CTPPH_8a_1_CtE_Hep2_rRNAdepl
Simultaneous Transcriptional Profiling Of Obligate Intracellular Pathogens And Their Host Cells
Chlamydia are obligate intracellular bacterial pathogens of humans responsible for significant disease worldwide. Chlamydia is intractable to most molecular biology techniques as it cannot be grown outside of the host cell and there is no system for laboratory genetic manipulation. These limitations continue to restrict basic understanding of this widespread pathogen and development of therapeutic interventions.  As an obligate intracellular pathogen, Chlamydia cannot be understood without accounting for its dynamic interaction with the host cell. In this GSCID-funded work, we describe a transformative RNA-Seq-based approach to determine the total transcriptome of the Chlamydia-infected cell. The primary experimental challenge of applying RNA-Seq to commingled populations of bacteria and human cells is obtaining sufficient RNA for both from a complex m?lange of RNA moieties. In the absence of a direct selection step for bacterial mRNA, we use serial depletion to remove mammalian and chlamydial non-mRNA moieties, enriching all mRNA irrespective of the source organism. This mixture of chlamydial and human mRNA can then be sequenced to produce the infected cell transcriptome. We coin the term "Heterogeneous RNA-Seq" to describe this novel approach.  Heterogeneous RNA-Seq reveals a fresh view of the Chlamydia-infected cell from both sides of the interaction simultaneously. One immediate application is the examination of host-Chlamydia interactions, including attachment and infection processes in vivo and in vitro over time and under various experimental conditions. The identification of chlamydial genes that are transcribed during specific infection time points and developmental milestones limits the number of genetic features that will need to be targeted for therapeutic development; similarly identification of host cell genes responding to infection by one or many chlamydial species or strains will greatly focus research onto relevant targets. While we cannot yet modify the pathogen through typical genetic manipulations, many tools exists for modifying mammalian cells - heterogenous RNA-Seq would enable appropriate host cell targets to be identified.  In summary, total transcriptomic snapshots of the dynamic host-Chlamydia relationship provided by heterogenous RNA-Seq will provide the detailed information required for the development of the next generation of therapeutics and vaccines. This project is led by Garry Myers, Ph.D., Institute for Genome Sciences, in collaboration with Dr. Patrik Bavoil, University of Maryland School of Medicine.