Mycobacterium smegmatis MfpC is a GEF that regulates mfpA translationally to alter the fluoroquinolone efficacy
Ontology highlight
ABSTRACT:
SUBMITTER: Huang Y
PROVIDER: S-EPMC11343762 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The fluoroquinolones (FQ) are used in the treatment of Mycobacterium tuberculosis, but the development of resistance could limit their effectiveness. FQ resistance (FQ(R)) is a multistep process involving alterations in the type II topoisomerases and perhaps in the regulation of efflux pumps, but several of the steps remain unidentified. Recombinant plasmid pGADIV was selected from a genomic library of wild-type (WT), FQ-sensitive M. smegmatis by its ability to confer low-level resistance to sparfloxacin (SPX). In WT M. smegmatis, pGADIV increased the MICs of ciprofloxacin (CIP) by fourfold and of SPX by eightfold, and in M. bovis BCG it increased the MICs of both CIP and SPX by fourfold. It had no effect on the accumulation of (14)C-labeled CIP or SPX. The open reading frame responsible for the increase in FQ(R), mfpA, encodes a putative protein belonging to the family of pentapeptides, in which almost every fifth amino acid is either leucine or phenylalanine. Very similar proteins are also present in M. tuberculosis and M. avium. The MICs of CIP and SPX were lower for an M. smegmatis mutant strain lacking an intact mfpA gene than for the WT strain, suggesting that, by some unknown mechanism, the gene product plays a role in determining the innate level of FQ(R) in M. smegmatis.
| S-EPMC90842 | biostudies-literature
Project description:The absence of the Holliday-junction Ruv resolvase of Mycobacterium smegmatis increased the bacteriostatic and bactericidal activities of the fluoroquinolone moxifloxacin, an important antituberculosis agent. The treatment of ruvAB-deficient cells with thiourea and 2,2'-bipyridyl lowered moxifloxacin lethality to wild-type levels, indicating that the absence of ruvAB stimulates a lethal pathway involving reactive oxygen species. A hexapeptide that traps the Holliday junction substrate of RuvAB potentiated moxifloxacin-mediated lethality, supporting the development of small-molecule enhancers for moxifloxacin activity against mycobacteria.
| S-EPMC4325773 | biostudies-literature
Project description:Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.
| S-EPMC9769933 | biostudies-literature
Project description:Quinazolinediones (diones) are fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV. To assess activity against mycobacteria, C-8-methoxy dione derivatives were compared with cognate fluoroquinolones by using cultured Mycobacterium smegmatis. Diones exhibited higher MIC values than fluoroquinolones; however, MICs for fluoroquinolone-resistant gyrA mutants, normalized to the MIC for wild-type cells, were lower. Addition of a 3-amino group to the 2,4-dione core increased relative activity against mutants, while alteration of the 8-methoxy group to a methyl or of the 2,4-dione core to a 1,3-dione core lowered activity against mutants. A GyrA G89C bacterial variant was strikingly susceptible to most of the diones tested; in contrast, low susceptibility to fluoroquinolones was observed. Many of the bacteriostatic differences between diones and fluoroquinolones were explained by interactions at the N terminus of GyrA helix IV revealed by recently published X-ray structures of drug-topoisomerase-DNA complexes. When lethal activity was normalized to the MIC in order to minimize the effects of drug uptake, efflux, and ternary complex formation, a 3-amino-2,4-dione exhibited killing activity comparable to that of a cognate fluoroquinolone. Surprisingly, the lethal activity of the dione was inhibited less by chloramphenicol than that of the cognate fluoroquinolone. This observation adds the 2,4-dione structural motif to the list of structural features known to impart lethality to fluoroquinolone-like compounds in the absence of protein synthesis, a phenomenon that is not explained by X-ray structures of drug-enzyme-DNA complexes.
| S-EPMC3088267 | biostudies-literature
Project description:Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, of Streptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.
| S-EPMC40238 | biostudies-other
Project description:Conjugal DNA transfer occurs by an atypical mechanism in Mycobacterium smegmatis. The transfer system is chromosomally encoded and requires recipient recombination functions for both chromosome and plasmid transfer. Cis-acting sequences have been identified that confer mobility on nontransferable plasmids, but these are larger and have different properties to canonical oriT sites found in bacterial plasmids. To identify trans-acting factors required for mediating DNA transfer, a library of transposon insertion mutants was generated in the donor strain, and individual mutants were screened for their effect on transfer. From this screen, a collection of insertion mutants was isolated that increased conjugation frequencies relative to wild type. Remarkably, the mutations map to a 25-kb region of the M. smegmatis chromosome that is syntenous with the RD1 region of Mycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. The genes of the RD1 region encode a secretory apparatus responsible for exporting Cfp10- and Esat-6, both potent antigens and virulence factors. In crosses using two M. smegmatis donors, we show that wild-type cells can suppress the elevated transfer phenotype of mutant donors, which is consistent with the secretion of a factor that suppresses conjugation. Most importantly, the RD1 region of M. tuberculosis complements the conjugation phenotype of the RD1 mutants in M. smegmatis. Our results indicate that the M. tuberculosis and M. smegmatis RD1 regions are functionally equivalent and provide a unique perspective on the role of this critical secretion apparatus.
| S-EPMC515103 | biostudies-literature
Project description:Sphingosine kinase-1 is known to mediate Mycobacterium smegmatis induced inflammatory responses in macrophages, but its role in controlling infection has not been reported to date. We aimed to unravel the significance of SphK-1 in controlling M. smegmatis infection in RAW 264.7 macrophages. Our results demonstrated for the first time that selective inhibition of SphK-1 by either D, L threo dihydrosphingosine (DHS; a competitive inhibitor of Sphk-1) or Sphk-1 siRNA rendered RAW macrophages sensitive to M. smegmatis infection. This was due to the reduction in the expression of iNOs, p38, pp-38, late phagosomal marker, LAMP-2 and stabilization of the RelA (pp-65) subunit of NF-kappaB. This led to a reduction in the generation of NO and secretion of TNF-alpha in infected macrophages. Congruently, overexpression of SphK-1 conferred resistance in macrophages to infection which was due to enhancement in the generation of NO and expression of iNOs, pp38 and LAMP-2. In addition, our results also unraveled a novel regulation of p38MAPK by SphK-1 during M. smegmatis infection and generation of NO in macrophages. Enhanced NO generation and expression of iNOs in SphK-1++ infected macrophages demonstrated their M-1(bright) phenotype of these macrophages. These findings thus suggested a novel antimycobacterial role of SphK-1 in macrophages.
| S-EPMC2871783 | biostudies-literature
Project description:The pentapeptide repeat is a recently discovered protein fold. Mycobacterium tuberculosis MfpA is a founding member of the pentapeptide repeat protein (PRP) family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and inhibiting its activity. The size, shape, and surface potential of MfpA mimics duplex DNA. As an initial step in a comprehensive biophysical analysis of the role of PRPs in the regulation of cellular topoisomerase activity and conferring antibiotic resistance, we have explored the solution structure and refolding of MfpA by fluorescence spectroscopy, CD, and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat fold is described. This spectrum reveals a native structure whose beta-strands and turns within the right-handed quadrilateral beta-helix that define the PRP fold differ from canonical secondary structure types. MfpA refolded from urea or guanidium by dialysis or dilution forms stable aggregates of monomers whose secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel "time-dependent renaturation" protocol yields protein with native secondary, tertiary, and quaternary structure. The generality of "time-dependent renaturation" to other proteins and denaturation methods is discussed.
| S-EPMC2606005 | biostudies-literature
Project description:Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the serine/threonine phosphatase PstP, in the model organism Mycobacterium smegmatis We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phosphomimetic mutation, pstP T171E, slows growth, misregulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis.IMPORTANCE Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.
| S-EPMC7847547 | biostudies-literature
Project description:Mycobacterium smegmatis is a commonly used mycobacterial model system. Here, we show that M. smegmatis protects itself against elevated salinity by synthesizing ectoine and hydroxyectoine and characterize the phenotype of a nonproducing mutant. This is the first analysis of M. smegmatis halotolerance and of the molecular mechanism that supports it.
| S-EPMC3457100 | biostudies-literature