Frequently arising ESX-1 associated phase variants influence Mycobacterium tuberculosis fitness in the presence of host and antibiotic pressures

Mycobacterium tuberculosis (Mtb) exhibits an impressive ability to adapt to rapidly changing environments, despite its genome’s apparent stability. Recently, phase variation through indel formation in homopolymeric tracts (HT) has emerged as potentially important mechanism promoting adaptation in Mtb. This study examines the impact of common phase variants associated with the ESX1 Type VII secretion system, focusing on a highly variable HT upstream of the ESX1 regulatory factor, espR. By engineering this frequently observed indel into an isogenic background, we demonstrate a single basepair insertion in the espR 5’ UTR causes post-transcriptional upregulation of EspR protein abundance and corresponding alterations in the EspR regulon. Consequently, this mutation increases expression of ESX1 components in the espACD operon and enhances ESX1 substrate secretion. We find that this indel specifically increases isoniazid resistance without impacting the effectiveness of other drugs tested. Furthermore, we show that commonly observed HT indels that regulate either espR translation or espACD transcription increase bacterial fitness in a mouse infection model. The presence of multiple ESX1 associated HTs provides a mechanism to combinatorially tune protein secretion, drug sensitivity, and host-pathogen interactions. More broadly, these findings support emerging data that Mtb utilizes HT-mediated phase variation to strategically direct genetic variation to certain sites across the genome in order to adapt to changing pressures. RNAseq was performed on mid-log phase Mycobacterium tuberculosis H37Rv. Expression levels in ESX mutant backgrounds were compared to a wild type control