RNase HI Is Essential for Survival of Mycobacterium smegmatis

RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.The study was supported by POIG.01.01.02-10-107/09 project implemented under Innovative Economy Operational Programme, years 2007–2013 "Studies of the molecular mechanisms at the interface the human organism - the pathogen - environmental factors" and by grant of Polish National Center of Science 2011/01/N/NZ6/04186 “Identification of a novel mechanism of initiation of DNA replication in Mycobacterium smegmatis”

Genetic evaluation of relationship between mutations in rpoB and resistance of Mycobacterium tuberculosis to rifampin

<p>Abstract</p> <p>Background</p> <p>Rifampin is a first line antituberculosis drug active against bacilli in logarithmic and stationary phase, which interferes with RNA synthesis by binding to bacterial RNA polymerase. Tubercle bacilli achieve resistance to rifampin by accumulation of mutations in a short-81 bp region of the <it>rpoB </it>gene. Among many mutations identified in the <it>rpo</it>B gene, few were verified by molecular genetic methods as responsible for resistance to rifampin (RMP).</p> <p>Results</p> <p>In this study eight different mutations identified in an 81 bp section of a "hot spot" region of the <it>rpo</it>B gene of RMP resistant <it>Mycobacterium tuberculosis </it>clinical strains were evaluated in respect to drug resistance. It was found that: mutations in positions 526 (H/D), 516 (D/V) and 531 (S/L) result in high level resistance to rifampin; mutations in positions 516 (D/Y), 515 (M/I), 510 (Q/H) or a double mutation in codons 512 (S/I) and 516 (D/G) relate to low level of resistance. Gene <it>rpo</it>B carrying mutations in codon 513 (Q/L) introduced into an <it>M. tuberculosis </it>laboratory strain did not cause resistance to rifampin, however the same gene introduced into two different clinical strains did, with the level of resistance depending on the host strain.</p> <p>Conclusion</p> <p>Mutations in an 81 bp "hot spot" region of the <it>rpoB </it>of <it>M. tuberculosis </it>lead to different levels of resistance to rifampin. Some mutations in this "hot spot" region of <it>rpoB </it>require a specific genetic background for the host strain to develop resistance to rifampin. Therefore, the identification of such mutations in a clinical <it>M. tuberculosis </it>strain is not enough to classify the given strain as resistant to rifampin.</p

Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway

Evaluation of DNA primase DnaG as a potential target for antibiotics

Mycobacteria contain genes for several DNA-dependent RNA primases, including dnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. An in silico analysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-type dnaG cannot be deleted from the chromosome of Mycobacterium smegmatis without disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identified M. smegmatis AEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of native dnaG in M. smegmatis was tolerated only after the integration of an extra intact copy of the M. smegmatis or Mycobacterium tuberculosis dnaG gene, under the control of chemically inducible promoters, into the attB site of the chromosome. M. tuberculosis and M. smegmatis DnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably, M. smegmatis bacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target

Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division

Identification of 2-(N-aryl-1,2,3-triazol-4-yl) quinoline derivatives as antitubercular agents endowed with InhA inhibitory activity

The spread of drug-resistant tuberculosis strains has become a significant economic burden globally. To tackle this challenge, there is a need to develop new drugs that target specific mycobacterial enzymes. Among these enzymes, InhA, which is crucial for the survival of Mycobacterium tuberculosis, is a key target for drug development. Herein, 24 compounds were synthesized by merging 4-carboxyquinoline with triazole motifs. These molecules were then tested for their effectiveness against different strains of tuberculosis, including M. bovis BCG, M. tuberculosis, and M. abscessus. Additionally, their ability to inhibit the InhA enzyme was also evaluated. Several molecules showed potential as inhibitors of M. tuberculosis. Compound 5n displayed the highest efficacy with a MIC value of 12.5 μg/mL. Compounds 5g, 5i, and 5n exhibited inhibitory effects on InhA. Notably, 5n showed significant activity compared to the reference drug Isoniazid. Molecular docking analysis revealed interactions between these molecules and their target enzyme. Additionally, the molecular dynamic simulations confirmed the stability of the complexes formed by quinoline-triazole conjugate 5n with the InhA. Finally, 5n underwent in silico analysis to predict its ADME characteristics. These findings provide promising insights for developing novel small compounds that are safe and effective for the global fight against tuberculosis

DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection

Mycobacteria, including most of all MTB (Mycobacterium tuberculosis), cause pathogenic infections in humans and, during the infectious process, are exposed to a range of environmental insults, including the host's immune response. From the moment MTB is exhaled by infected individuals, through an active and latent phase in the body of the new host, until the time they reach the reactivation stage, MTB is exposed to many types of DNA-damaging agents. Like all cellular organisms, MTB has efficient DNA repair systems, and these are believed to play essential roles in mycobacterial pathogenesis. As different stages of infection have great variation in the conditions in which mycobacteria reside, it is possible that different repair systems are essential for progression to specific phases of infection. MTB possesses homologues of DNA repair systems that are found widely in other species of bacteria, such as nucleotide excision repair, base excision repair and repair by homologous recombination. MTB also possesses a system for non-homologous end-joining of DNA breaks, which appears to be widespread in prokaryotes, although its presence is sporadic within different species within a genus. However, MTB does not possess homologues of the typical mismatch repair system that is found in most bacteria. Recent studies have demonstrated that DNA repair genes are expressed differentially at each stage of infection. In the present review, we focus on different DNA repair systems from mycobacteria and identify questions that remain in our understanding of how these systems have an impact upon the infection processes of these important pathogens

Influence of quorum sensing signal molecules on biofilm formation in Proteus mirabilis O18

The influence of basis of quorum sensing molecules on Proteus strains is much less known as compared to Pseudomonas or Escherichia. We have previously shown that a series of acylated homoserine lactones (acyl-HSL) does not influence the ureolytic, proteolytic, or hemolytic abilities, and that the swarming motility of Proteus mirabilis rods is strain specific. The aim of the presented study was to find out if the presence of a series of acyl-HSL influences biofilm formation of P. mirabilis laboratory strain belonging to O18 serogroup. This serogroup is characterized by the presence of a unique non-carbohydrate component, namely phosphocholine. Escherichia coli and P. mirabilis O18 strains used in this work contains cloned plasmids encoding fluorescent protein genes with constitutive gene expression. In mixed biofilms in stationary and continuous flow conditions, P. mirabilis O18 overgrow whole culture. P. mirabilis O18 strain has genetically proved a presence of AI–2 quorum sensing system. Differences in biofilm structure were observed depending on the biofilm type and culture methods. From tested acylated homoserine lactones (BHL, HHL, OHL, DHL, dDHL, tDHL), a significant influence had BHL on thickness, structure, and the amount of exopolysaccharides produced by biofilms formed by P. mirabilis O18 pDsRed2

Genetic Evidence for Inhibition of Bacterial Division Protein FtsZ by Berberine

Background: Berberine is a plant alkaloid that is widely used as an anti-infective in traditional medicine. Escherichia coli exposed to berberine form filaments, suggesting an antibacterial mechanism that involves inhibition of cell division. Berberine is a DNA ligand and may induce filamentation through induction of the SOS response. Also, there is biochemical evidence for berberine inhibition of the cell division protein FtsZ. Here we aimed to assess possible berberine mechanism(s) of action in growing bacteria using genetics tools. Methodology/Principal Findings: First, we tested whether berberine inhibits bacterial growth through DNA damage and induction of the SOS response. The SOS response induced by berberine was much lower compared to that induced by mitomycin C in an SOS response reporter strain. Also, cell filamentation was observed in an SOS-negative E. coli strain. To test whether berberine inhibits FtsZ, we assessed its effects on formation of the cell division Z-rings, and observed a dramatic reduction in Z-rings in the presence of berberine. We next used two different strategies for RNA silencing of ftsZ and both resulted in sensitisation of bacteria to berberine, visible as a drop in the Minimum Inhibitory Concentration (MIC). Furthermore, Fractional Inhibitory Concentration Indices (FICIs) showed a high level of synergy between ftsZ silencing and berberine treatment (FICI values of 0.23 and 0.25 for peptide nucleic acid- and expressed antisense RNA-based silencing of ftsZ, respectively). Finally, over-expression of ftsZ led to a mild rescue effect in berberine-treated cells