Multiple genetic paths including massive gene amplification allow <i>Mycobacterium tuberculosis</i> to overcome loss of ESX-3 secretion system substrates.

Abstract

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. <i>Mtb</i> strains deleted for the genes encoding substrates of the ESX-3 T7SS, <i>esxG</i> or <i>esxH</i>, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of <i>esxG</i> or <i>esxH</i> deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of <i>Mtb</i> chromosome evolution involving "accordion-type" amplification.