Zafirlukast inhibits complexation of Lsr2 with DNA and growth of Mycobacterium tuberculosis.
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ABSTRACT:
SUBMITTER: Pinault L
PROVIDER: S-EPMC3632936 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bacterial nucleoid-associated proteins, such as H-NS-like proteins in Enterobacteriaceae, are abundant DNA-binding proteins that function in chromosomal DNA organization and gene transcription regulation. The Mycobacterium tuberculosis Lsr2 protein has been proposed to be the first identified H-NS analogue in Gram-positive bacteria based on its capability to complement numerous in vivo functions of H-NS. Here, we report that Lsr2 cooperatively binds to DNA forming a rigid Lsr2 nucleoprotein complex that restricts DNA accessibility, similar to H-NS. On large DNA, the rigid Lsr2 nucleoprotein complexes can mediate DNA condensation into highly compact DNA conformations. In addition, the responses of Lsr2 nucleoprotein complex to environmental factors (salt concentration, temperature and pH) were studied over physiological ranges. These results provide mechanistic insights into how Lsr2 may mediate its gene silencing, genomic DNA protection and organization functions in vivo. Finally, our results strongly support that Lsr2 is an H-NS-like protein in Gram-positive bacteria from a structural perspective.
| S-EPMC3664827 | biostudies-other
Project description:Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H-NS, Lsr2 binds DNA in sequence-dependent and non-specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA-binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP-sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.
| S-EPMC6906086 | biostudies-literature
Project description:Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a novel nucleoid-associated protein that specifically binds AT-rich regions of the genome, including regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM/PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain swamping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA binding domain. NMR analysis of the DNA binding domain of Lsr2 and its interaction with DNA reveals a novel structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve an equivalent function as the Enterobacteriaceae H-NS, coordinating global gene regulation and virulence in this group of medically important bacteria.
2009-11-15 | GSE18652 | GEO
Project description:Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a novel nucleoid-associated protein that specifically binds AT-rich regions of the genome, including regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM/PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain swamping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA binding domain. NMR analysis of the DNA binding domain of Lsr2 and its interaction with DNA reveals a novel structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve an equivalent function as the Enterobacteriaceae H-NS, coordinating global gene regulation and virulence in this group of medically important bacteria. Comparison of Lsr2 chromatin-immunoprecipitated DNA sequences to total reference DNA
2010-06-19 | E-GEOD-18652 | biostudies-arrayexpress
Project description:Lung cancer has the highest mortality among cancers worldwide due to its high incidence and lack of the effective cures. We have previously demonstrated that the membrane ion channel TMEM16A is a potential drug target for the treatment of lung adenocarcinoma and have identified a pocket of inhibitor binding that provides the basis for screening promising new inhibitors. However, conventional drug discovery strategies are lengthy and costly, and the unpredictable side effects lead to a high failure rate in drug development. Therefore, finding new therapeutic directions for already marketed drugs may be a feasible strategy to obtain safe and effective therapeutic drugs. Here, we screened a library of over 1400 Food and Drug Administration-approved drugs through virtual screening and activity testing. We identified a drug candidate, Zafirlukast (ZAF), clinically approved for the treatment of asthma, that could inhibit the TMEM16A channel in a concentration-dependent manner. Molecular dynamics simulations and site-directed mutagenesis experiments showed that ZAF can bind to S387/N533/R535 in the nonselective inhibitor binding pocket, thereby blocking the channel pore. Furthermore, we demonstrate ZAF can target TMEM16A channel to inhibit the proliferation and migration of lung adenocarcinoma LA795 cells. In vivo experiments showed that ZAF can significantly inhibit lung adenocarcinoma tumor growth in mice. Taken together, we identified ZAF as a novel TMEM16A channel inhibitor with excellent anticancer activity, and as such, it represents a promising candidate for future preclinical and clinical studies.
| S-EPMC8931426 | biostudies-literature
Project description:Mycobacterium abscessus, a pathogen responsible for severe lung infections in cystic fibrosis patients, exhibits either smooth (S) or rough (R) morphotypes. The S-to-R transition correlates with inhibition of the synthesis and/or transport of glycopeptidolipids (GPLs) and is associated with an increase of pathogenicity in animal and human hosts. Lsr2 is a small nucleoid-associated protein highly conserved in mycobacteria, including M. abscessus, and is a functional homolog of the heat-stable nucleoid-structuring protein (H-NS). It is essential in Mycobacterium tuberculosis but not in the non-pathogenic model organism Mycobacterium smegmatis. It acts as a master transcriptional regulator of multiple genes involved in virulence and immunogenicity through binding to AT-rich genomic regions. Previous transcriptomic studies, confirmed here by quantitative PCR, showed increased expression of lsr2 (MAB_0545) in R morphotypes when compared to their S counterparts, suggesting a possible role of this protein in the virulence of the R form. This was addressed by generating lsr2 knock-out mutants in both S (Δlsr2-S) and R (Δlsr2-R) variants, demonstrating that this gene is dispensable for M. abscessus growth. We show that the wild-type S variant, Δlsr2-S and Δlsr2-R strains were more sensitive to H2O2 as compared to the wild-type R variant of M. abscessus. Importantly, virulence of the Lsr2 mutants was considerably diminished in cellular models (macrophage and amoeba) as well as in infected animals (mouse and zebrafish). Collectively, these results emphasize the importance of Lsr2 in M. abscessus virulence.
| S-EPMC6503116 | biostudies-literature
Project description:Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112
| PRJEB31102 | ENA
Project description:Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a unique nucleoid-associated protein that binds AT-rich regions of the genome, including genomic islands acquired by horizontal gene transfer and regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM and PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain-swapping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA-binding domain. Nuclear magnetic resonance analysis of the DNA-binding domain of Lsr2 and its interaction with DNA reveals a unique structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve a function similar to the Enterobacteriaceae H-NS, likely coordinating global gene regulation and virulence in this group of medically important bacteria.
| S-EPMC2841939 | biostudies-literature
Project description:UNLABELLED:To survive a dynamic host environment, Mycobacterium tuberculosis must endure a series of challenges, from reactive oxygen and nitrogen stress to drastic shifts in oxygen availability. The mycobacterial Lsr2 protein has been implicated in reactive oxygen defense via direct protection of DNA. To examine the role of Lsr2 in pathogenesis and physiology of M. tuberculosis, we generated a strain deleted for lsr2. Analysis of the M. tuberculosis ?lsr2 strain demonstrated that Lsr2 is not required for DNA protection, as this strain was equally susceptible as the wild type to DNA-damaging agents. The lsr2 mutant did display severe growth defects under normoxic and hyperoxic conditions, but it was not required for growth under low-oxygen conditions. However, it was also required for adaptation to anaerobiosis. The defect in anaerobic adaptation led to a marked decrease in viability during anaerobiosis, as well as a lag in recovery from it. Gene expression profiling of the ?lsr2 mutant under aerobic and anaerobic conditions in conjunction with published DNA binding-site data indicates that Lsr2 is a global transcriptional regulator controlling adaptation to changing oxygen levels. The ?lsr2 strain was capable of establishing an early infection in the BALB/c mouse model; however, it was severely defective in persisting in the lungs and caused no discernible lung pathology. These findings demonstrate M. tuberculosis Lsr2 is a global transcriptional regulator required for control of genes involved in adaptation to extremes in oxygen availability and is required for persistent infection. IMPORTANCE:M. tuberculosis causes nearly two million deaths per year and infects nearly one-third of the world population. The success of this aerobic pathogen is due in part to its ability to successfully adapt to constantly changing oxygen availability throughout the infectious cycle, from the high oxygen tension during aerosol transmission to anaerobiosis within necrotic lesions. An understanding of how M. tuberculosis copes with these changes in oxygen tension is critical for its eventual eradication. Using a mutation in lsr2, we demonstrate that the Lsr2 protein present in all mycobacteria is a global transcriptional regulator in control of genes required for adaptation to changes in oxygen levels. M. tuberculosis lacking lsr2 was unable to adapt to both high and very low levels of oxygen and was defective in long-term anaerobic survival. Lsr2 was also required for disease pathology and for chronic infection in a mouse model of TB.
| S-EPMC4049101 | biostudies-literature
Project description:Previously, we found that interleukin 22 (IL-22) inhibits intracellular growth of Mycobacterium tuberculosis in human monocyte-derived macrophages (MDMs). In the current study, we determined the mechanisms underlying these effects. We found that W7, a phagolysosomal fusion inhibitor, abrogates IL-22-dependent M. tuberculosis growth inhibition in MDMs, suggesting that IL-22 acts through enhanced phagolysosomal fusion. Our microarray analysis indicated that recombinant IL-22 (rIL-22) enhances the expression of an intracellular signaling molecule, calgranulin A. This was confirmed by real-time polymerase chain reaction, Western blot, and confocal microscopy. Calgranulin A small interfering RNA (siRNA) abrogated rIL-22-dependent growth inhibition of M. tuberculosis in MDMs. IL-22 enhanced Rab7 expression and downregulated Rab14 expression of M. tuberculosis-infected MDMs, and these effects were reversed by calgranulin A siRNA. These results suggest that M. tuberculosis growth inhibition by IL-22 depends on calgranulin A and enhanced phagolysosomal fusion, which is associated with increased Rab7 and reduced Rab14 expression.
| S-EPMC3903372 | biostudies-literature