TraSH analysis of Mycobacterium tuberculosis transposon mutants with reduced bodipy-palmitate uptake on the third day of resting bone marrow-derived macrophage infection

Mycobacterium tuberculosis transposon mutants with reduced bodipy-palmitate intake on the third day of resting bone marrow-derived macrophage infection are identified by TraSH. Discovery of genetic requirements for fatty acid assimilation by the pathogen is essential for understanding its metabolism during host infection. The screen was performed with transposon (Himar) mutant library generated in M. tuberculosis Erdman strain pDE43-MCH (smyc'::mCherry). Bone marrow-derived macrophages were infected with this transposon library (~2X104) at MOI 4:1, metabolically labeled with Bodipy-palmitate on the third day of infection. After isolation mutants with low Bodipy signal were sorted, plated, and pooled for DNA extraction. This selection was repeated for enrichment. TraSH analysis is performed to compare pools of mutants after 1st and 2nd rounds of selection relative to the input pool (Input). 3 technical replicates were used for each of the rounds of enrichment.