The ESX-4 substrates, EsxU and EsxT, modulate Mycobacterium abscessus fitness

Abstract

ESX type VII secretion systems are complex secretion machineries spanning across the mycobacterial membrane and play an important role in pathogenicity, nutrient uptake and conjugation. We previously reported the role of ESX-4 in modulating Mycobacterium abscessus intracellular survival. The loss of EccB4 was associated with limited secretion of two effector proteins belonging to the WXG-100 family, EsxU and EsxT, and encoded by the esx-4 locus. This prompted us to investigate the function of M. abscessus EsxU and EsxT in vitro and in vivo. Herein, we show that EsxU and EsxT are substrates of ESX-4 and form a stable 1:1 heterodimer that permeabilizes artificial membranes. While expression of esxU and esxT was up-regulated in M. abscessus-infected macrophages, their absence in an esxUT deletion mutant prevented phagosomal membrane disruption while maintaining M. abscessus in an unacidified phagosome. Unexpectedly, the esxUT deletion was associated with a hyper-virulent phenotype, characterised by increased bacterial loads and mortality in mouse and zebrafish infection models. Collectively, these results demonstrate that the presence of EsxU and EsxT dampens survival and persistence of M. abscessus during infection.

The cell envelope of Mycobacterium abscessus and its role in pathogenesis

“…The effects of ESX-4 are known to be substrate dependent since deletion of ESX-4 core complex genes results in lower expression of EsxU and EsxT (ESX-4 substrates) as well as PE-PPE proteins (ESX-3 substrates) [4]. In line with these findings, a recent study shows that EsxU and EsxT from M. abscessus form a heterodimer involved in phagosome membrane damage of macrophages [65]. Therefore, the EsxU/EsxT pair is crucial for inducing membrane permeability, which is advantageous during early stages of bacterial infection.…”

“…In line with these findings, a recent study shows that EsxU and EsxT from M . abscessus form a heterodimer involved in phagosome membrane damage of macrophages [ 65 ]. Therefore, the EsxU/EsxT pair is crucial for inducing membrane permeability, which is advantageous during early stages of bacterial infection.…”

“…Therefore, the EsxU/EsxT pair is crucial for inducing membrane permeability, which is advantageous during early stages of bacterial infection. However, when tested in vivo in zebrafish and mice, knockout mutants of EsxU/EsxT induced hypervirulence via increased bacterial growth and granuloma/abscess formation [ 65 ]. These findings from 2 different experimental settings imply that the ESX-4 substrates may be essential during the initial stages of infection and down-regulated subsequently to induce hypervirulence and spread of the infection.…”

Role of the Mycobacterium tuberculosis ESX-4 Secretion System in Heme Iron Utilization and Pore Formation by PPE Proteins

“…It has been suggested that EsxUT is the primary secretion substrate of ESX-4, but previous studies in Mtb and M. smegmatis have only examined esxUT gene expression through sigma factor overexpression ( 57 , 58 ) and esxUT gene transcript levels in the context of mycobacterial distributive conjugal transfer ( 34 , 44 ). While EsxUT secretion has been observed in M. abscessus ( Mab ), it should be noted that the Mab EsxT homolog contains the H xxxD/Exx h xxx H motif necessary for secretion ( 59 ). To surmise, we do not actually know if EsxUT is secreted in Mtb , which may explain why EsxUT was not detected in our proteomic analysis.…”

The small non-coding RNA B11 regulates multiple facets of Mycobacterium abscessus virulence

“…This initially suggested that part of the explanation for the increased virulence could be increased ESX-4 activity. However, a recent paper found that specific deletion of the secretion substrates alone ( esxU and esxT ) caused a hypervirulent phenotype in mice, suggesting the role of ESX-4 in virulence is complex and still poorly understood [ 85 ]. The exact interplay, therefore, of B11, ESX-4 as a whole, specifically the ESX-4 secretion substrates and bacterial virulence should continue to be a focus of intense research.…”