A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies WhiB6 as a novel ESX-1 component

Abstract

The 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.

FIG 1

The M. tuberculosis whiB6 gene is differentially regulated by PhoP, depending on the genetic background. (A) Schematic representation of ESX-1 and extended ESX-1 genes from M. tuberculosis. The diagram shows genetic regions from whiB6 (rv3862c) to mycP1 (rv3883c), espD (rv3614c) to espA (rv3616c), and espR (rv3849). Genes are represented by filled arrows and colored according to their putative function. Predicted operons are represented by thin arrows. The RD1 region deleted in M. bovis BCG is also indicated. (B) Heat map from microarray comparisons of H37Rv and GC1237 with their respective phoPR mutants. Only genes involved in ESAT-6 production and secretion are reported for clarity. The GC1237 phoPR mutant displays downregulation of most ESX-1 genes. In contrast, the H37Rv reference strain show divergent PhoP regulation of whiB6, esxB (rv3874), esxA (rv3875), eccD1 (rv3877), and espJ (rv3878) (indicated by asterisks) compared to strain GC1237. Note that whiB6 shows the most divergent PhoP regulation between both strains. (C) qRT-PCR analyses showing PhoP regulation over whiB6 in the H37Rv strain compared to those of MT103 and GC1237 clinical isolates. Note the divergent PhoP regulation in clinical strains compared to that of the reference standard H37Rv. Bars indicate fold changes in gene expression relative to the sigA gene used as endogenous control. Results represent the average of three independent experiments, and error bars indicate the standard deviation (SD) of the mean.

FIG 2

Expression profiles of ESX-1 genes show differential PhoP regulation in clinical and laboratory strains of M. tuberculosis. qRT-PCR measurements of representative genes from ESX-1 and extended ESX-1 regions involved in ESAT-6 secretion, showing PhoP-dependent expression in the H37Rv, MT103, and GC1237 strains. Expression of esxA and esxB genes correlates with that of the whiB6 gene in Fig. 1C, showing a divergent PhoP regulation in H37Rv compared to those of the MT103 and GC1237 clinical strains. In contrast, PhoP regulation of the espA, espC, and espD genes is independent of the genetic background. Bars indicate fold change in gene expression relative to the sigA gene used as an endogenous control. Results represent the average of three independent experiments, and error bars indicate the SD of the mean.

FIG 3

Production and secretion of ESAT-6 vary between clinical isolates and the H37Rv reference strain. (A) Immunoblots against ESAT-6 and GroEL2 in whole-cell extracts of MT103, H37Rv and GC1237 parent strains and their respective phoP mutants. MT103 and GC1237 phoP mutants show decreased production of ESAT-6 with respect to their WT strains. The H37Rv WT strain produces a smaller amount of ESAT-6 than the MT103 and GC1237 clinical strains. GroEL2 was used as a control of protein loading. (B) Immunoblots against ESAT-6 and GroEL2 in the secreted fraction of the MT103, H37Rv, and GC1237 WT strains. Note the decreased secretion of ESAT-6 in H37Rv compared to that in the clinical isolates. GroEL2 was used as a control for absence of cell lysis. (C) Immunoblots against ESAT-6 and SigA in the whole-cell lysate and secreted fraction of M. tuberculosis H37Rv, CDC1551, and Erdman strains, grown in Sauton medium for 7 days. Note the decreased amount of ESAT-6 in the whole-cell lysate and secreted fraction of H37Rv compared to those in the two other reference strains. Bars represent densitometric analysis of band intensity and are referred to the H37Rv strain. Results are representative of three independent experiments.

FIG 4

Study of M. tuberculosis complex strains reveals a point mutation in the whiB6 promoter exclusive of the H37Rv strain. (A) The whiB6 gene and flanking sequences were aligned with 100 available mycobacterial genomes. The image shows a single nucleotide insertion (labeled as a G in boldface) upstream of the whiB6 gene exclusively present in H37Rv/Ra, being absent in other M. tuberculosis complex strains. Note the presence of inverted repeats (depicted by arrows) that originated as a consequence of the guanine insertion in H37Rv. (B) Immunoprecipitation of M. tuberculosis DNA with anti-PhoP antibodies and subsequent qRT-PCR quantification of the region adjacent to the G-insertion in the whiB6 promoter. The results show an enrichment of immunoprecipitated DNA in the MT103 and H37Rv WT strains relative to their phoP mutants, indicative of PhoP binding to the whiB6 promoter. (C) Inverted repeats in panel A probably result in formation of the indicated stem-loop structure.

FIG 5

Extensive sequence analysis of M. tuberculosis clinical isolates reveals that whiB6 mutation is exclusive of H37Rv and independent of the M. tuberculosis lineage. The whiB6 promoter region of 76 clinical isolates from our laboratory collection was sequenced. The dendrogram based on spoligotyping of the clinical isolates reveals that the G insertion in H37Rv is independent of the lineage. Spoligotyping is further detailed in Fig. S1 in the supplemental material.

FIG 6

Reintroduction of a WT whiB6 allele in H37Rv restores ESAT-6 production and secretion to the levels of clinical strains. (A) Immunoblots against ESAT-6 and GroEL2 in whole-cell extracts of H37Rv-derived strains containing either a WT (KIMt) or a mutated (KIRv) whiB6 allele. MT103 and its phoP mutant were used as controls for ESAT-6 phenotypes in clinical strains. Introduction of a WT copy of whiB6 in H37Rv restores ESAT-6 production to levels comparable to those in the MT103 clinical isolate. Introduction of either a WT or mutated whiB6 allele in H37Rv phoP mutant does not significantly impact ESAT-6 production. GroEL2 was used as a control of protein loading. (B) Immunoblots against ESAT-6 and GroEL2 in the secreted fraction from strains used in panel A. The whiB6 WT allele is able to enhance ESAT-6 secretion in H37Rv to the levels of the MT103 clinical strain. The phoP mutants show absence of ESAT-6 secretion, independent of whiB6 expression. GroEL2 was used as a control for absence of cell lysis. Note that introduction of an additional “mutated” allele into H37Rv (H37Rv::KIRv) has no effect over ESAT-6 production or secretion. Bars represent densitometric analysis of band intensity and are referred to the H37Rv strain. (C) qRT-PCR measurements of the esxA, esxB, and espJ genes in KIMt and KIRv derivatives of H37Rv and its phoP mutant. Note that esxA, esxB, and, to a lesser extent, espJ require both WT alleles of phoP and whiB6 in order to reach expression levels comparable to those of the clinical isolates in Fig. 2. Bars indicate fold changes in gene expression relative to the sigA gene used as an endogenous control. Results are representative of three independent experiments, and error bars indicate the SD of the mean.