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

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

Simple Resazurin-Based Microplate Assay for Measuring Chlamydia Infections

This is the publisher's version. Copyright 2013 by the American Society for Microbiology.The conventional method for quantification of Chlamydia infection using fluorescence microscopy typically involves time- and labor-intensive manual enumeration, which is not applicable for a large-scale analysis required for an inhibitory compound screen. In this study, an alamarBlue (resazurin) assay was adopted to measure Chlamydia infection by measuring the redox capability of infected host cells in a 96-well format. The assay provided measurements comparable to those of the conventional microscopy method while drastically reducing the time required for analysis

Lipopolysaccharide-Binding Alkylpolyamine DS-96 Inhibits Chlamydia trachomatis Infection by Blocking Attachment and Entry

This is the published version. Copyright © 2014, American Society for Microbiology. All Rights Reserved.Vaginally delivered microbicides are being developed to offer women self-initiated protection against transmission of sexually transmitted infections such as Chlamydia trachomatis. A small molecule, DS-96, rationally designed for high affinity to Escherichia coli lipid A, was previously demonstrated to bind and neutralize lipopolysaccharide (LPS) from a wide variety of Gram-negative bacteria (D. Sil et al., Antimicrob. Agents Chemother. 51:2811–2819, 2007, doi:10.1128/AAC.00200-07). Aside from the lack of the repeating O antigen, chlamydial lipooligosaccharide (LOS) shares general molecular architecture features with E. coli LPS. Importantly, the portion of lipid A where the interaction with DS-96 is expected to take place is well conserved between the two organisms, leading to the hypothesis that DS-96 inhibits Chlamydia infection by binding to LOS and compromising the function. In this study, antichlamydial activity of DS-96 was examined in cell culture. DS-96 inhibited the intercellular growth of Chlamydia in a dose-dependent manner and offered a high level of inhibition at a relatively low concentration (8 μM). The data also revealed that infectious elementary bodies (EBs) were predominantly blocked at the attachment step, as indicated by the reduced number of EBs associated with the host cell surface following pretreatment. Of those EBs that were capable of attachment, the vast majority was unable to gain entry into the host cell. Inhibition of EB attachment and entry by DS-96 suggests that Chlamydia LOS is critical to these processes during the developmental cycle. Importantly, given the low association of host toxicity previously reported by Sil et al., DS-96 is expected to perform well in animal studies as an active antichlamydial compound in a vaginal microbicide

An Automated Image-Based Method for Rapid Analysis of Chlamydia Infection as a Tool for Screening Antichlamydial Agents

This is the published version. Copyright American Society for MicrobiologyA major limitation in the identification of novel antichlamydial compounds is the paucity of effective methods for large-scale compound screening. The immunofluorescence assay is the preferred approach for accurate quantification of the intracellular growth of Chlamydia. In this study, an immunofluorescence image-based method (termed image-based automated chlamydial identification and enumeration [iBAChIE]) was customized for fully automated quantification of Chlamydia infection using the freely available open-source image analysis software program CellProfiler and the complementary data exploration software program CellProfiler Analyst. The method yielded enumeration of different species and strains of Chlamydia highly comparably to the conventional manual methods while drastically reducing the analysis time. The inhibitory capability of established antichlamydial activity was also evaluated. Overall, these data support that iBAChIE is a highly effective tool for automated quantification of Chlamydia infection and assessment of antichlamydial activities of molecules. Furthermore, iBAChIE is expected to be amenable to high-throughput screening studies for inhibitory compounds and fluorescently labeled molecules to study host-pathogen interactions

Small-Molecule Inhibitor of the Shigella flexneri Master Virulence Regulator VirF

This is the publisher's version, also available electronically from http://iai.asm.org/content/81/11/4220VirF is an AraC family transcriptional activator that is required for the expression of virulence genes associated with invasion and cell-to-cell spread by Shigella flexneri, including multiple components of the type three secretion system (T3SS) machinery and effectors. We tested a small-molecule compound, SE-1 (formerly designated OSSL_051168), which we had identified as an effective inhibitor of the AraC family proteins RhaS and RhaR, for its ability to inhibit VirF. Cell-based reporter gene assays with Escherichia coli and Shigella, as well as in vitro DNA binding assays with purified VirF, demonstrated that SE-1 inhibited DNA binding and transcription activation (likely by blocking DNA binding) by VirF. Analysis of mRNA levels using real-time quantitative reverse transcription-PCR (qRT-PCR) further demonstrated that SE-1 reduced the expression of the VirF-dependent virulence genes icsA, virB, icsB, and ipaB in Shigella. We also performed eukaryotic cell invasion assays and found that SE-1 reduced invasion by Shigella. The effect of SE-1 on invasion required preincubation of Shigella with SE-1, in agreement with the hypothesis that SE-1 inhibited the expression of VirF-activated genes required for the formation of the T3SS apparatus and invasion. We found that the same concentrations of SE-1 had no detectable effects on the growth or metabolism of the bacterial cells or the eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to inhibit transcription activation by VirF, exhibits selectivity toward AraC family proteins, and has the potential to be developed into a novel antibacterial agent