Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis

Abstract

bMore people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB. In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis. T he intracellular pathogen Mycobacterium tuberculosis survives within phagocytic immune cells such as macrophages and dendritic cells (1).M. tuberculosis evades degradation by the endolysosomal pathway, growing in an early endosome-like compartment or escaping into the cytosol (2, 3). Acidified lysosomes are just one obstacle for the bacilli to overcome as the host also regulates metals such as iron, zinc, copper, and manganese to create an uninhabitable microenvironment (4). These metals are essential but at the same time can be toxic, so both host and pathogen tightly regulate them. For example, macrophages can increase zinc levels in M. tuberculosis-containing phagosomes to induce toxicity (5). Calprotectin, on the other hand, is released at sites of infection to bind and withhold zinc and manganese from bacteria (6). Similarly, host iron is bound to glycoproteins such as transferrin and lactoferrin, and during infection the host further limits available iron by reducing plasma iron levels via ferroportins (7-9). In addition, lipocalin 2-mediated sequestration of iron has been shown to be an important antimycobacterial innate immune response (10). To compete for iron, M. tuberculosis produces siderophores, mycobactin and carboxymycobactin, which are highaffinity iron chelators (11). Since iron is a strong redox catalyst that can be toxic to cel...

Mycobacterium tuberculosis EsxH inhibits ESCRT-dependent CD4+ T-cell activation

“…EsxH forms a heterodimer with EsxG (EsxGH Mt ), which is secreted by the Esx-3 type VII secretion system. EsxGH Mt is involved in iron and zinc acquisition 21–25 , and recent work has demonstrated that EsxGH Mt also plays an additional role in virulence 20,26,27 . EsxGH Mt binds the host protein hepatocyte growth factor-regulated tyrosine kinase substrate (HGS, also known as HRS), a component of the ESCRT machinery 20 .…”

Rerouting of an Esx substrate pair from the ESX-1 type VII secretion system to ESX-5 by modifying a PE/PPE substrate pair

“…Mycobacteria employ specialized machineries, called type VII secretion systems (T7SSs) to secrete proteins across their complex cell envelope (1,2). Mycobacterium tuberculosis possesses five of such T7SSs, named ESX-1 to ESX-5 (1,2), of which ESX-1, ESX-3 and ESX-5 have been functionally analysed (3)(4)(5)(6)(7)(8)(9)(10). Each of these systems plays a different role in the mycobacterial life cycle.…”

“…and ESX-5 are necessary for iron and fatty acid uptake, respectively, making them essential for bacterial viability (7)(8)(9)(10). Besides their roles in nutrient and metabolite acquisition, ESX-3 and ESX-5 have also been shown to be involved in immune modulation of the host (9,17,18).…”

Virulence Factors and Pathogenicity of Mycobacterium

“…ESX-3 proteins: EsxG and EsxH are associated with the (proline-glutamic acid, proline-proline-glutamic acid) PE and PPE secretion [52]. The EsxG and EsxH heterodimer, which harms macrophage phagosome maturation, is secreted by the ESX-3 system [53] and inhibits the endosomal-sorting complex required for transport (ESCRT) impairing MTB antigen-specific CD4 + activation by macrophages and DC [54]. In M. abscessus ESX3 is composed by the genes esxH, esxG, esx-3, EsxG and EsxG proteins are related with enhancement of inflammatory cytokine generation in macrophages, M. abcessus esx3 mutant resulted in less inflammatory response [55].…”