Mechanism of Chlamydia-induced centrosome amplification

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DESCRIPTION (provided by applicant): Chlamydia infections are the most commonly reported infectious disease in the U.S. Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, and it has been associated with cervical cancer, both as an independent risk factor and as a putative co-factor for human papillomavirus. However, the mechanisms by which Chlamydia may cause or contribute to cancer are not understood. Recently, C. trachomatis has been shown to induce centrosome abnormalities in an infected cell, which is intriguing as centrosome abnormalities can lead to genomic instability and have been detected in the majority of human cancers. We have made a novel finding that a key regulator of centrosome duplication, HsSAS-6, is cleaved during a C. trachomatis infection. Furthermore, our studies provide indirect evidence that cleavage removes a degradation motif at the C-terminus of HsSAS-6 that is necessary for regulating HsSAS-6 protein levels so that centrosome duplication only occurs once in a cell cycle. Based on these findings, our hypothesis is that HsSAS- 6 is proteolytically cleaved by a chlamydial protease to produce a truncated but stabilized form of the protein that causes centrosome amplification by dysregulation of normal centrosome duplication. This hypothesis will be tested with 2 specific aims: In the first aim, the consequences of HsSAS-6 cleavage will be studied to understand how a chlamydial infection leads to centrosome amplification. The truncated HsSAS-6 product detected in Chlamydia-infected cells will be examined to determine if it is a stabilized form that does not undergo degradation during the cell cycle like full length HsSAS-6. We will also examine whether expression of truncated HsSAS-6 is sufficient to cause centrosome amplification in the absence of a Chlamydia infection. In the second aim, the cleavage of HsSAS-6 will be investigated. The cleavage site in HsSAS-6 will be determined by protein sequencing and mass spectrometry. An in vitro cleavage assay that can cleave recombinant HsSAS-6 to produce large amounts of the cleavage product for analysis will be established. A well-characterized chlamydial protease, CPAF, will be tested to determine if it is the protease that cleaves HsSAS-6. CPAF is a promising candidate because it is known to cleave several host proteins in a Chlamydia-infected cell over the same time course as cleavage of HsSAS-6. Successful completion of these studies will help us understand the underlying mechanisms that lead to centrosome amplification in Chlamydia-infected cells and may provide a pathophysiologic explanation for the increased incidence of cervical cancer associated with C. trachomatis infection. PUBLIC HEALTH RELEVANCE: The centrosome, which through its role in cell division and chromosome segregation has effects on genomic stability, is dysregulated in many human cancers. In addition, centrosome abnormalities have been detected during infections with the intracellular bacterium, Chlamydia trachomatis, which has been associated with cervical cancer. As we have found that the key regulator of centrosome number, HsSAS-6, is cleaved during a chlamydial infection, we wish to test whether the dysregulation of HsSAS-6 function in the control of centrosome number links Chlamydia infections to genomic instability and cervical cancer. It is the goal of this project to examine the effect of HsSAS-6 cleavage on the function and regulation of this protein and to determine if the HsSAS-6 cleavage products seen during a chlamydial infection are sufficient to induce centrosome abnormalities. We also aim to determine the site and molecular requirements for this cleavage event during an infection with this epidemiologically significant bacterium.