Global assessment of host cell functions involved in the intracellular survival and replication of Chlamydia using RNA interference in human cells

Chlamydia trachomatis is a Gram-negative obligate intracellular bacterial pathogen. It is associated with significant human illness, including preventable blindness, reactive arthritis. Furthermore, it is the most common agent of bacterial sexually transmitted diseases with potentially serious sequel in women that include pelvic inflammatory disease, ectopic pregnancy and sterility. As an obligate intracellular pathogen, Chlamydia rely on host cell for all aspects of their survival, from the initial attachment with the host cell membranes, to cellular invasion, acquisition of host cell metabolites and intracellular replication. Unique and largely uncharacterized intracellular host-pathogen interactions are critical for successful chlamydial infection. Despite the prevalence of Chlamydia spp. and their role in human disease, little is known about the mechanisms underlying the infection process, the host pathogen interactions, and the intracellular survival and replication of Chlamydia. To gain a comprehensive understanding of the disease pathogenesis, it is equally important to understand the contribution of the host as well as the pathogen to these complex host pathogen interactions. Therefore in this study I decided to focus on the host factors involved in the Chlamydia infections. For this purpose, silencing of gene expression by RNA interference (RNAi) has proven to be a robust and straight forward technique for gene function analysis in eukaryotic cells. This has become a method of choice for loss of function studies and can serve to unravel the contribution of host cell in the pathogenesis during Chlamydia infection. This study aims to obtain deeper insights into infection processes of Chlamydia by using RNAi based loss-of-function screen of host cell determinants. As a readout Chlamydia number and inclusion size for the primary infection as well as quantification of Chlamydia progeny (infectivity) were measured to monitor entry, survival and replication of the pathogen within the host. Therefore, a screen was performed in 96 well format, analyzing the impact of ~1500 different human genes mainly covering the human kinome, apoptosis as well as trafficking related genes. With help of various robust statistical analysis methods 134 primary hits were selected for further analysis. To rule out possible off-target effects, which are inherent to the current status of RNAi technology, the primary hits were further validated using 4 new siRNA sequences for each of the identified primary hits. This resulted