TnSeq study evaluating the impact of Atg5 and Atg7 on survivial of Mycobacterium tuberculosis transposon mutants in IFN-gamma-activated murine bone marrow-derived macrophages

Mycobacterium tuberculosis impairs host lysosomal trafficking pathways. Xenophagy and LC3-associated phagocytosis (LAP) are lysosomal trafficking pathways that normally clear intracellular microbes. The xenophagy and LAP pathways depend upon ATG5 and ATG7, which result in the lipidation of LC3-I to LC3-II. How Mtb undermines these innate immune defenses of the host in not well understood. We used a transposon sequencing (Tn-seq) screen to identify bacterial factors that are required for the bacilli to resist ATG5 and ATG7-dependent processes. We found that that mutants defective in production of PDIM are attenuated in murine macrophages, and they are able to survive in macrophages lacking ATG5 or ATG7. Overall design: To identified Mtb factors which are required to resist xenophagy and/or LAP, we infected IFN-g-activated BMDMs that were WT (Atg5fl/fl Cre- and Atg7fl/fl Cre-) or Atg-deficient (Atg5fl/fl LysM-Cre+ and Atg7fl/fl LysM-Cre+) with a pool of saturated Himar1 transposon (Tn) mutants. After 5 days of infection, colony forming units (CFU) were collected for isolation of genomic DNA from surviving bacilli, subject to library preparation, sequenced by Illumina HiSeq, and analyzed using TRANSIT.