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

ABSTRACT 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. IMPORTANCE ESX-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.