Temporal genome-wide fitness analysis ofMycobacterium marinumduring infection reveals genetic requirement for virulence and survival in amoebae and microglial cells

Abstract

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple or even fully drug-resistant strains increases the risk and emphasizes the need for more efficient and better drug treatments. A key feature of mycobacteria pathogenesis is the metabolic switch during infection and expression of virulence genes is often adapted to specific infection conditions. This study aims to identify genes that are involved in the establishment and maintenance of the infection. To answer these questions, we have applied Transposon Sequencing (Tn-Seq) inM. marinum, an unbiased genome-wide strategy that combines saturation insertional mutagenesis and high throughput sequencing. This approach allowed us to precisely identify the localization and relative abundance of insertions in pools of Tn mutants. The essentiality and fitness cost, in terms of growth advantage and disadvantage of over 105 mutants were quantitatively compared between in vitro and different stages of infection in two evolutionary distinct hosts,D. discoideumand BV2 microglial cells. We found that 57% of TA sites in theM. marinumgenome were disrupted and that 568 genes (10.2%) are essential forM. marinum, which is comparable to previous Tn-Seq studies onM. tuberculosis. The major pathways involved in the survival ofM. marinumduring infection ofD. discoideumwere related to vitamin metabolism, theesx-1operon, as well as themce1operon.