% of cells presented nuclear fragmentation. To further characterize apoptosis we carried out western blotting analysis and our results showed that P. asymbiotica activates the caspase 3 pathways with a subsequent activation of caspase 7 and release of PARPs. From ours results, we can conclude that P. asymbiotica is an intracellular bacteria that posses an active process that provokes apoptosis, through caspase 3 pathway. The precise mechanism for host immune suppression and consequent human pathogenicity of P. asymbiotica is currently under investigation