Phosphorylation of PhoP Protein Plays Direct Regulatory Role in Lipid Biosynthesis of Mycobacterium tuberculosis

Goyal, Reena; Das, Arijit; Singh, Ranjeet; Singh, Pradip Kumar; Korpole, Suresh; Sarkar, Dibyendu

doi:10.1074/jbc.m111.307447

Cited by 41 publications

(63 citation statements)

References 51 publications

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“…Cultures of M. smegmatis strains harboring indicated lacZ fusions and the PhoP expression construct (or no expression plasmid as control) were grown in the absence or in presence of 50 ng/ml anhydrotetracycline, used as an inducer of PhoP expression. Promoter activation with induction of PhoP expression was assessed by measuring ␤-galactosidase activity (in Miller units) of crude cell lysates as detailed earlier (54). TetR-controlled PhoP expression in crude lysates of M. smegmatis (Ϸ5 g of protein) was verified by Western blot analysis using anti-PhoP antibody (Alpha Omega Sciences, India).…”

Section: Methodsmentioning

confidence: 99%

“…Reporter Assays in M. smegmatis-Electro-competent M. smegmatis transformed with pME1mL1-phoP expressed M. tuberculosis PhoP from the P myc1 tetO promoter under the control of the TetR repressor as described previously (54). Cultures of M. smegmatis strains harboring indicated lacZ fusions and the PhoP expression construct (or no expression plasmid as control) were grown in the absence or in presence of 50 ng/ml anhydrotetracycline, used as an inducer of PhoP expression.…”

Section: Methodsmentioning

confidence: 99%

“…Cloning, Bacteria Preparation, and Culture Filtrate Analysis-Plasmid DNA isolation, restriction digestion, and electrophoresis by agarose gels were carried out by standard protocol as described previously (54,55). To express phoP, espB, espR, and espACD in ⌬phoP M. tuberculosis, appropriate ORFs were cloned in p19KPro (56) using primer pairs FPphoP19K/RPphoP19K, FPespB19K/RPespB19K, FPespR19K/ RPespR19K, and PespACD19K/RPespACD19K, respectively (supplemental Table S1).…”

Section: Methodsmentioning

confidence: 99%

“…H, PhoP regulates expression of espAup by specific recognition of the direct repeat motif. WT M. smegmatis harboring espAup-lacZ and espAupmut-lacZ fusions were grown, and ␤-galactosidase activities with or without inducing M. tuberculosis PhoP expression were measured at 24-h time point as described earlier (54). The results show the average values with standard error of mean (S.E.)…”

Section: Ectopic Expression Of Espacd Bypasses Phop Functioning In Phmentioning

confidence: 99%

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EspR-dependent ESAT-6 Protein Secretion of Mycobacterium tuberculosis Requires the Presence of Virulence Regulator PhoP

Kumar

Goyal

Bansal

et al. 2016

Journal of Biological Chemistry

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Attenuation of Mycobacterium bovis BCG strain is related to the loss of the RD1-encoded ESX-1 secretion system. The ESX-1 system secretes virulence factor ESAT-6 that plays a critical role in modulation of the host immune system, which is essential for establishment of a productive infection. Previous studies suggest that among the reasons for attenuation of Mycobacterium tuberculosis H37Ra is a mutation in the phoP gene that interferes with the ESX-1 secretion system and inhibits secretion of ESAT-6. Here, we identify a totally different and distinct regulatory mechanism involving PhoP and transcription regulator EspR on transcriptional control of the espACD operon, which is required for ESX-1-dependent ESAT-6 secretion. Although both of these regulators are capable of influencing espACD expression, we show that activation of espACD requires direct recruitment of both PhoP and EspR at the espACD promoter. The most fundamental insights are derived from the inhibition of EspR binding at the espACD regulatory region of the phoP mutant strain because of PhoP-EspR protein-protein interactions. Based on these results, a model is proposed suggesting how PhoP and EspR protein-protein interactions contribute to activation of espACD expression and, in turn, control ESAT-6 secretion, an essential pathogenic determinant of M. tuberculosis. Together, these results have significant implications on the mechanism of virulence regulation of M. tuberculosis.Mycobacterium tuberculosis uses the ESX-1 secretion system to transport virulence factors in host cells (1-5). ESX-1 secretion system is encoded by the genes of the esx-1 locus, which is highly conserved in members of the M. tuberculosis complex and in other pathogenic mycobacteria (5-9). The best known ESX-1 substrates, the secreted proteins EsxA (ESAT-6) and EsxB (CFP10), have been implicated in the majority of the ESX-1-dependent modulations of host cell defense (10 -19) and therefore are considered to be essential for virulence (12)(13)(14). Consistently, deletion of the esx-1 locus abrogates ESX-1-dependent secretion and strongly attenuates M. tuberculosis (15,20). Notably, the genes encoding esx-1, located within the M. tuberculosis RD1 locus, are absent in the attenuated Mycobacterium bovis BCG and the potential vaccine strain Mycobacterium microti (15,17). However, a chromosomally unlinked non-RD1 locus (Rv3616c-Rv3615c-Rv3614c, also designated as espA, espC, and espD) is essential for ESX-1 function (13, 21). In fact, EspA and EspC proteins themselves are substrates of the ESX-1 secretion system, thus constituting mutually dependent secretion of substrates, a notable feature of this secretion system. More recently, EspA of the tubercle bacilli has been implicated as a critical mediator of bacterial cell wall integrity and ESX-1-dependent virulence regulation (22).ESX-1 represents the first and the most characterized member of the ESX secretion family. Although intracellular concentrations of ESAT-6 are similar in both virulent M. tuberculosis H37Rv and the attenuated...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ectopic Expression Of Espacd Bypasses Phop Functioning In Phmentioning

confidence: 99%

See 2 more Smart Citations

EspR-dependent ESAT-6 Protein Secretion of Mycobacterium tuberculosis Requires the Presence of Virulence Regulator PhoP

Kumar

Goyal

Bansal

et al. 2016

Journal of Biological Chemistry

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“…Deletion of phoPR resulted in decreased expression of espA, blocked secretion of ESAT-6 and attenuated virulence (Frigui et al, 2008;Pang et al, 2013;Ryndak et al, 2008;Walters et al, 2006). PhoP recognizes a sequence consisting of two or three direct repeats, which have been identified in its own promoter and several others (Cimino et al, 2012;Goyal et al, 2011;Gupta et al, 2006Gupta et al, , 2009Pathak et al, 2010). A point mutation in S219A in the DNA-binding motif of PhoP in M. tuberculosis strain H37Ra decreases the binding affinity of PhoP for the phoP promoter (Lee et al, 2008), and results in the attenuation of this strain (Frigui et al, 2008;Lee et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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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Transcriptional activation of the Mycobacterium tuberculosis virulence‐associated small RNA MTS1338 by the response regulators DosR and PhoP

Kumar,

Dutta

2024

FEBS Letters

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MTS1338, a distinctive small RNA in pathogenic mycobacteria, plays a crucial role in host–pathogen interactions during infection. Mycobacterial cells encounter heterogeneous stresses in macrophages, which highly upregulate MTS1338. A dormancy regulatory factor DosR regulates the intracellular abundance of MTS1338. Herein, we investigated the interplay of DosR and a low pH‐inducible gene regulator PhoP binding to the MTS1338 promoter. We identified that DosR strongly binds to two regions upstream of the MTS1338 gene. The proximal region possesses a threefold higher affinity than the distal site, but the presence of both regions increased the affinity for DosR by > 10‐fold. PhoP did not bind to the MTS1338 gene but binds to the DosR‐bound MTS1338 gene, suggesting a concerted mechanism for MTS1338 expression.

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Phosphorylation of PhoP Protein Plays Direct Regulatory Role in Lipid Biosynthesis of Mycobacterium tuberculosis

Cited by 41 publications

References 51 publications

EspR-dependent ESAT-6 Protein Secretion of Mycobacterium tuberculosis Requires the Presence of Virulence Regulator PhoP

EspR-dependent ESAT-6 Protein Secretion of Mycobacterium tuberculosis Requires the Presence of Virulence Regulator PhoP

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

Transcriptional activation of the Mycobacterium tuberculosis virulence‐associated small RNA MTS1338 by the response regulators DosR and PhoP

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