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Microbiology Spectrum Feb 2023In order to adapt in host tissues, microbial pathogens regulate their gene expression through a variety of transcription factors. Here, we have functionally...
In order to adapt in host tissues, microbial pathogens regulate their gene expression through a variety of transcription factors. Here, we have functionally characterized Rv0792c, a HutC homolog from Mycobacterium tuberculosis. In comparison to the parental strain, a strain of M. tuberculosis with a Rv0792c mutant was compromised for survival upon exposure to oxidative stress and infection in guinea pigs. RNA sequencing analysis revealed that Rv0792c regulates the expression of genes involved in stress adaptation and virulence of M. tuberculosis. Solution small-angle X-ray scattering (SAXS) data-steered model building confirmed that the C-terminal region plays a pivotal role in dimer formation. Systematic evolution of ligands by exponential enrichment (SELEX) resulted in the identification of single-strand DNA (ssDNA) aptamers that can be used as a tool to identify small-molecule inhibitors targeting Rv0792c. Using SELEX and SAXS data-based modeling, we identified residues essential for Rv0792c's aptamer binding activity. In this study, we also identified I-OMe-Tyrphostin as an inhibitor of Rv0792c's aptamer and DNA binding activity. The identified small molecule reduced the growth of intracellular M. tuberculosis in macrophages. The present study thus provides a detailed shape-function characterization of a HutC family of transcription factor from M. tuberculosis. Prokaryotes encode a large number of GntR family transcription factors that are involved in various fundamental biological processes, including stress adaptation and pathogenesis. Here, we investigated the structural and functional role of Rv0792c, a HutC homolog from M. tuberculosis. We demonstrated that Rv0792c is essential for M. tuberculosis to adapt to oxidative stress and establish disease in guinea pigs. Using a systematic evolution of ligands by exponential enrichment (SELEX) approach, we identified ssDNA aptamers from a random ssDNA library that bound to Rv0792c protein. These aptamers were thoroughly characterized using biochemical and biophysical assays. Using SAXS, we determined the structural model of Rv0792c in both the presence and absence of the aptamers. Further, using a combination of SELEX and SAXS methodologies, we identified I-OMe-Tyrphostin as a potential inhibitor of Rv0792c. Here we provide a detailed functional characterization of a transcription factor belonging to the HutC family from M. tuberculosis.
Topics: Animals; Guinea Pigs; Mycobacterium tuberculosis; Tyrphostins; Scattering, Small Angle; Aptamers, Nucleotide; X-Ray Diffraction; Transcription Factors; Tuberculosis; DNA
PubMed: 36507689
DOI: 10.1128/spectrum.01973-22 -
Microbiology (Reading, England) Mar 2021The success of as a pathogen is well established: tuberculosis is the leading cause of death by a single infectious agent worldwide. The threat of multi- and... (Review)
Review
The success of as a pathogen is well established: tuberculosis is the leading cause of death by a single infectious agent worldwide. The threat of multi- and extensively drug-resistant bacteria has renewed global concerns about this pathogen and understanding its virulence strategies will be essential in the fight against tuberculosis. The current review will focus on phthiocerol dimycocerosates (PDIMs), a long-known and well-studied group of complex lipids found in the cell envelope. Numerous studies show a role for PDIMs in several key steps of pathogenesis, with recent studies highlighting its involvement in bacterial virulence, in association with the ESX-1 secretion system. Yet, the mechanisms by which PDIMs help to control macrophage phagocytosis, inhibit phagosome acidification and modulate host innate immunity, remain to be fully elucidated.
Topics: Animals; Cell Membrane; Host-Pathogen Interactions; Humans; Lipids; Macrophages; Mycobacterium tuberculosis; Phagocytosis; Tuberculosis; Virulence
PubMed: 33629944
DOI: 10.1099/mic.0.001042 -
Briefings in Bioinformatics Jul 2021Whole genome sequencing (WGS) is increasingly used for Mycobacterium tuberculosis (Mtb) research. Countries with the highest tuberculosis (TB) burden face important... (Review)
Review
BACKGROUND
Whole genome sequencing (WGS) is increasingly used for Mycobacterium tuberculosis (Mtb) research. Countries with the highest tuberculosis (TB) burden face important challenges to integrate WGS into surveillance and research.
METHODS
We assessed the global status of Mtb WGS and developed a 3-week training course coupled with long-term mentoring and WGS infrastructure building. Training focused on genome sequencing, bioinformatics and development of a locally relevant WGS research project. The aim of the long-term mentoring was to support trainees in project implementation and funding acquisition. The focus of WGS infrastructure building was on the DNA extraction process and bioinformatics.
FINDINGS
Compared to their TB burden, Asia and Africa are grossly underrepresented in Mtb WGS research. Challenges faced resulted in adaptations to the training, mentoring and infrastructure building. Out-of-date laptop hardware and operating systems were overcome by using online tools and a Galaxy WGS analysis pipeline. A case studies approach created a safe atmosphere for students to formulate and defend opinions. Because quality DNA extraction is paramount for WGS, a biosafety level 3 and general laboratory skill training session were added, use of commercial DNA extraction kits was introduced and a 2-week training in a highly equipped laboratory was combined with a 1-week training in the local setting.
INTERPRETATION
By developing and sharing the components of and experiences with a sequencing and bioinformatics training program, we hope to stimulate capacity building programs for Mtb WGS and empower high-burden countries to play an important role in WGS-based TB surveillance and research.
Topics: Africa; Asia; Computational Biology; Cost of Illness; Genome, Bacterial; Humans; Mycobacterium tuberculosis; Tuberculosis; Whole Genome Sequencing
PubMed: 33009560
DOI: 10.1093/bib/bbaa246 -
The Indian Journal of Tuberculosis Oct 2020Tuberculosis is a global threat but in particular affects people from developing countries. It is thought that nearly a third of the population of the world live with... (Review)
Review
Tuberculosis is a global threat but in particular affects people from developing countries. It is thought that nearly a third of the population of the world live with its causative bacteria in a dormant form. Although tuberculosis is a curable disease, the chances of cure become slim as the disease becomes multidrug-resistant and the situation gets even worse as the disease becomes extensively drug-resistant. After approximately 5 decades without any new TB drug in the pipeline, there has been some good news in the recent years with the discovery of new drugs such as bedaquiline and delamanid as well as the discovery of new classes of anti-tubercular drugs. Some old drugs such as clofazimine, linezolid and many others which were not previously indicated for tuberculosis have been also repurposed for tuberculosis and they are performing well.
Topics: Antitubercular Agents; Drug Development; Humans; Mycobacterium tuberculosis; Tuberculosis; Tuberculosis, Multidrug-Resistant
PubMed: 33077057
DOI: 10.1016/j.ijtb.2020.07.017 -
Biomolecules Nov 2023() is an important and harmful intracellular pathogen that is responsible for the cause of tuberculosis (TB). capsular polysaccharides can misdirect the host's immune...
() is an important and harmful intracellular pathogen that is responsible for the cause of tuberculosis (TB). capsular polysaccharides can misdirect the host's immune response pathways, resulting in additional challenges in TB treatment. These capsule polysaccharides are biosynthesized by stealth proteins, including CpsY. The structure and functional mechanism of CpsY are not completely delineated. Here, we reported the crystal structure of CpsY at 1.64 Å. CpsY comprises three β-sheets with five α-helices on one side and three on the other. Four conserved regions (CR1-CR4) are located near and at the base of its catalytic cavity, and three spacer segments (S1-S3) surround the catalytic cavity. Site-directed mutagenesis demonstrated the strict conservation of R419 at CR3 and S1-S3 in regulating the phosphotransferase activity of CpsY. In addition, deletion of S2 or S3 (∆S2 or ∆S3) dramatically increased the activity compared to the wild-type (WT) CpsY. Results from molecular dynamics (MD) simulations showed that S2 and S3 are highly flexible. Our study provides new insights for the development of new vaccines and targeted immunotherapy against
Topics: Humans; Mycobacterium tuberculosis; Bacterial Proteins; Tuberculosis; Molecular Dynamics Simulation; Polysaccharides
PubMed: 38002293
DOI: 10.3390/biom13111611 -
Microbial Drug Resistance (Larchmont,... Sep 2021Shaanxi is the most highly populated province with high burdens of tuberculosis in northwestern China. The aim of this study was to investigate the molecular...
Shaanxi is the most highly populated province with high burdens of tuberculosis in northwestern China. The aim of this study was to investigate the molecular characteristics and drug resistance of isolates from Shaanxi province of China in 2018. Phenotypic drug susceptibility testing and spoligotyping methods were performed on 518 isolates; drug-resistant isolates were sequenced in 11 drug loci, including , , , , , , (), , , (), . The prevalences of isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and kanamycin resistance were 22.0%, 19.3%, 7.9%, 23.8%, 10.4%, and 3.3%, respectively. The Beijing family (82.8%) was the predominant genotype, followed by the T (9.3%), H (0.6%), CAS (0.4%), LAM (0.4%), and U (0.4%) families. The percentage of Beijing genotype in a central area (88.1%) was higher than in the south (77.3%) and the north area (80.1%) ( < 0.05), while the sex, age, and treatment history between Beijing and non-Beijing family were not statistically different. Mutation analysis found that the most prevalent mutations were , , , , , and ; the Beijing family exhibited a high rate of isoniazid-resistant isolates carrying mutations ( < 0.05). Furthermore, compared with the phenotypic data, the sensitivities of isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and kanamycin resistance by sequencing base on 11 loci were 85.1%, 94.0%, 53.7%, 74.8%, 77.8%, and 64.7%, respectively. Shaanxi has a serious epidemic of drug-resistant tuberculosis, Beijing family is the predominant genotype, and the distribution showed geographic diversity. The prevalence of Beijing genotypes has a tendency to promote the transmission of high-level isoniazid-resistant . Besides, the hot spot regions localized in the , , and gene appear not to serve as excellent biomarkers for predicting ethambutol and kanamycin resistance in Shaanxi.
Topics: Adult; Aged; Antitubercular Agents; China; Female; Genes, Bacterial; Humans; Male; Microbial Sensitivity Tests; Middle Aged; Mycobacterium tuberculosis; Phenotype; Sequence Analysis, DNA; Tuberculosis, Multidrug-Resistant
PubMed: 33794134
DOI: 10.1089/mdr.2020.0496 -
Tuberculosis (Edinburgh, Scotland) Dec 2019Mycobacterium tuberculosis (MTB) serine proteases are important pathogen-associated virulence factors that are involved in the invasion, bacterial persistence, and...
Mycobacterium tuberculosis (MTB) serine proteases are important pathogen-associated virulence factors that are involved in the invasion, bacterial persistence, and degradation of host defense factors. The current study identified and characterized a novel serine protease, Rv3194c, of MTB. A heterologous Rv3194c protein, purified from Escherichia coli, possessed proteolytic activity that could hydrolyze bovine serum albumin (BSA), milk, casein, and gelatin at an optimal temperature of 40 °C and a pH of 8.0. Furthermore, the divalent metal ions Ca and Mn increased the activity of Rv3194c. Betulinic acid, a Traditional Chinese Medicine (TCM) monomer; PMSF, a chemical inhibitor; and the Roche inhibitor cocktail inhibited proteolytic activity. Site-directed mutagenesis demonstrated that D308 and particularly S309 play a crucial role in the catalytic activity of Rv3194c protease. The cellular assays revealed that Rv3194c inhibits THP1-derived macrophage migration. Moreover, Rv3194c degraded the complement components, C3b and C5a, causing inhibition of phagocytosis and chemotaxis. In mice, Rv3194c enhanced the persistence of Mycobacterium smegmatis (Ms) in the lung, induced lung lesions, and promoted the release of inflammatory cytokines. The results of this study indicate that Rv3194c may play an important role in the pathogenicity of mycobacteria.
Topics: Animals; Disease Models, Animal; Female; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mycobacterium tuberculosis; Serine Proteases; Virulence
PubMed: 31731061
DOI: 10.1016/j.tube.2019.101880 -
Journal of Immunology (Baltimore, Md. :... Nov 2021In ∼10% of its genome encodes the proline-glutamic acid and proline-proline-glutamic acid (PPE) family of proteins, some of which were recently established to be key... (Review)
Review
In ∼10% of its genome encodes the proline-glutamic acid and proline-proline-glutamic acid (PPE) family of proteins, some of which were recently established to be key players in mycobacterial virulence. PPE2 (Rv0256c) is one among these proteins that we found to have pleiotropic effects during mycobacterial infection. PPE2 weakens the innate immune system by disturbing NO and reactive oxygen species production and myeloid hematopoiesis. We showed that PPE2 is unique for having nuclear localization signal, DNA binding domain, and SRC homology 3 (PXXP) binding domain, which enable it to interfere with the host immune system. Interestingly, PPE2 is a secretary protein, expressed during active tuberculosis (TB) infection, and is involved in facilitating survival of Thus, PPE2 could be a valuable drug target for developing effective therapeutics against TB. In this article, we describe possible roles of PPE2 in TB pathogenesis and the importance of PPE2 as a novel therapeutic target against TB.
Topics: Animals; Antigens, Bacterial; Bacterial Proteins; Humans; Mycobacterium tuberculosis; Virulence
PubMed: 34750243
DOI: 10.4049/jimmunol.2100212 -
Microbiological Research Jul 2024Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), mainly transmitted through droplets to infect the lungs, and... (Review)
Review
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), mainly transmitted through droplets to infect the lungs, and seriously affecting patients' health and quality of life. Clinically, anti-TB drugs often entail side effects and lack efficacy against resistant strains. Thus, the exploration and development of novel targeted anti-TB medications are imperative. Currently, protein-protein interactions (PPIs) offer novel avenues for anti-TB drug development, and the study of targeted modulators of PPIs in M. tuberculosis has become a prominent research focus. Furthermore, a comprehensive PPI network has been constructed using computational methods and bioinformatics tools. This network allows for a more in-depth analysis of the structural biology of PPIs and furnishes essential insights for the development of targeted small-molecule modulators. Furthermore, this article provides a detailed overview of the research progress and regulatory mechanisms of PPI modulators in M. tuberculosis, the causative agent of TB. Additionally, it summarizes potential targets for anti-TB drugs and discusses the prospects of existing PPI modulators.
Topics: Mycobacterium tuberculosis; Bacterial Proteins; Antitubercular Agents; Humans; Tuberculosis; Protein Interaction Maps; Computational Biology
PubMed: 38636239
DOI: 10.1016/j.micres.2024.127675 -
Disease Models & Mechanisms Mar 2020Lactate dehydrogenase A (LDHA) mediates interconversion of pyruvate and lactate, and increased lactate turnover is exhibited by malignant and infected immune cells....
Lactate dehydrogenase A (LDHA) mediates interconversion of pyruvate and lactate, and increased lactate turnover is exhibited by malignant and infected immune cells. Hypoxic lung granuloma in -infected animals present elevated levels of and lactate. Such alterations in the metabolic milieu could influence the outcome of host- interactions. Given the central role of LDHA for tumorigenicity, targeting lactate metabolism is a promising approach for cancer therapy. Here, we sought to determine the importance of LDHA for tuberculosis (TB) disease progression and its potential as a target for host-directed therapy. To this end, we orally administered FX11, a known small-molecule NADH-competitive LDHA inhibitor, to -infected C57BL/6J mice and mice with hypoxic necrotizing lung TB lesions. FX11 did not inhibit growth in aerobic/hypoxic liquid culture, but modestly reduced the pulmonary bacterial burden in C57BL/6J mice. Intriguingly, FX11 administration limited replication and onset of necrotic lung lesions in mice. In this model, isoniazid (INH) monotherapy has been known to exhibit biphasic killing kinetics owing to the probable selection of an INH-tolerant bacterial subpopulation. However, adjunct FX11 treatment corrected this adverse effect and resulted in sustained bactericidal activity of INH against As a limitation, LDHA inhibition as an underlying cause of FX11-mediated effect could not be established as the on-target effect of FX11 was unconfirmed. Nevertheless, this proof-of-concept study encourages further investigation on the underlying mechanisms of LDHA inhibition and its significance in TB pathogenesis.
Topics: Animals; Disease Models, Animal; Female; Host-Pathogen Interactions; Isoniazid; Mice, Inbred C57BL; Mycobacterium tuberculosis; Naphthalenes
PubMed: 32034005
DOI: 10.1242/dmm.041954