Granuloma Dual RNA-Seq Reveals Composite Transcriptional Programs Driven by Neutrophils and Necrosis within Tuberculous Granulomas

Mycobacterial granulomas lie at the center of tuberculosis (TB) pathogenesis and represent a unique niche where infecting bacteria survive in nutrient-restricted conditions and in the face of a host immune response. The granuloma's necrotic core, where bacteria reside extracellularly in humans, is difficult to assess in many experimentally tractable models. Here, using necrotic mycobacterial granulomas in adult zebrafish, we develop dual RNA-seq across different host genotypes to profile transcriptional alterations that enable bacteria to survive and take advantage of host immune responses. Using both pharmacological and genetic interventions, we find that neutrophils within mature, necrotic granulomas promote bacterial growth, in part through upregulation of the bacterial devR regulon. Finally, we identify conserved transcriptional programs related to K+ transport and the rpf genes induced only in the context of this unique necrotic extracellular niche. Analysis of Mycobacterium tuberculosis strains across diverse lineages suggests that these modules represent targets for bacterial genetic adaptation in the context of human infection. Overall design: Dual host-pathogen RNA-Seq profiling of necrotic granulomas containing Mycobacterium marinum (M. marinum) isolated from wild-type and neutrophil deficient Rac2D57N zebrafish. Total RNA isolated from these granulomas was divided into two portions. One portion was processed without any enrichment strategy and used to derive the host (Zebrafish) transcriptome. The second portion was enriched for M. marinum transcripts using oligo-dT beads, which selectively bind and deplete host RNAs. The enriched fraction was further used to derive bacterial transcripts from the granulomas. Additionally, bulk RNA-Seq was performed on in vitro grown log-phase M. marinum cultures. Comparitive analyses were conducted between the host and bacterial transcriptomes of wild-type and Rac2D57N granulomas to identify transcriptional changes under neutrophil depleted conditions. Further, comparisons were made between the bacterial transcriptome from wild-type granulomas and in vitro grown log-phase cultures to identify mycobacterial transcripts specifically required for survival in wild-type granulomas.