Analysis and role of the inhibition of apoptosis by the Chlamydia trachomatis OmpA protein

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Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium: it replicates within a vacuole in infected human epithelial cells. Ctr is known to protect infected human cells against numerous experimental pro-apoptotic stimuli. The molecular mechanism of apoptosis protection is contentious. We had observed, using Bcl-2-family inhibitors on infected human cells, that Ctr can block apoptosis through a protease-sensitive factor at the level of the pro-apoptotic Bcl-2-family effector protein Bak. Inhibition thus occurs upstream of cytochrome c-release but downstream of anti-apoptotic Bcl-2-like proteins. As no human protein is regularly known to act at this step of apoptotic signaling, this strongly suggested a chlamydial protein interfering with apoptosis. We found that the chlamydial major outer membrane porin, OmpA, translocates from the bacteria to mitochondria where it is found membrane-inserted and in the proximity of Bak. We mapped the anti-apoptotic activity of Ctr onto the known activation steps of Bak during apoptosis and were able to monitor Bak inhibition. Expression of OmpA in uninfected cells precisely phenocopied Bak-inhibition by Ctr-infection. Bak is in resting cells bound to a mitochondrial outer membrane porin, VDAC2, from where it is released upon apoptosis-induction. Our results suggest that OmpA sequesters and inhibits Bak upon its release from VDAC2, and we have identified a related protein from Simkania negevensis (SnOmpA) with functionally similar activity. ‘Chlamydia-like’, protozoa-associated bacteria such as Parachlamydia induce rather than inhibit apoptosis in human cells. We have found that Parachlamydia can grow in human cells if mitochondrial apoptosis is blocked, and that OmpA can support the growth of Parachlamydia. In this project we will first conduct a comparative study of Ctr and Sn OmpA to understand the degree of conservation of the anti-apoptotic mechanism. How OmpA translocates to mitochondria during chlamydial infection is unclear. We hypothesize that it occurs in outer membrane vesicles that are shed by the bacteria and propose to test this mechanism. Lastly, we aim at a molecular understanding of the anti-apoptotic effect of OmpA We will first compare the anti-apoptotic activity of OmpA and VDAC2. We will further follow up on our finding that OmpA can reduce mitochondrial concentration of Bax, very likely through enhancing the retro-translocation of Bax into the cytosol, an established anti-apoptotic mechanism. These experiments will help understand not only the function of OmpA in apoptosis inhibition but also provide information on activation and activity of Bax and Bak. Our data suggest that Chlamydiales exploit their evolutionary relationship to mitochondria to block mitochondrial apoptosis of an infected human cell. We expect that the results of this project will shed light on a novel facet of interaction of Gram-negative bacteria and human cells.