Virulence Regulator EspR of Mycobacterium tuberculosis Is a Nucleoid-Associated Protein

Abstract

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.

Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

“…This could indicate that EspR regulates ponA1 transcription. Indeed, we found multiple canonical EspR binding sites (23) in the ponA1 promoter region (Fig. S3).…”

“…Allelic exchange occurs as described (16,23). The ΔponA1 L5::ponA1 wt loxed strain wherein ponA2, rv1086, or ldtB was deleted was used for allelic exchange.…”

EspR, a regulator of the ESX-1 secretion system in Mycobacterium tuberculosis, is directly regulated by the two-component systems MprAB and PhoPR

“…To define the role of MprA Region-2, the BOX2/3-M type mutation was introduced into pXP-1 to obtain pXP-11(BOX2/3-M), which was used to generate the complemented strain C11(BOX2/3-M). In pXP-12, MprA Region-1 was removed, leaving a shortened (Blasco et al, 2012) are underlined. Numbers are given relative to the start codon, which is marked by a bent arrow.…”

“…Higher-order oligomerization of EspR appears to be involved in cooperatively linking distant DNA sites, such as the three EspR binding sites in the espA promoter (Blasco et al, 2011; Rosenberg et al, 2011). Three EspR sites were also identified in the espR promoter (Blasco et al, 2012). However, these EspR sites are much closer than in the espA promoter, suggesting that EspR may have a…”

A Novel Mechanism of Growth Phase-dependent Tolerance to Isoniazid in Mycobacteria

“…Immunoblotting was carried out after SDS-PAGE and transfer of proteins to a PVDF membrane by incubation with anti-MDP1 and anti-KatG antibodies, as described previously (25). Anti-heat shock protein Hsp65 (GroEL2) antibody was used as a loading control (26,27). The expression levels of different proteins were analyzed using the public domain software Image J (a Java image processing program developed by the National Institutes of Health Image for Macintosh).…”