IN VITRO SUSCEPTIBILITY OF CHLAMYDIA TRACHOMATIS TO LPS-BINDING POLYAMINES AND CELLULOSE ETHER POLYMERS: TOWARDS THE DEVELOPMENT OF A MICROBICIDE AGAINST CHLAMYDIA INFECTION

Abstract

Chlamydia trachomatis is the causative agent of the most prevalent sexually transmitted bacterial infection in the United States. Antibiotic therapy is currently effective in treating Chlamydia infection; however, the vast majority of infected individuals are asymptomatic. In women, untreated cases of Chlamydia infection can lead to serious reproductive health consequences. In the current absence of a safe and effective vaccine, my study focused on development of a vaginally-delivered topical microbicide as an alternative strategy for prevention of Chlamydia. The paucity of a well-established method suitable for large-scale analysis of in vitro Chlamydia infection is a major limitation in the development of novel anti-Chlamydia compounds. In my effort to identify compounds that can be considered as microbicide candidates, I developed two automated methods for enumeration of Chlamydiathat are amenable to a large-scale study. The automated immunofluorescence image based assay uses computational analysis of microscopic images, and allows automated phenotypic characterization and classification of Chlamydia-infected host cells. The second method utilizes a host cell viability assay, and offers a facile approach with reduced liquid-handling requirements, as well as the ability to simultaneously assess anti-Chlamydia and cytotoxic properties of compounds. Both methods yielded enumeration of Chlamydia infection that is comparable to the conventional manual microscopy while drastically reducing the time requirements for analysis. Using the automated image-based method, I performed a compound screen to test in vitro susceptibility of C. trachomatis to small subsets of compounds for the two main components of microbicides; excipient and active ingredient. Excipient candidates were cellulose ether polymers, commonly used in vaginal gels and have pharmaceutical properties that favor their use in the preparation of controlled-release formulations for long-term administration of vaginal microbicide. Candidate active ingredients were analogues of an antibiotic polymyxin B (PMB) rationally designed to target lipid A portion of Gram-negative lipopolysaccharide (LPS). A wide range of anti chlamydial activity was observed among cellulose ether polymers, and 14 out of 18 PMB analogues exhibited greater than 60% inhibition of Chlamydia growth. Additionally, I was interested in studying the biological role of chlamydial lipooligosaccharide (LOS), using small molecule DS-96 targeting lipid A as a chemical probe. DS-96 effectively blocked chlamydial attachment and entry steps, suggesting that chlamydial LOS plays a role in these steps. These data were supported by the observation that a high level of inhibition by DS-96 was maintained through centrifugation, which is known to enhance Chlamydia infection but thought to override the attachment and entry mechanisms. Together, these data demonstrated that targeting chlamydial LOS is effective in blocking Chlamydia infection prior to the bacterial entry, and therefore, has a high potential to be a suitable approach for prevention of Chlamydia infection. Furthermore, utilizing DS-96 as chemical tool, my study expanded upon the current understanding of the biological significance of chlamydial LOS.