Defining the persistence associated transcriptome in Chlamydia trachomatis

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Project Summary Chlamydia trachomatis is a Gram-negative, obligate intracellular bacterium that continues to be the world’s most common bacterial sexually transmitted infection year after year. A combination of a high rate of asymptomatic cases, resistance to natural clearance, and slow progress in the development of a vaccine has allowed C. trachomatis to maintain its status as a significant public health threat. As an intracellular pathogen, C. trachomatis is insulated from many host immune effectors. Additionally, downstream effects of IFN, a cytokine released by host immune cells to combat infection, cause Chlamydia to transition into a persistent state – a viable yet non- replicative and non-infectious state that specializes in survival rather than growth. In the absence of growth, immune pressure dissipates, allowing C. trachomatis to revert back to its developmentally competent state and continue the infection cycle. Considering Chlamydia’s asymptomatic nature and the difficulty in developing an effective vaccine, a prophylactic drug treatment may be the best alternative – one specifically designed to inhibit chlamydial persistence. Unfortunately, developing a prophylactic to target the persistent response is not currently plausible since the molecular mechanisms involved in entering, maintaining, and exiting persistence are largely unknown. Current knowledge is limited to broad observations in the persistent response, such as the dysregulation of transcription and a decrease in translation. We hypothesize that a decrease in translation results in the loss of transcriptional repressors, allowing sigma factors to become overactive in initiating transcription. This proposal aims to characterize transcriptional changes associated with the initiation and maintenance of the persistent state through combined RNA sequencing and chromatin immunoprecipitation and sequencing (ChIP seq) approaches. IFN and two additional models shown to induce persistence will be used to observe transcriptional changes associated with persistence via RNA seq. To gain further insight into the dynamics of transcriptional changes, ChIP seq will be performed during persistence using multiple strains of C. trachomatis, each harboring an endogenous polyhistidine tag on a different sigma factor. In addition to providing the first genome wide analysis of sigma factor activity in C. trachomatis during standard growth and persistence, transcriptional changes observed by RNA seq will be linked to specific sigma factors. This high resolution map of transcriptional changes that occur during persistence will direct future investigations aimed at uncovering the molecular mechanisms involved in the persistent response. Training under this fellowship will provide extensive experience in state-of-the-art chlamydial molecular techniques, next generation sequencing, analysis of large data sets, and scientific writing and presentation. Home to both a Genomics Core Facility and Bioinformatics and Systems Biology Core Facility, the University of Nebraska Medical Center is fully equipped to support the goals outlined in this proposal. Furthermore, several experts in the chlamydial field are faculty members at UNMC, each capable of providing various perspectives and expertise relevant to the proposal.