EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in Mycobacterium tuberculosis by Affecting Bacterial Cell Wall Integrity

Garces, Alejandra; Atmakuri, Krishnamohan; Chase, Michael R.; Woodworth, Joshua S.; Krastins, Bryan; Rothchild, Alissa C.; Ramsdell, Talia L.; Lopez, Mary F.; Behar, Samuel M.; Sarracino, David; Fortune, Sarah M.

doi:10.1371/journal.ppat.1000957

Cited by 94 publications

(125 citation statements)

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“…EspR regulates multiple genes associated with the synthesis of cell-wall lipids, including phthiocerol dimycocerosate, phenol glycolipid and mycolic acid (Blasco et al, 2012), and the ESX-1 system itself is important for the stability of the cell wall (Garces et al, 2010). Notably, both MprAB and PhoPR are also associated with regulation of the response to cell-wall damage and/or regulation of lipid synthesis (He et al, 2006;Walters et al, 2006), and we identified several genes that are shared by the EspR and PhoP regulons (Walters et al, 2006).…”

Section: Discussionmentioning

confidence: 89%

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

et al. 2015

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EspR, a regulator of the ESX-1 secretion system in Mycobacterium tuberculosis, is directly regulated by the two-component systems MprAB and PhoPR INTRODUCTIONESX-1 (ESAT-6 secretion system-1) is a major virulence determinant of Mycobacterium tuberculosis, the causative agent of human tuberculosis. ESX-1 exports pathogenic and immunogenic proteins such as ESAT-6 (EsxA), EspA and other substrate proteins into host cells (Simeone et al., 2009). The secretion of EspA and ESAT-6 is mutually dependent, with deletion of espA resulting in loss of ESAT-6 secretion, and vice versa (Fortune et al., 2005). EspA is encoded within the espA operon, while esxA is located in RD-1 (Region of difference 1), which contains most ESX-1 genes (Mahairas et al., 1996). The intergenic region upstream of espA contains known or predicted binding sites for several different regulatory factors (Hunt et al., 2012;Pang et al., 2013;Rickman et al., 2005; Rosenberg et al., 2011), suggesting that ESX-1 activity could be modulated via altering espA expression.EspR, a key transcriptional regulator of ESX-1, directly regulates espA (Raghavan et al., 2008; Rosenberg et al., 2011). An espR mutant exhibited decreased transcription of the espA operon, loss of ESAT-6 secretion and reduced virulence (Raghavan et al., 2008). 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 aAbbreviations: CRP, cAMP receptor protein; EMSA, electrophoresis mobility shift assay; ESX-1, ESAT-6 secretion system-1; L6, lineage 6; RD-1, Region of difference 1; TCS, two-component system.

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Section: Discussionmentioning

confidence: 89%

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

et al. 2015

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“…These findings augment the growing evidence that genes associated with the ESX-1 system contribute to the structure and integrity of the cell envelope. The espA operon is activated by thioridazine, an agent that damages the cell envelope (42), and an espA mutant was more susceptible to the detergent SDS (19). Furthermore, a recent paper reports that EspR regulates numerous genes involved in cell wall function and that it binds within the Rv0986-to-Rv0989c region (24), part of the in vivo genomic island containing mprAB (43).…”

Section: Discussionmentioning

confidence: 99%

“…The basis for this hypergrowth phenotype is unknown and is surprising in view of the decreased secretion of ESAT-6, EspA, and EspB, all virulence factors (5,11,19,53). However, Rv-D981 secretes low levels of these proteins, and perhaps this is sufficient for some functions.…”

Section: Fig 5 Secretion Of Esx-1 Substrate Proteins Is Dysregulated mentioning

confidence: 99%

“…Secretion of EspA and ESAT-6 is mutually dependent, with deletion of espA resulting in loss of ESAT-6 secretion and attenuation (17). Disruption of disulfide bond formation in EspA retains ESX-1 function but produces a severely attenuated strain, indicating that EspA is itself a major virulence determinant (19). EspC contains a signal sequence required for secretion of EspA and ESAT-6 (20), and EspD stabilizes cellular levels of EspA and EspC (16).…”

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confidence: 99%

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MprAB Regulates theespAOperon in Mycobacterium tuberculosis and Modulates ESX-1 Function and Host Cytokine Response

et al. 2013

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dThe ESX-1 secretion system exports the immunomodulatory protein ESAT-6 and other proteins important in the pathogenesis of Mycobacterium tuberculosis. Components and substrates of ESX-1 are encoded at several loci, but the regulation of the encoding genes is only partially understood. In this study, we investigated the role of the MprAB two-component system in the regulation of ESX-1 activity. We determined that MprAB directly regulates the espA gene cluster, a locus necessary for ESX-1 function. Transcript mapping determined that the five genes in the cluster form an operon with two transcriptional start points, and several MprA binding sites were detected in the espA promoter. Expression analyses and promoter constructs indicated that MprAB represses the espA operon. However, the MprAB mutant Rv-D981 secreted lower levels of EspA, ESAT-6, and the ESX-1 substrate EspB than control strains. Secretion of CFP10, which is normally cosecreted with ESAT-6, was similar in Rv-D981 and control strains, further demonstrating aberrant ESX-1 activity in the mutant. ESAT-6 induces proinflammatory cytokines, and macrophages infected with Rv-D981 elicited lower levels of interleukin 1␤ (IL-1␤) and tumor necrosis factor alpha (TNF-␣), consistent with the reduced levels of ESAT-6. These findings indicate that MprAB modulates ESX-1 function and reveal a new role for MprAB in host-pathogen interactions.

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“…Because ESX-1 is a transport system lacking in BCG, lack of export of an ESX-1-dependent lipid synthase might account for the loss of substance A. However, ESX-1-deficient M. tuberculosis lacking either the espA gene (Rv3616c) or the entire RD1 locus (17), which are both necessary for ESX-1 function, produces substance A at normal levels (SI Appendix, Fig. S1C).…”

Section: Resultsmentioning

confidence: 99%

Molecular profiling ofMycobacterium tuberculosisidentifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

Layre

Lee

Young

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.TbAd | terpenyl transferase W ith a mortality rate exceeding 1.5 million deaths annually, Mycobacterium tuberculosis remains one of the world's most important pathogens (1). M. tuberculosis succeeds as a pathogen because of productive infection of the endosomal network of phagocytes. Its residence within the phagosome protects it from immune responses during its decades long infection cycle. However, intracellular survival depends on active inhibition of pH-dependent killing mechanisms, which occurs for M. tuberculosis but not species with low disease-causing potential (2). Intracellular survival is also enhanced by an unusually hydrophobic and multilayered protective cell envelope. Despite study of this pathogen for more than a century, the spectrum of natural lipids within M. tuberculosis membranes is not yet fully defined. For example, the products of many genes annotated as lipid synthases remain unknown (3), and mass spectrometry detects hundreds of ions that do not correspond to known lipids in the MycoMass and LipidDB databases (4, 5).To broadly compare the lipid profiles of virulent and avirulent mycobacteria, we took advantage of a recently validated metabolomics platform (4). This high performance liquid chromatography-mass spectrometry (HPLC-MS)...

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EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in Mycobacterium tuberculosis by Affecting Bacterial Cell Wall Integrity

Cited by 94 publications

References 54 publications

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

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

MprAB Regulates theespAOperon in Mycobacterium tuberculosis and Modulates ESX-1 Function and Host Cytokine Response

Molecular profiling ofMycobacterium tuberculosisidentifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

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