The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes. A major infection mechanism employed by the causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), is the ESX-1 secretion system. It has been postulated that the DNA-binding protein EspR controls the virulence of Mtb by specifically regulating expression of the exported EspA protein, which is required for ESX-1 to function. Previous structural studies indicated that EspR forms dimers capable of multimerizing on DNA and forming loop structures, thus bringing together otherwise distant chromosomal regions. Such characteristics are reminiscent of nucleoid-associated proteins (NAPs), the histone equivalent in bacteria. Here we use ChIP-Seq technology to map EspR binding sites on the Mtb chromosome in living bacterial cells. Genome-wide analysis of EspR identified hundreds of binding-sites, with almost equal inter- and intra-genic distribution, and mostly found in proximity to genes associated with cell wall function. We validated a subset of EspR-binding sites experimentally and identified a consensus motif required for optimal binding affinity. Moreover, our study reveals that EspR expression varies with bacterial growth and that intracellular levels are not linked to EspR secretion. These findings corroborate the NAP nature of EspR and its dual roles, architectural and regulatory, that impact the Mtb chromosome and pathogenesis globally rather than the ESX-1 loci specifically.

The lipid-rich cell wall is a defining feature ofMycobacteriumspecies. Individual cell wall components affect diverse mycobacterial phenotypes including colony morphology, biofilm formation, antibiotic resistance, and virulence. In this study, we describe a transposon insertion mutant ofMycobacterium smegmatismc²155 that exhibits altered colony morphology and defects in biofilm formation. The mutation was localized to thelsr2gene. First identified as an immunodominant T-cell antigen ofMycobacterium leprae,lsr2orthologs have been identified in all sequenced mycobacterial genomes, and homologs are found in many actinomycetes. Although its precise function remains unknown, localization experiments indicate that Lsr2 is a cytosolic protein, and cross-linking experiments demonstrate that it exists as a dimer. Characterization of cell wall lipid components reveals that theM. smegmatis lsr2mutant lacks two previously unidentified apolar lipids. Characterization by mass spectrometry and thin-layer chromatography indicate that these two apolar lipids are novel mycolate-containing compounds, called mycolyl-diacylglycerols (MDAGs), in which a mycolic acid (α- or α′-mycolate) molecule is esterified to a glycerol. Upon complementation with an intactlsr2gene, the mutant reverts to the parental phenotypes and MDAG production is restored. This study demonstrates that due to its impact on the biosynthesis of the hydrophobic MDAGs, Lsr2 plays an important role in the colony morphology and biofilm formation ofM. smegmatis.

Lsr2 is a small, basic protein present in Mycobacterium and related actinomycetes. Recent studies suggest that Lsr2 is a regulatory protein involved in multiple cellular processes including cell wall biosynthesis and antibiotic resistance. However, the underlying molecular mechanisms remain unknown. In this article, we performed biochemical studies of Lsr2–DNA interactions and structure–function analysis of Lsr2. Analysis by atomic force microscopy revealed that Lsr2 has the ability to bridge distant DNA segments, suggesting that Lsr2 plays a role in the overall organization and compactness of the nucleoid. Mutational analysis identified critical residues and selection of dominant negative mutants demonstrated that both DNA binding and protein oligomerization are essential for the normal functions of Lsr2 in vivo . These results provide strong evidence that Lsr2 is a DNA bridging protein, which represents the first identification of such proteins in bacteria phylogenetically distant from the Enterobacteriaceae. DNA bridging by Lsr2 also provides a mechanism of transcriptional regulation by Lsr2.

ESAT-6 system 1 (ESX-1)-mediated secretion in Mycobacterium tuberculosis is dependent on proteins encoded by the cotranscribed espA-espC-espD gene cluster. While the roles of EspA and EspC with respect to the ESX-1 secretion system have been actively investigated, the function of EspD remains unknown. We show that EspD is secreted by M. tuberculosis , but unlike EspA and EsxA, its export does not exclusively require the ESX-1 system. Evidence for stabilization of cellular levels of EspA and EspC by EspD is presented, and depletion of EspD results in loss of EsxA secretion. Site-directed mutagenesis of EspD reveals that its role in the maintenance of cellular levels of EspA in M. tuberculosis is distinct from its facilitation of EsxA secretion. The same mutagenesis experiments have also shown that secretion of EspD is not required for the secretion of EsxA. Our findings highlight a critical and complex role for EspD in modulating the ESX-1 secretion system in M. tuberculosis .

Background. The pathogenesis of Mycobacterium tuberculosis largely depends on the secretion of the 6-kD early secreted antigenic target ESAT-6 (EsxA) and the 10-kD culture filtrate protein CFP-10 (EsxB) via the ESX-1/typeVII secretion system. Although gene products from the core RD1 region have been shown to be deeply implicated in this process, less is known about proteins encoded further upstream in the 5′ region of the ESX-1 cluster, such as the ESX-1 secretion–associated proteins (Esps) EspF or EspG₁. Methods. To elucidate the role of EspF/G₁, whose orthologs in Mycobacterium marinum and Mycobacterium smegmatis are reportedly involved in EsxA/B secretion, we constructed 3 M. tuberculosis knockout strains deleted for espF, espG₁ or the segment corresponding to the combined RD1^bcg-RD1^mic region of bacille Calmette-Guérin (BCG) and Mycobacterium microti, which also contains espF and espG₁. Results. Analysis of these strains revealed that, unlike observations with the model organisms M. smegmatis or M. marinum, disruption of espF and espG₁ in M. tuberculosis did not impact the secretion and T cell recognition of EsxA/B but still caused severe attenuation. Conclusions. The separation of the 2 ESX-1–connected phenotypes (ie, EsxA/B secretion and virulence) indicates that EsxA/B secretion is not the only readout for a functional ESX-1 system and suggests that other processes involving EspF/G₁ also play important roles in ESX-1–mediated pathogenicity.