The ESX-2 type VII secretion system is required for Mycobacterium tuberculosis survival under nutrient starvation and...

ESX type VII secretion systems of Mycobacterium tuberculosis (Mtb) export proteins that are essential for bacterial virulence and adaptation to the host. While ESX-1, ESX-3, and ESX-5 have been extensively characterized, the physiological role of ESX-2 and its impact on host-pathogen interactions remain largely unexplored. Experimental evidence suggests that the esx-2 expression is regulated by WhiB5, a transcriptional regulator induced in in vitro nutrient-starvation conditions, and required for Mtb reactivation in the mouse infection model. This study aimed at characterizing the ESX-2 secretion system, with a focus on its role in Mtb adaptation/survival under nutrient-related stress conditions and in virulence. The analysis of the esx-2 gene expression profiles revealed an upregulation of selected esx-2 genes during Mtb cultivation in nutrient-depleted media or upon reactivation from a hypoxia-induced dormancy status. Consistently, the characterization of a panel of Mtb ESX-2 mutant strains (inactivated for selected esx-2 genes encoding ESX-2 components or deleted for the entire esx-2 locus) demonstrated an involvement of ESX-2 in Mtb survival in nutrient starvation and in promoting Mtb growth after reactivation from dormancy. Moreover, the intracellular growth kinetics of ESX-2 mutants in ex vivo models (THP-1 and A549 cells) and their attenuation profiles in the severe combined immunodeficient mice indicated that a functional ESX-2 is required for optimal Mtb replication inside the host. Overall, these findings identify ESX-2 as a modulator of Mtb survival under selected nutrient-starvation-related stress conditions peculiar to intracellular environments, thus contributing to the intracellular and in vivo Mtb growth.IMPORTANCEESX-2 is the second most recently evolved ESX secretion system in mycobacteria, largely conserved in Mycobacterium tuberculosis (Mtb) and other slow-growing pathogenic mycobacteria. Among the ESX secretion systems, ESX-2 is the least extensively characterized, and to date, its function is largely unknown. This study provides novel experimental evidence of the impact of ESX-2 on Mtb physiology and host-pathogen interaction. A functional ESX-2 is required for Mtb survival under hostile conditions, such as nutrient limitation conditions, and is involved in Mtb growth reactivation from a dormant state in vitro. Moreover, the observed role of ESX-2 in promoting Mtb replication in cellular and animal models emphasizes the impact of this secretion system in Mtb adaptation to the human host.