C. trachomatis is the leading cause of bacerially-acquired sexually transmitted infections in the USA and is responsible for 25-50% of the estimate 1 million cases per year of pelvic inflammatory disease. Sequelae include ectopic pregnancy and sterility. To begin to dissect the destructive versus the protective response as well as identify potential vaccine candidates, our overall goal is to understand the cell and molecular biology of chlamydial antigen sorting and secretion in infected polarized human endometrial epithelial cells. We have detected escape of chlamydial LPS and the major outer membrane protein (MOMP) to the surface of infected cells prior to the natural release of chlamydiae at the end of the developmental cycle. Do chlamydial stress response proteins also escape to the infected cell surface: What pathway do these antigens take from the inclusion and how are the surface exposed antigens perceived by the host immune response: We shall use the powerful technique of post-embedding immunoelectron microscopy on Lowicryl-processed samples to determine in Specific Aim 1 if C. trachomatis serovar E GroEL and DnaK are also secreted to the surface of normally infected and penicillin and interferon gamma-induced persistently infected human endometrial epithelial cells, and in Specific Aim 2 to continue to assess whether or not chlamydial antigens are secreted in a polarized direction. Primary antibodies (ABY) directed against chlamydia specific histones will serve as a control marker for antigens which do not escape the inclusion and mABY directed against Class 1 molecules will serve as a marker for the basolateral domain. Digitized fluorescence microscopy of duplicate samples will give a more quantitative assessment of the pattern of directed antigen secretion.
In Specific Aim 3, we shall investigate if chlamydial surface antigen secretion is modulated by estrogen and progesterone. Since the trans- golgi excytosis inhibitor Brefeldin A reduces escape of LPS and MOMP from inclusions, chlamydial antigens may exploit the secretory pathway to reach the infected cell surface.
In Specific Aim 4, we shall analyze the pathway of chlamydial antigen sorting by co-localizing escaped chlamydial antigen with the golgi markers beta-COP, p58 and C6-NBD-ceramide. Finally, to begin to examine immune control of chlamydial growth, we have established an in vitro co-cultivation model to assess host effector PMN and T cell responses to infected human epithelial cells.
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