Mycobacterium tuberculosis HN878 transcriptomes from replicating broth culture, THP1 macrophage-like cells, caseum surrogate, and rabbit caseum

Among the many influences affecting patient outcomes, the heterogeneity of Mtb populations in remote lesion compartments and the nature of pathogen physiology in these stressful environments are increasingly appreciated as factors that limit the efficacy of current therapies. The transcriptional state of Mtb during infection has been difficult to study and represents a potentially game-changing area for intervention. Limited studies in humans and animals have suggested that subpopulations of Mtb persist in the caseum (soft necrotic debris) that accumulates in the center of necrotic TB granulomas. Stress factors in the caseous foci include hypoxia, iron deprivation, and nutrient limitation. To persist in this environment, it is likely that this population elicits sweeping, transcriptionally coordinated molecular changes that are reflected in their gene expression profiles. Determining the basis of Mtb's ability to persist in this environment is a critical hurdle in addressing whether intracaseum Mtb are the rate-limiting step for successfully curing TB infection and designing interventions specific to this subpopulation. To address this population, we are adapting the developing GenomeCaptureSeq technology to isolate TB transcripts, determine the transcriptional profile of Mtb in caseum and characterize caseum-specific molecular programs that are utilized by Mtb in this environment to generate targets for intervention. This process involves fragmenting Mtb genomic DNA, linking to beads and then fishing Mtb transcripts from caseous material. Our approach has been applied to caseum samples from Mtb-infected rabbits and we have successfully sequenced TB-specific transcripts from multiple infected lesions. We aim to define a caseum-specific transcriptional signature that will help direct future studies of this subpopulation of interest. Overall design: Replicating broth cultures of Mtb strain HN878, infected THP1 macrophages, and Mtb allowed to acclimate to surrogate caseum are compared with freshly harvested rabbit caseum samples. RNAseq was performed using hybridization capture with Transcript Capture Sequencing probes made from H37Rv gDNA.