Characterization of Mycobacterium abscessus EsxU/EsxT complex

Abstract

Mycobacterium abscessus (Mab) is a nontuberculous mycobacterium capable of producing pulmonary and disseminated infections in humans. These infections are very difficult to treat due to innate and acquired antibiotic resistance that render a scenario of limited and suboptimal therapeutic options, the majority of which significantly reduce the patient’s quality of life. The resistance mechanisms combined with the ability of Mab to evade the immune system via the formation of giant cords, to shield itself from antibiotic therapy though the induction of granulomas and biofilms, and to hide inside host phagocytes, represent a formidable set of virulence and defence mechanisms that turn Mab into one of the most challenging human pathogens. Understanding the foundations of its virulence and pathogenicity could unveil new ways for fighting Mab infections.

Secretion systems are associated with the transport of virulence factors and the persistence of mycobacteria within the immune cells. In particular, some Type VII secretion systems (T7SS or ESX) are involved in host-pathogen interactions. Mab possesses only ESX-3 and ESX-4. ESX-4 contains 5 genes encoding proteins that form a structure that spans the inner membrane, and 3 additional genes which products have not been previously characterized. However, a recent study proposed that ESX-4 was associated to Mab virulence. In this study, we have characterized two proteins from Mab ESX-4 locus, EsxU and EsxT, previously thought to be secreted factors.

Our results show that Mab EsxU and EsxT are helical proteins that form a 1:1 soluble heterodimer, as previously reported for other Esx protein pairs. Through cloning, expression and purification experiments we found that EsxU/EsxT is a substrate of Mab ESX-4 and that this system is functional. Subcellular localization suggests that the heterodimer associates with the mycomembrane, maintaining a weak bond with the inner membrane complex. Cytokine secretion and expression experiments indicate that this heterodimer does not activate an immune response on its own, nor induce lysis or apoptosis in human phagocytes, nor act as an efflux-pump protecting the bacteria from antibiotics. However, experiments conducted in collaboration with research groups at IRIM and UVSQ show that EsxU/EsxT has a role in host-pathogen interaction and in the fate of the mycobacteria inside the phagosome. RT-PCR experiments showed that both esxU and esxT are expressed in larger quantities than the ESX-4 structural components in the Mab ATCC 19977 type strain. Their expression levels are even greater in clinical Mab isolates, suggesting a critical role during infection. Nevertheless, esxU and esxT are not essential as evidenced by the successful generation of double knock-out strains by our collaborators. Biophysical experiments indicated that the EsxU/EsxT heterodimer interacts with membranes, probably forming a high-order porin-like structure, and that this interaction depends on the pH of the environment, the protein concentration, the transmembrane voltage, and the lipid composition of the membrane. By performing similar biophysical experiments with an EsxU/EsxTA43W mutant, we found that the WXG motif plays and important role in porin formation.

The present study presents, for the first time, an overview of the localization, roles and structure of the Mab EsxU/EsxT heterodimer, with insight on how mutations on the WXG motif affect the protein interaction with membranes. Further research is needed to clarify EsxU/EsxT function and relevance during infection. Likewise, a deeper investigation is required to fully understand the role of the WXG motifs and secretion signals in the localization and function of Esx proteins, and to study how these proteins evolved to fulfil different roles.