Molecular analysis of apoptosis inhibition by Chlamydia trachomatis

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Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that develops in a cytoplasmic vacuole in human epithelial cells. Medically, Ctr is important especially as a pathogen of the eye and of the female genital tract. Scientifically, the interplay of host cell and bacterium is intriguing and in large parts not understood. One prominent way how Ctr manipulates the host cell is the inhibition of apoptosis: infected cells are strongly resistant to stimuli that would induce mitochondrial apoptosis in uninfected cells. This inhibition is very impressive but not understood; three molecular models have been put forward in the past but are incompatible with each other. The work in the current project has focused on a stringent experimental testing of these three hypotheses, applying recent advancements in the understanding of the apoptosis apparatus. Surprisingly, all three hypotheses turned out to be incorrect, including the one we had proposed ourselves some time ago. We moved on to map the anti-apoptotic activity. Using assays with intact cells and with in vitro reconstituted mitochondrial apoptosis systems we found evidence that the anti-apoptotic activity involves the direct inhibition of the two effectors of mitochondrial apoptosis, Bax and Bak. This inhibition may be the result of chemical modification or of the attachment of a binding partner. In this continuation of the project we will pursue the two goals of first identifying the molecular alterations on Bax and Bak associated with this inhibition and secondly identifying the modifications or binding partner of the two proteins. Using HeLa cells deficient for Bax (and thus initially focusing on Bak) we will, in the first part of the project, map Bak-alterations by limited proteolysis, native gel electrophoresis and cross-linking studies. Intramolecular changes in Bak that occur during its activation have been reported, and these studies will therefore pin down the step where Bak-activation is blocked by Ctr-infection. In the second part of the project we will attempt specifically to identify modifications and/or binding partners of Bak. Through proteomic analysis of immuno-precipitated Bak or through the detection of proteins biotinylated because of their proximity to Bak we will endeavour to identify potential binding partners. Through approaches also encompassing proteomic analyses we will search for chemical modifications of Bak. Results obtained with Bak will be tested on Bax. Identified underlying mechanisms of bacterial or host cell origin will be specifically targeted to test their relevance for both inhibition of apoptosis and development of Ctr. We believe that with these approaches we have the chance of understanding how an important bacterial pathogen manipulates human cells as well as delineating new details of the activation of the core effectors of mitochondrial apoptosis, Bax and Bak.