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

Abstract

ABSTRACTThe ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors ofMycobacterium tuberculosis, given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates ofM. tuberculosisproduce and secrete larger amounts of ESAT-6 than the widely usedM. tuberculosisH37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed thatwhiB6(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 availableM. tuberculosiscomplex genomes or in any of the 76 clinicalM. tuberculosisisolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulateswhiB6expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy ofwhiB6in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that inM. 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.