A Specific Polymorphism in Mycobacterium tuberculosis H37Rv Causes Differential ESAT-6 Expression and Identifies WhiB6 as a Novel ESX-1 Component

Abstract

dThe ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors of Mycobacterium tuberculosis, given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates of M. tuberculosis produce and secrete larger amounts of ESAT-6 than the widely used M. tuberculosis H37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed that whiB6 (rv3862c), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of the ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publicly available M. tuberculosis complex genomes or in any of the 76 clinical M. tuberculosis isolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulates whiB6 expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy of whiB6 in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that in M. tuberculosis-with the exception of the H37Rv strain-ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.

MTBVAC from discovery to clinical trials in tuberculosis-endemic countries

“…For example, a single-nucleotide insertion in the whiB6 promoter in M. tuberculosis H37Rv has shifted the role of PhoP from transcriptional activator to transcriptional repressor, resulting in lower levels of ESAT-6 expression and secretion in H37Rv [43]. This polymorphism was shown to be absent in any of the other publicly available MTBC genomes or in 76 clinical M. tuberculosis isolates (including MTBVAC parental strain Mt103) analyzed [43].…”

“…This isolate belongs to the modern M. tuberculosis Lineage 4, which together with Lineage 2 (Beijing strains), represents the most geographically widespread lineages of MTBC transmitted by the aerosol route between humans [40]. The primary reasons for using a clinical strain for MTBVAC design and construction were to achieve a representative antigen repertoire of the clinical M. tuberculosis strains transmitted between humans and at the same time to avoid the inherent effects that come with laboratory strains (such as H37Rv) as result of repeated sub-cultivation in vitro [41][42][43][44], the same process that lead to BCG attenuation (between 1908 and 1921) and strain diversity afterwards [1]. For example, a single-nucleotide insertion in the whiB6 promoter in M. tuberculosis H37Rv has shifted the role of PhoP from transcriptional activator to transcriptional repressor, resulting in lower levels of ESAT-6 expression and secretion in H37Rv [43].…”

“…The gene phoP encodes the transcription factor of the two-component virulence system PhoPR and the gene fadD26 is one of the 13 genes responsible for biosynthesis and export of phthiocerol dimycocerosates (PDIM), the main virulence-associated cell-wall lipids of M. tuberculosis [54][55][56]. PhoP has been shown to regulate more than 2% of M. tuberculosis genome, most of which implicated in virulence [57,58], and this regulation could be mediated through other transcription factors, for example, Whib6 of the ESX-1 system [43] or via noncoding RNAs (ncRNAs), such as the recently described mcr7 and its role in activating the TAT secretion system in MTBVAC leading to over secretion of major antigens such as the Ag85 complex [59]. Other relevant virulence genes regulated by PhoP include lipid metabolism genes (e.g.…”

“…The primary reasons for using a clinical strain for MTBVAC design and construction were to achieve a representative antigen repertoire of the clinical M. tuberculosis strains transmitted between humans and at the same time to avoid the inherent effects that come with laboratory strains (such as H37Rv) as result of repeated sub-cultivation in vitro [41][42][43][44], the same process that lead to BCG attenuation (between 1908 and 1921) and strain diversity afterwards [1]. For example, a single-nucleotide insertion in the whiB6 promoter in M. tuberculosis H37Rv has shifted the role of PhoP from transcriptional activator to transcriptional repressor, resulting in lower levels of ESAT-6 expression and secretion in H37Rv [43]. This polymorphism was shown to be absent in any of the other publicly available MTBC genomes or in 76 clinical M. tuberculosis isolates (including MTBVAC parental strain Mt103) analyzed [43].…”

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

“…PhoP is part of a two-component system that regulates several virulence pathways in M. tuberculosis, including biosynthetic genes of the M. tuberculosis-specific lipids, sulfolipid, DAT, and PAT (103). In addition to regulating lipid biosynthesis genes, PhoP also regulates whiB6 and works directly with EspR, and both regulate ESX-1 gene expression (96,104,105). Therefore, under many of the conditions in which cell envelope remodeling may be occurring or where cell wall genes are regulated, ESX genes are also regulated.…”