ESX-5 paralog deletions in Mycobacterium tuberculosis alter macrophage cytokine signaling and bacterial heavy metal response

The ESX-5 secretion system is critical for Mycobacterium tuberculosis (Mtb) viability and pathogenesis. However, a functional understanding of its impact on the host is unknown. ESX-5 is encoded at a single site with small putative virulence paralogs (ESX-5a, ESX-5b, and ESX-5c) spaced throughout the genome. To examine host-pathogen interactions of these putative virulence clusters, we made deletion mutants lacking these paralogs and infected primary human macrophages. Independent infection with any deletion mutant led to reduced cytokine secretion. This defect was dependent on viable bacteria and is mediated by an unresolved post-transcriptional mechanism. In bacterial transcriptomic analyses, each mutant downregulated heavy metal response genes compared with the wild type. Treatment of wild-type Mtb with heavy metals led to increased ESX-5a and ESX-5c expression, driving concurrent TNF and IL-6 secretion in macrophages compared with untreated bacilli, indicating a putative link between ESX-5 cargo export and cellular cytokine levels.