Transcriptional regulator-induced phenotype screen reveals drug potentiators in Mycobacterium tuberculosis.
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ABSTRACT:
SUBMITTER: Ma S
PROVIDER: S-EPMC8331221 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.
| S-EPMC4815240 | biostudies-literature
Project description:The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.
| S-EPMC3970893 | biostudies-literature
Project description:Recent work demonstrates that the MmpL (mycobacterial membrane protein large) transporters are dedicated to the export of mycobacterial lipids for cell wall biosynthesis. An MmpL transporter frequently works with an accessory protein, belonging to the MmpS (mycobacterial membrane protein small) family, to transport these key virulence factors. One such efflux system in Mycobacterium tuberculosis is the MmpS5-MmpL5 transporter. The expression of MmpS5-MmpL5 is controlled by the MarR-like transcriptional regulator Rv0678, whose open reading frame is located downstream of the mmpS5-mmpL5 operon. To elucidate the structural basis of Rv0678 regulation, we have determined the crystal structure of this regulator, to 1.64 Å resolution, revealing a dimeric two-domain molecule with an architecture similar to members of the MarR family of transcriptional regulators. Rv0678 is distinct from other MarR regulators in that its DNA-binding and dimerization domains are clustered together. These two domains seemingly cooperate to bind an inducing ligand that we identified as 2-stearoylglycerol, which is a fatty acid glycerol ester. The structure also suggests that the conformational change leading to substrate-mediated derepression is primarily caused by a rigid body rotational motion of the entire DNA-binding domain of the regulator toward the dimerization domain. This movement results in a conformational state that is incompatible with DNA binding. We demonstrate using electrophoretic mobility shift assays that Rv0678 binds to the mmpS5-mmpL5, mmpS4-mmpL4, and the mmpS2-mmpL2 promoters. Binding by Rv0678 was reversed upon the addition of the ligand. These findings provide new insight into the mechanisms of gene regulation in the MarR family of regulators.
| S-EPMC4047419 | biostudies-literature
Project description:BackgroundTreatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli.MethodsM. tuberculosis gene expression was quantified via reverse-transcription polymerase chain reaction in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis.ResultsWithin 4 days, bacterial mRNA abundance declined >98%, indicating rapid killing. Thereafter, the rate of decline slowed >94%, indicating drug tolerance. After 14 days, 16S ribosomal RNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked downregulation of genes associated with growth, metabolism, and lipid synthesis and upregulation in stress responses and key regulatory categories-including stress-associated sigma factors, transcription factors, and toxin-antitoxin genes. Drug efflux pumps were upregulated. The isoniazid stress signature was induced by initial drug exposure, then disappeared after 4 days.ConclusionsTranscriptional patterns suggest that drug-tolerant bacilli in sputum are in a slow-growing, metabolically and synthetically downregulated state. Absence of the isoniazid stress signature in drug-tolerant bacilli indicates that physiological state influences drug responsiveness in vivo. These results identify novel drug targets that should aid in development of novel shorter tuberculosis treatment regimens.
| S-EPMC4548467 | biostudies-literature
Project description:The pathogenic mycobacterium Mycobacterium tuberculosis encodes two members of the DtxR/MntR family of metalloregulators, IdeR and SirR. IdeR represses gene expression in response to ferrous iron, and we here demonstrate that SirR (Rv2788), although also annotated as an iron-dependent repressor, functions instead as a manganese-dependent transcriptional repressor and is therefore renamed MntR. MntR regulates transporters that promote manganese import and genes that respond to metal ion deficiency such as the esx3 system. Repression of manganese import by MntR is essential for survival of M. tuberculosis under conditions of high manganese availability, but mntR is dispensable during infection. In contrast, manganese import by MntH and MntABCD was found to be indispensable for replication of M. tuberculosis in macrophages. These results suggest that manganese is limiting in the host and that interfering with import of this essential metal may be an effective strategy to attenuate M. tuberculosis.
| S-EPMC5157835 | biostudies-literature
Project description:CarD is an essential global transcription regulator from Mycobacterium tuberculosis (Mtb) that binds RNA polymerase and activates transcription by stabilizing the transcription initiation complex. Available crystal structures have captured two distinct, monomeric and domain-swapped homodimeric, oligomeric states of CarD. However, the actual oligomeric state of CarD in solution and its biological relevance has remained unclear. Here, we confirm the presence of the homodimeric state of CarD in solution by using synchrotron-based small-angle X-ray scattering. Furthermore, by using biochemical and biophysical experiments, in addition to mass-spectrometry, transmission electron microscopy, and confocal imaging, we show that CarD is the first soluble cytosolic protein in Mtb which displays the tendency to form amyloid-like fibrils both in vitro as well as in vivo. We demonstrate that the deletion of the fourteen N-terminal residues involved in domain-swapping hampers amyloid formation, thus, suggesting that domain-swapping is crucial in amyloidogenesis. The discovery of the amyloidogenic property of an essential cytosolic global transcription regulator, CarD, in a pathogenic bacteria will further open up new frontiers in research.
| S-EPMC6031611 | biostudies-literature
Project description:The Mmr multidrug efflux pump recognizes and actively extrudes a broad range of antimicrobial agents, and promotes the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Mmr is controlled by the TetR-like transcriptional regulator Rv3066, whose open reading frame is located downstream of the mmr operon. To understand the structural basis of Rv3066 regulation, we have determined the crystal structures of Rv3066, both in the absence and presence of bound ethidium, revealing an asymmetric homodimeric two-domain molecule with an entirely helical architecture. The structures underscore the flexibility and plasticity of the regulator essential for multidrug recognition. Comparison of the apo-Rv3066 and Rv3066-ethidium crystal structures suggests that the conformational changes leading to drug-mediated derepression is primarily due to a rigid body rotational motion within the dimer interface of the regulator. The Rv3066 regulator creates a multidrug-binding pocket, which contains five aromatic residues. The bound ethidium is found buried within the multidrug-binding site, where extensive aromatic stacking interactions seemingly govern the binding. In vitro studies reveal that the dimeric Rv3066 regulator binds to a 14-bp palindromic inverted repeat sequence in the nanomolar range. These findings provide new insight into the mechanisms of ligand binding and Rv3066 regulation.
| S-EPMC3467072 | biostudies-literature
Project description:The proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, a Mycobacterium tuberculosis protein belonging to this superfamily. A null mutant was constructed in M. tuberculosis H37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, the whiB5 mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain to S-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, including sigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.
| S-EPMC3418748 | biostudies-literature
Project description:Catabolism of host cholesterol is critical to the virulence of Mycobacterium tuberculosis and is a potential target for novel therapeutics. KstR2, a TetR family repressor (TFR), regulates the expression of 15 genes encoding enzymes that catabolize the last half of the cholesterol molecule, represented by 3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indane-dione (HIP). Binding of KstR2 to its operator sequences is relieved upon binding of HIP-CoA. A 1.6-Å resolution crystal structure of the KstR2(Mtb)·HIP-CoA complex reveals that the KstR2(Mtb) dimer accommodates two molecules of HIP-CoA. Each ligand binds in an elongated cleft spanning the dimerization interface such that the HIP and CoA moieties interact with different KstR2(Mtb) protomers. In isothermal titration calorimetry studies, the dimer bound 2 eq of HIP-CoA with high affinity (K(d) = 80 ± 10 nm) but bound neither HIP nor CoASH. Substitution of Arg-162 or Trp-166, residues that interact, respectively, with the diphosphate and HIP moieties of HIP-CoA, dramatically decreased the affinity of KstR2(Mtb) for HIP-CoA but not for its operator sequence. The variant of R162M that decreased the affinity for HIP-CoA (ΔΔG = 13 kJ mol(-1)) is consistent with the loss of three hydrogen bonds as indicated in the structural data. A 24-bp operator sequence bound two dimers of KstR2. Structural comparisons with a ligand-free rhodococcal homologue and a DNA-bound homologue suggest that HIP-CoA induces conformational changes of the DNA-binding domains of the dimer that preclude their proper positioning in the major groove of DNA. The results provide insight into KstR2-mediated regulation of expression of steroid catabolic genes and the determinants of ligand binding in TFRs.
| S-EPMC4294509 | biostudies-literature
Project description:Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of Mycobacterium tuberculosis revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (mce1) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in Mycobacterium smegmatis, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (tgs1)] and intracellular survival (e.g., mce1), among other genes. Interestingly, the colonization level of the DeltamosR mutant strain was significantly lower than that of the wild-type strain of M. tuberculosis, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed mosR for regulator of mycobacterial operons of survival.
| S-EPMC2747884 | biostudies-other