Dual-active oxadiazoles targeting the Mycobacterium tuberculosis ESX-1 secretion system and boosting ethionamide activity
Ontology highlight
ABSTRACT:
ORGANISM(S): Mycobacterium tuberculosis
PROVIDER: GSE226474 | GEO | 2024/01/01
REPOSITORIES: GEO
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Project description: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.
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Project description:Mycobacterium tuberculosis uses the ESX-1 secretion system to deliver virulence proteins during infection of host cells. Here we report a mechanism of posttranscriptional control of ESX-1 mediated by MycP1, a M. tuberculosis serine protease. We show that MycP1 is required for ESX-1 secretion but that, unexpectedly, genetic inactivation of MycP1 protease activity increases secretion of ESX-1 substrates. We demonstrate that EspB, an ESX-1 substrate required for secretion, is a target of MycP1 in vitro and in vivo. During macrophage infection, an inactive MycP1 protease mutant causes hyperactivation of ESX-1-stimulated innate signaling pathways. MycP1 is required for growth in mice during acute infection, while loss of its protease activity leads to attenuated virulence during chronic infection. As the key ESX-1 substrates ESAT-6 and CFP-10 are highly immunogenic, fine-tuning of their secretion by MycP1 may balance virulence and immune detection and be essential for successful maintenance of long-term M. tuberculosis infection.
| S-EPMC3121311 | biostudies-literature
Project description:Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE-PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25-PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB's role in M. tuberculosis pathogenesis.
| S-EPMC4520771 | biostudies-literature
Project description:ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.
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Project description:The function of EspI, a 70 kDa protein in Mycobacterium tuberculosis, has remained unclear. Although EspI is encoded by a gene within the esx-1 locus, in this study we clarify previous conflicting results and show that EspI is not essential for ESX-1-mediated secretion or virulence in M. tuberculosis. We also provide evidence that reduction of cellular ATP levels in wild-type M. tuberculosis using the drug bedaquiline completely blocks ESX-1-mediated secretion. Remarkably, M. tuberculosis lacking EspI fails to exhibit this phenotype. Furthermore, mutagenesis of a highly conserved ATP-binding motif in EspI renders M. tuberculosis incapable of shutting down ESX-1-mediated secretion during ATP depletion. Collectively these results show that M. tuberculosis EspI negatively regulates the ESX-1 secretion system in response to low cellular ATP levels and this function requires the ATP-binding motif. In light of our results the potential significance of EspI in ESX-1 function during latent tuberculosis infection and reactivation is also discussed.
| S-EPMC4150839 | biostudies-literature
Project description:Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF form a water-soluble heterodimer (EsxEF) that assembles into oligomers and long filaments, binds to membranes, and forms stable membrane-spanning channels. Electron microscopy of EsxEF reveals mainly pentameric structures with a central pore. Mutations of both WXG motifs and of a GXW motif do not affect dimerization, but abolish pore formation, membrane deformation and TNT secretion. The WXG/GXW mutants are locked in conformations with altered thermostability and solvent exposure, indicating that the WXG/GXW motifs are molecular switches controlling membrane interaction and pore formation. EsxF is accessible on the bacterial cell surface, suggesting that EsxEF form an outer membrane channel for toxin export. Thus, our study reveals a protein secretion mechanism in bacteria that relies on pore formation by small WXG proteins.
| S-EPMC7810871 | biostudies-literature
Project description:Rv3868, a conserved hypothetical protein of the ESAT-6 secretion system of Mycobacterium tuberculosis, is essential for the secretion of at least four virulence factors. Each protein chain is approximately 63 kDa and assembles into a hexamer. Limited proteolysis demonstrates that it consists of two domains joined by a linker. The N-terminal domain is a compact, helical domain of approximately 30 kDa and apparently functions to regulate the ATPase activity of the C-terminal domain and the oligomerization. The nucleotide binding site is situated in the C-terminal domain, which exhibits ATP-dependent self-association. It is also the oligomerization domain. Dynamic fluorescence quenching studies demonstrate that the domain is proximal to the C terminus in the apoprotein and exhibits a specific movement upon ATP binding. In silico modeling of the domains suggests that Arg-429 of a neighboring subunit forms a part of the binding site upon oligomerization. Mutational analysis of binding site residues demonstrates that the Arg-429 functions as the important "sensor arginine" in AAA-ATPases. Protein NMR experiments involving CFP-10 and activity assays rule out a general chaperone-like function for Rv3868. On the other hand, ATP-dependent "open-close" movements of the individual domains apparently enable it to interact and transfer energy to co-proteins in the ESX-1 pathway.
| S-EPMC2662308 | biostudies-literature
Project description:Background:It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis (TB). The BCG vaccine is widely used to fight against TB; however, many question its ability to provide complete protection from Mtb. Recently, the "Region of Difference 1" (RD1) set of genes were shown to be involved in the pathogenesis of Mtb. Downstream of RD1 transcription region, two proteins are encoded, known as EspB and EspC, which were found to contribute to Mtb virulence.In this study these two proteins are targeted as potential vaccine candidates against TB. Methods:The EspB and EspC Mtb genes were codon-optimized for expression and synthesis in Escherichia coli (E. coli). The amplicons were cloned into a pET21a expression vector and transformed into E. coli BL21(DE3). The expression and purity of the expressed proteins (i.e. rEspC, rEspB and rEspC/EspB) were confirmed by SDS-PAGE and Western blotting. Moreover, BALB/c mice were immunized against Mtb using the recombinant proteins. Finally, the mice sera were analyzed via Western blotting. Results:EspC, EspB, and EspC/EspB fusion genes were cloned and expressed in E. coli. Both SDS-PAGE and Western blots confirmed the presence and successful purification of the desired proteins. Moreover, antisera produced against the purified recombinant proteins reacted with Mtb proteins. Conclusion:rEspC, rEspB, and rEspC/EspB could be expressed and purified using an E. coli expression system. The recombinant proteins induced the production of antibodies in BALB/c mice that reacted with Mtb proteins.
| S-EPMC7275833 | biostudies-literature
Project description:Fluoroquinolone resistance in Mycobacterium tuberculosis is often conferred by DNA gyrase mutations. However, a substantial proportion of fluoroquinolone-resistant M. tuberculosis isolates do not have such mutations.Ofloxacin-resistant and lineage-matched ofloxacin-susceptible M. tuberculosis isolates underwent WGS. Novel candidate resistance mutations were confirmed by Sanger sequencing and conferral of resistance was assessed via site-directed mutagenesis and allelic exchange. Ofloxacin MIC was determined by resazurin microtitre assay (REMA) and the effects on MICs of efflux pump inhibitors (CCCP, reserpine and verapamil) were determined.Of 26 ofloxacin-resistant isolates, 8 (31%) did not have resistance-conferring DNA gyrase mutations. The V762G mutation in Rv1783 (eccC5, encoding a protein in the ESX-5 membrane complex secretion system) was present on WGS in 8/26 (31%) resistant isolates and 0/11 susceptible isolates (P?=?0.005). The mutation was identified in five isolates without DNA gyrase mutations and three isolates with such mutations; it was identified in both European-American and East Asian M. tuberculosis lineages. The ofloxacin MIC increased from 1 to 32 mg/L after introduction of the V762G mutation into M. tuberculosis H37Rv. In this strain with the V762G mutation, ofloxacin MIC did not change in the presence of efflux pump inhibitors.A novel V762G mutation in Rv1783 conferred ofloxacin resistance in M. tuberculosis by a mechanism other than drug efflux. This occurred in a substantial proportion of resistant isolates, particularly those without DNA gyrase mutations.
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Project description:Mycobacterium tuberculosis uses the Type VII ESX secretion systems to transport proteins across its complex cell wall. ESX-5 has been implicated in M. tuberculosis virulence, but the regulatory mechanisms controlling ESX-5 secretion were unknown. Here we uncover a link between ESX-5 and the Pst/SenX3-RegX3 system that controls gene expression in response to phosphate availability. The DNA-binding response regulator RegX3 is normally activated by phosphate limitation. Deletion of pstA1, which encodes a Pst phosphate uptake system component, causes constitutive activation of RegX3. A ?pstA1 mutant exhibited RegX3-dependent overexpression of esx-5 genes and hyper-secretion of the ESX-5 substrates EsxN and PPE41 when the bacteria were grown in phosphate-rich medium. In wild-type M. tuberculosis, phosphate limitation activated esx-5 transcription and secretion of both EsxN and PPE41, and this response required RegX3. Electrophoretic mobility shift assays revealed that RegX3 binds directly to a promoter within the esx-5 locus. Remarkably, phosphate limitation also induced secretion of EsxB, an effector of the virulence-associated ESX-1 secretion system, though this induction was RegX3 independent. Our work demonstrates that the Pst/SenX3-RegX3 system directly regulates ESX-5 secretion at the transcriptional level in response to phosphate availability and defines phosphate limitation as an environmental signal that activates ESX-5 secretion.
| S-EPMC4863468 | biostudies-literature