Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator.

Abdallah M Abdallah,Sabir A Adroub,Eveline M Weerdenburg,Wilbert Bitter,Qingtian Guan,Huoming Zhang,Stephanie Borggreve,Roy Ummels,Raeece Naeem,Tareq B Malas,Arnab Pain,Thomas D Otto

doi:10.1371/journal.pone.0211003

Abstract

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

Highlights

Mycobacteria use several different type VII secretion systems (T7S) to transport proteins across their thick and waxy cell envelopes
We determined the transcriptomes of the M. marinum E11 wild-type and the double-auxotrophic M. tuberculosis mc26020 mutant strains and compared these transcriptomes with those of the corresponding isogenic esx-1 mutants
We found that during the growth of M. marinum in 7H9 culture medium, genes encoding ESX-1 substrates, such as EsxA and other ESX-1-associated proteins, were down-regulated in the mutant strains, whereas the transcription of genes encoding several structural components of the ESX-1 system remained unaffected

Summary

Introduction

Mycobacteria use several different type VII secretion systems (T7S) to transport proteins across their thick and waxy cell envelopes One of these T7S systems, ESX-1, is responsible for the transport of a number of important virulence factors. In the case of pathogenic mycobacteria, such as M. tuberculosis and the fish pathogen Mycobacterium marinum, ESX-1 is responsible for the translocation of the bacteria from the phagolysosomal compartments to the cytosols of macrophages [3,4,5]. This translocation activity has been attributed to the ESX-1 substrate EsxA (previously known as ESAT-6) [6, 7]. These other substrates could be involved in other proposed functions of ESX-1 in pathogenic mycobacterial species, including host cell entry and intercellular spread [11,12,13]

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Results

Conclusion