Mechanistic Studies Of Host Pathogen Interaction During Chlamydia Trachomatis Infection Of Epithelial Cells

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes human diseases.Ocular infection by C.trachomatis causes the most preventable blindness,while urogenital track infection by C.trachomatis is the most common cause of sexually transmitted disease(STD),causing male urethritis,pelvic inflammatory disease,tubal blockage,ectopic pregnancy and infertility in women.A hallmark of Chlamydia infection is the asymptomatic nature of the infection since up to 70%patients do not realize that they have an infection.Chlamydiae are obligate intracellular bacterial pathogens that reside in membrane bound vacuoles for replication and infection.In addition to the inclusion membrane that may provide a niche to shield the bacteria from host recognition by pattern recognition receptors(PRRs),the bacteria may have evolved sophisticated mechanisms to evade host antimicrobial defense.For example,we and several other groups have reported that the proteases encoded by the chlamydial genomes had the ability to degrade or cleave host proteins of immune response,although detailed mechanisms that regulate protease release and translocation remain unknown.In this study,we focused on the induction of RIG-I,an intracellular PRR that recognizes 5'-uncapped RNA and certain DNA,and the cleavage of mitochondria antiviral signaling protein(MAVS)during Chlamydia infection of epithelial cells and tried to relate RIG-I induction and MAVS cleavage to host-pathogen interactions.We found that C.trachomatis infection strongly induced RIG-I expression,independent of Chlamydia replication since antibiotics that block Chlamydia replication showed little effect on RIG-I induction.Instead,we found that transfection of purified nucleic acid extracts to HeLa 229 cells promoted RIG-I expression.RIG-I is an important member of the RLR family proteins that modulate innate immune response of non-immune cells to recognizes viral infection.We found that overexpression of RIG-I did not suppress Chlamydia infection,although suppression of RIG-I induction by siRNA treatment resulted in reduced induction of IFN? and interferon-stimulated genes.The action of RIG-I in modulating innate immune response requires protein-protein interaction of RIG-I with the mitochondria-anchored MAVS protein.The anchorage of MAVS on mitochondria is essential for its activity.Therefore,some viral pathogens have been observed to target RIG-I and MAVS to evade host antimicrobial response.We investigated whether Chlamydia spp.had the ability to target proteins of the RIG-I/MAVS pathway and found that purified Chlamydia possessed activity of cleaving MAVS protein.Using immunodepletion combined with molecular biological approaches,we identified the enzyme that was responsible for MAVS cleavage as CPAF,a protease encoded by CT858 gene of Chlamydia trachomatis.In addition,we also characterized the cleavage site of MAVS.Overexpression of the cleavage fragments failed to induce luciferase expression under the promoter of NF-?B and IFN?,respectively.Although we were unable to demonstrate CPAF cleavage during Chlamydia infection,those results nonetheless demosntrated that Chlamydia spp.may utilize the encoded proteases for evasion of host antimicrobial response.