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1.
Methods Mol Biol ; 2833: 109-119, 2024.
Article in English | MEDLINE | ID: mdl-38949705

ABSTRACT

Tuberculosis (TB) is the most common cause of death from an infectious disease. Although treatment has been available for more than 70 years, it still takes too long and many patients default risking relapse and the emergence of resistance. It is known that lipid-rich, phenotypically antibiotic-tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse necessitating extended therapy. Using a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis we can perform optical analysis in the form of wavelength-modulated Raman spectroscopy (WMRS) on the trapped organisms. This system can allow observations of the mycobacteria for up to 8 h. By adding antibiotics, it is possible to study the effect of antibiotics in real-time by comparing the Raman fingerprints in comparison to the unstressed condition. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to many conditions including antibiotic stress, and changes in the growth media. This opens the possibility of understanding better the stimuli that trigger the lipid-rich downregulated and phenotypically antibiotic-resistant cell state.


Subject(s)
Mycobacterium smegmatis , Spectrum Analysis, Raman , Spectrum Analysis, Raman/methods , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/growth & development , Microfluidics/methods , Microfluidics/instrumentation , Anti-Bacterial Agents/pharmacology , Acoustics/instrumentation , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Humans
2.
Sci Rep ; 14(1): 14660, 2024 06 25.
Article in English | MEDLINE | ID: mdl-38918410

ABSTRACT

The emergence of drug-resistant Mycobacterium tuberculosis strains is a threat to global health necessitating the discovery of novel chemotherapeutic agents. Natural products drug discovery, which previously led to the discovery of rifamycins, is a valuable approach in this endeavor. Against this backdrop, we set out to investigate the in vitro antimycobacterial properties of medicinal plants from Ghana and South Africa, evaluating 36 extracts and their 252 corresponding solid phase extraction (SPE) generated fractions primarily against the non-pathogenic Mycobacterium smegmatis and Mycobacterium aurum species. The most potent fraction was further evaluated in vitro against infectious M. tuberculosis strain. Crinum asiaticum (bulb) (Amaryllidaceae) emerged as the most potent plant species with specific fractions showing exceptional, near equipotent activity against the non-pathogenic Mycobacterium species (0.39 µg/ml ≤ MIC ≤ 25 µg/ml) with one fraction being moderately active (MIC = 32.6 µg/ml) against M. tuberculosis. Metabolomic analysis led to the identification of eight compounds predicted to be active against M. smegmatis and M. aurum. In conclusion, from our comprehensive study, we generated data which provided an insight into the antimycobacterial properties of Ghanaian and South African plants. Future work will be focused on the isolation and evaluation of the compounds predicted to be active.


Subject(s)
Microbial Sensitivity Tests , Mycobacterium tuberculosis , Plant Extracts , Plants, Medicinal , Plants, Medicinal/chemistry , South Africa , Plant Extracts/pharmacology , Plant Extracts/chemistry , Ghana , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/pharmacology , Antitubercular Agents/chemistry , Mycobacterium/drug effects , Mycobacterium smegmatis/drug effects , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry
3.
Int J Biol Macromol ; 272(Pt 1): 132727, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38823743

ABSTRACT

Due to the uniqueness and essentiality of MEP pathway for the synthesis of crucial metabolites- isoprenoids, hopanoids, menaquinone etc. in mycobacterium, enzymes of this pathway are considered promising anti-tubercular drug targets. In the present study we seek to understand the consequences of downregulation of three of the essential genes- DXS, IspD, and IspF of MEP pathway using CRISPRi approach combined with transcriptomics in Mycobacterium smegmatis. Conditional knock down of either DXS or IspD or IspF gene showed strong bactericidal effect and a profound change in colony morphology. Impaired MEP pathway due to downregulation of these genes increased the susceptibility to frontline anti-tubercular drugs. Further, reduced EtBr accumulation in all the knock down strains in the presence and absence of efflux inhibitor indicated altered cell wall topology. Subsequently, transcriptional analysis validated by qRT-PCR of +154DXS, +128IspD, +104IspF strains showed that modifying the expression of these MEP pathway enzymes affects the regulation of mycobacterial core components. Among the DEGs, expression of small and large ribosomal binding proteins (rpsL, rpsJ, rplN, rplX, rplM, rplS, etc), essential protein translocases (secE, secY and infA, infC), transcriptional regulator (CarD and SigB) and metabolic enzymes (acpP, hydA, ald and fabD) were significantly depleted causing the bactericidal effect. However, mycobacteria survived under these damaging conditions by upregulating mostly the genes needed for the repair of DNA damage (DNA polymerase IV, dinB), synthesis of essential metabolites (serB, LeuA, atpD) and those strengthening the cell wall integrity (otsA, murA, D-alanyl-D-alanine dipeptidase etc.).


Subject(s)
Bacterial Proteins , Gene Expression Regulation, Bacterial , Mycobacterium smegmatis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/drug effects , Gene Expression Regulation, Bacterial/drug effects , Antitubercular Agents/pharmacology , Microbial Viability/drug effects , Microbial Viability/genetics , Metabolic Networks and Pathways
4.
Nat Commun ; 15(1): 4065, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38744895

ABSTRACT

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.


Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Mycobacterium smegmatis , Mycobacterium tuberculosis , Proteolysis , Proteolysis/drug effects , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Anti-Bacterial Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Humans , Microbial Sensitivity Tests , Machine Learning
5.
mSphere ; 9(5): e0012224, 2024 May 29.
Article in English | MEDLINE | ID: mdl-38591887

ABSTRACT

Antibiotic resistance in Mycobacterium tuberculosis exclusively originates from chromosomal mutations, either during normal DNA replication or under stress, when the expression of error-prone DNA polymerases increases to repair damaged DNA. To bypass DNA lesions and catalyze error-prone DNA synthesis, translesion polymerases must be able to access the DNA, temporarily replacing the high-fidelity replicative polymerase. The mechanisms that govern polymerase exchange are not well understood, especially in mycobacteria. Here, using a suite of quantitative fluorescence imaging techniques, we discover that in Mycobacterium smegmatis, as in other bacterial species, the replicative polymerase, DnaE1, exchanges at a timescale much faster than that of DNA replication. Interestingly, this fast exchange rate depends on an actinobacteria-specific nucleoid-associated protein (NAP), Lsr2. In cells missing lsr2, DnaE1 exchanges less frequently, and the chromosome is replicated more faithfully. Additionally, in conditions that damage DNA, cells lacking lsr2 load the complex needed to bypass DNA lesions less effectively and, consistently, replicate with higher fidelity but exhibit growth defects. Together, our results show that Lsr2 promotes dynamic flexibility of the mycobacterial replisome, which is critical for robust cell growth and lesion repair in conditions that damage DNA. IMPORTANCE: Unlike many other pathogens, Mycobacterium tuberculosis has limited ability for horizontal gene transfer, a major mechanism for developing antibiotic resistance. Thus, the mechanisms that facilitate chromosomal mutagenesis are of particular importance in mycobacteria. Here, we show that Lsr2, a nucleoid-associated protein, has a novel role in DNA replication and mutagenesis in the model mycobacterium Mycobacterium smegmatis. We find that Lsr2 promotes the fast exchange rate of the replicative DNA polymerase, DnaE1, at the replication fork and is important for the effective loading of the DnaE2-ImuA'-ImuB translesion complex. Without lsr2, M. smegmatis replicates its chromosome more faithfully and acquires resistance to rifampin at a lower rate, but at the cost of impaired survival to DNA damaging agents. Together, our work establishes Lsr2 as a potential factor in the emergence of mycobacterial antibiotic resistance.


Subject(s)
Bacterial Proteins , DNA Replication , DNA-Directed DNA Polymerase , Drug Resistance, Bacterial , Mycobacterium smegmatis , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/enzymology , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Resistance, Bacterial/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Antigens, Bacterial
6.
Mol Pharm ; 21(5): 2238-2249, 2024 May 06.
Article in English | MEDLINE | ID: mdl-38622497

ABSTRACT

Tuberculosis (TB) is a chronic disease caused byMycobacterium tuberculosis (Mtb), which shows a long treatment cycle often leads to drug resistance, making treatment more difficult. Immunogens present in the pathogen's cell membrane can stimulate endogenous immune responses. Therefore, an effective lipid-based vaccine or drug delivery vehicle formulated from the pathogen's cell membrane can improve treatment outcomes. Herein, we extracted and characterized lipids fromMycobacterium smegmatis, and the extracts contained lipids belonging to numerous lipid classes and compounds typically found associated with mycobacteria. The extracted lipids were used to formulate biomimetic lipid reconstituted nanoparticles (LrNs) and LrNs-coated poly(lactic-co-glycolic acid) nanoparticles (PLGA-LrNs). Physiochemical characterization and results of morphology suggested that PLGA-LrNs exhibited enhanced stability compared with LrNs. And both of these two types of nanoparticles inhibited the growth of M. smegmatis. After loading different drugs, PLGA-LrNs containing berberine or coptisine strongly and synergistically prevented the growth of M. smegmatis. Altogether, the bacterial membrane lipids we extracted with antibacterial activity can be used as nanocarrier coating for synergistic antibacterial treatment of M. smegmatis─an alternative model of Mtb, which is expected as a novel therapeutic system for TB treatment.


Subject(s)
Mycobacterium smegmatis , Nanoparticles , Polylactic Acid-Polyglycolic Acid Copolymer , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Nanoparticles/chemistry , Mycobacterium smegmatis/drug effects , Lipids/chemistry , Drug Synergism , Cell Membrane/drug effects , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/pharmacology , Antitubercular Agents/chemistry , Antitubercular Agents/administration & dosage , Mycobacterium/drug effects , Berberine/pharmacology , Berberine/chemistry , Drug Carriers/chemistry , Tuberculosis/drug therapy
7.
Biochemistry (Mosc) ; 89(3): 407-416, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38648761

ABSTRACT

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.


Subject(s)
Bacterial Proteins , Ligases , Mycobacterium tuberculosis , Bacterial Proteins/metabolism , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Kinetics , Ligases/antagonists & inhibitors , Ligases/metabolism , Molecular Docking Simulation , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/drug effects , Naphthalenes/pharmacology , Naphthalenes/chemistry , Diterpenes/pharmacology
8.
Int J Antimicrob Agents ; 63(6): 107158, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38537722

ABSTRACT

Rifampicin is the most powerful first-line antibiotic for tuberculosis, which is caused by Mycobacterium tuberculosis. Although accumulating evidence from sequencing data of clinical M. tuberculosis isolates suggested that mutations in the rifampicin-resistance-determining region (RRDR) are strongly associated with rifampicin resistance, the comprehensive characterisation of RRDR polymorphisms that confer this resistance remains challenging. By incorporating I-SceI sites for I-SceI-based integrant removal and utilizing an L5 swap strategy, we efficiently replaced the integrated plasmid with alternative alleles, making mass allelic exchange feasible in mycobacteria. Using this method to establish a fitness-related gain-of function screen, we generated a mutant library that included all single-amino-acid mutations in the RRDR, and identified the important positions corresponding to some well-known rifampicin-resistance mutations (Q513, D516, S522, H525, R529, S531). We also detected a novel two-point mutation located in the RRDR confers a fitness advantage to M. smegmatis in the presence or absence of rifampicin. Our method provides a comprehensive insight into the growth phenotypes of RRDR mutants and should facilitate the development of anti-tuberculosis drugs.


Subject(s)
Drug Resistance, Bacterial , Mycobacterium tuberculosis , Rifampin , Rifampin/pharmacology , Drug Resistance, Bacterial/genetics , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/drug effects , Mutation , Mutagenesis , Antitubercular Agents/pharmacology , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/drug effects , Microbial Sensitivity Tests , High-Throughput Screening Assays/methods , Humans
9.
J Biol Chem ; 300(1): 105567, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38103641

ABSTRACT

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.


Subject(s)
Bacterial Proteins , G-Quadruplexes , Gene Expression Regulation, Bacterial , Mycobacterium tuberculosis , Protein Biosynthesis , RNA, Bacterial , RNA, Messenger , Humans , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Genes, Bacterial/genetics , Inflammation/microbiology , Ligands , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/growth & development , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/metabolism , Oligoribonucleotides/genetics , Oligoribonucleotides/metabolism , RNA Stability , RNA, Bacterial/genetics , RNA, Messenger/genetics , THP-1 Cells , Transcription, Genetic/drug effects
10.
Chem Biodivers ; 20(3): e202200691, 2023 Mar.
Article in English | MEDLINE | ID: mdl-36692091

ABSTRACT

Plectranthus amboinicus (Lour.) Spreng, known as the Indian borage or Mexican mint, is one of the most documented species in the family Lamiaceae for its therapeutic and pharmaceutical values. It is found in the tropical and subtropical regions of the world. The leaf essential oil has immense medicinal benefits like treating illnesses of the skin and disorders like colds, asthma, constipation, headaches, coughs, and fevers. After analyzing earlier reports with regard to the quantity and quality of leaf oil yield, we discovered that the germplasm taken from Odisha is preferable to other germplasms. The objective of the present work is to evaluate the free radical scavenging activity and bactericidal effect of leaf essential oil (EO) of Plectranthus amboinicus (Lour.) Spreng collected from the state of Odisha, India. The hydro distillation technique has been used for essential oil extraction. Upon GC/MS analysis, approximately 57 compounds were identified with Carvacrol as the major compound (peak area=20.25 %), followed by p-thymol (peak area=20.17 %), o-cymene (peak area=19.41 %) and carene (peak area=15.89 %). On evaluation of free radical scavenging activity, it was recorded that the best value of inhibitory concentration, was for DPPH with IC50 =18.64 ppm and for H2 O2 with IC50 =9.35 ppm. The EO showed efficient bactericidal effect against both gram positive (Mycobacterium smegmatis, Staphylococcus aureus, Enterococcus faecium) and gram negative (Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae) bacteria studied through well diffusion method. Fumigatory action of the essential oil was found against M. smegmatis, the model organism for tuberculosis study. Alamar Blue assay, gave a result with MIC value for M. smegmatis i. e., 0.12 µg/ml and the MBC value of 0.12 µg/ml. Hence, P. amboinicus found in Odisha can be suggested as an elite variety and should be further investigated for efficient administration in drug formulation.


Subject(s)
Oils, Volatile , Plectranthus , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/analysis , Free Radicals , Microbial Sensitivity Tests , Oils, Volatile/chemistry , Oils, Volatile/pharmacology , Plant Leaves/chemistry , Plectranthus/chemistry , Mycobacterium smegmatis/drug effects
11.
PLoS One ; 18(1): e0281170, 2023.
Article in English | MEDLINE | ID: mdl-36719870

ABSTRACT

BACKGROUND: Drug resistance is a prominent problem in the treatment of tuberculosis, so it is urgent to develop new anti- tuberculosis drugs. Here, we investigated the effects and mechanisms of cisplatin (DDP) on intracellular Mycobacterium smegmatis to tap the therapeutic potential of DDP in mycobacterial infection. RESULTS: Macrophages infected with Mycobacterium smegmatis were treated with DDP alone or combined with isoniazid or rifampicin. The results showed that the bacterial count in macrophages decreased significantly after DDP (≤ 6 µg/mL) treatment. When isoniazid or rifampicin was combined with DDP, the number of intracellular mycobacteria was also significantly lower than that of isoniazid or rifampicin alone. Apoptosis of infected cells increased after 24 h of DDP treatment, as shown by flow cytometry and transmission electron microscopy detection. Transcriptome sequencing showed that there were 1161 upregulated and 645 downregulated differentially expressed genes (DEGs) between the control group and DDP treatment group. A Trp53-centered protein interaction network was found based on the top 100 significant DEGs through STRING and Cytoscape software. The expression of phosphorylated p53, Bax, JAK, p38 MAPK and PI3K increased after DDP treatment, as shown by Western blot analysis. Inhibitors of JAK, PI3K or p38 MAPK inhibited the increase in cell apoptosis and the reduction in the intracellular bacterial count induced by DDP. The p53 promoter Kevetrin hydrochloride scavenges intracellular mycobacteria. If combined with DDP, Kevetrin hydrochloride could increase the effect of DDP on the elimination of intracellular mycobacteria. In conclusion, DDP at low concentrations could activate the JAK, p38 MAPK and PI3K pathways in infected macrophages, promote the phosphorylation of p53 protein, and increase the ratio of Bax to Bcl-2, leading to cell apoptosis, thus eliminating intracellular bacteria and reducing the spread of mycobacteria. CONCLUSION: DDP may be a new host-directed therapy for tuberculosis treatment, as well as the p53 promoter Kevetrin hydrochloride.


Subject(s)
Antitubercular Agents , Cisplatin , Drug Resistance, Bacterial , Macrophages , Mycobacterium smegmatis , Apoptosis/drug effects , bcl-2-Associated X Protein , Cell Proliferation/drug effects , Cisplatin/pharmacology , Isoniazid/pharmacology , Phosphatidylinositol 3-Kinases , Rifampin/pharmacology , Tumor Suppressor Protein p53/genetics , Antitubercular Agents/pharmacology , Drug Resistance, Bacterial/genetics , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/immunology , Macrophages/drug effects , Macrophages/immunology , Macrophages/microbiology , Nitriles/pharmacology , Thiourea/analogs & derivatives , Thiourea/pharmacology , Butanones/pharmacology
12.
J Biol Chem ; 298(4): 101752, 2022 04.
Article in English | MEDLINE | ID: mdl-35189142

ABSTRACT

RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (RPo). RbpA consists of four domains: an N-terminal tail (NTT), a core domain (CD), a basic linker, and a sigma interaction domain. We have previously shown that truncation of the RbpA NTT and CD increases RPo stabilization by RbpA, implying that these domains inhibit this activity of RbpA. Previously published structural studies showed that the NTT and CD are positioned near multiple RNAP-σA holoenzyme functional domains and predict that the RbpA NTT contributes specific amino acids to the binding site of the antibiotic fidaxomicin (Fdx), which inhibits the formation of the RPo complex. Furthermore, deletion of the NTT results in decreased Mycobacterium smegmatis sensitivity to Fdx, but whether this is caused by a loss in Fdx binding is unknown. We generated a panel of rbpA mutants and found that the RbpA NTT residues predicted to directly interact with Fdx are partially responsible for RbpA-dependent Fdx activity in vitro, while multiple additional RbpA domains contribute to Fdx activity in vivo. Specifically, our results suggest that the RPo-stabilizing activity of RbpA decreases Fdx activity in vivo. In support of the association between RPo stability and Fdx activity, we find that another factor that promotes RPo stability in bacteria, CarD, also impacts to Fdx sensitivity. Our findings highlight how RbpA and other factors may influence RNAP dynamics to affect Fdx sensitivity.


Subject(s)
Fidaxomicin , Mycobacterium smegmatis , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Fidaxomicin/pharmacology , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/genetics , Promoter Regions, Genetic , Sigma Factor/metabolism
13.
Microbiol Spectr ; 10(1): e0126221, 2022 02 23.
Article in English | MEDLINE | ID: mdl-35171048

ABSTRACT

The emergence of antimicrobial resistance warrants for the development of improved treatment approaches. In this regard, peptide nucleic acids (PNAs) have shown great promise, exhibiting antibiotic properties through the targeting of cellular nucleic acids. We aimed to study the efficacy of PNA as an anti-tuberculosis agent. Since the efficacy of PNA is limited by its low penetration into the cell, we also investigated combinatorial treatments using permeabilizing drugs to improve PNA efficacy. Various concentrations of anti-inhA PNA, permeabilizing drugs, and their combinations were screened against extracellular and intracellular mycobacteria.0.625 to 5 µM anti-inhA PNA was observed to merely inhibit the growth of extracellular M. smegmatis, while low intracellular bacterial load was reduced by 2 or 2.5 log-fold when treated with 2.5 or 5 µM PNA, respectively. Anti-inhA PNA against M. tuberculosis H37Ra exhibited bactericidal properties at 2.5 and 5 µM and enabled a slight reduction in intracellular M. tuberculosis at concentrations from 2.5 to 20 µM. Of the permeabilizing drugs tested, ethambutol showed the most permeabilizing potential and ultimately potentiated anti-inhA PNA to the greatest extent, reducing its efficacious concentration to 1.25 µM against both M. smegmatis and M. tuberculosis. Furthermore, an enhanced clearance of 1.3 log-fold was observed for ethambutol-anti-inhA PNA combinations against intracellular M. tuberculosis. Thus, permeabilizing drug-PNA combinations indeed exhibit improved efficacies. We therefore propose that anti-inhA PNA could improve therapy even when applied in minute doses as an addition to the current anti-tuberculosis drug regimen. IMPORTANCE Peptide nucleic acids have great potential in therapeutics as anti-gene/anti-sense agents. However, their limited uptake in cells has curtailed their widespread application. Through this study, we explore a PNA-drug combinatorial strategy to improve the efficacy of PNAs and reduce their effective concentrations. This work also focuses on improving tuberculosis treatment, which is hindered by the emergence of antimicrobial-resistant strains of Mycobacterium tuberculosis. It is observed that the antibacterial efficacy of anti-inhA PNA is enhanced when it is combined with permeabilizing drugs, particularly ethambutol. This indicates that the addition of even small concentrations of anti-inhA PNA to the current TB regimen could potentiate their therapeutic efficiency. We hypothesize that this system would also overcome isoniazid resistance, since the resistance mutations lie outside the designed anti-inhA PNA target site.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Ceftazidime/pharmacology , Colistin/pharmacology , Ethambutol/pharmacology , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , Oxidoreductases/genetics , Peptide Nucleic Acids/pharmacology , Bacterial Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane Permeability , Drug Synergism , Humans , Microbial Sensitivity Tests , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/growth & development , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/metabolism , Oxidoreductases/metabolism , Peptide Nucleic Acids/genetics , Tuberculosis/drug therapy , Tuberculosis/microbiology
14.
J Antibiot (Tokyo) ; 75(2): 72-76, 2022 02.
Article in English | MEDLINE | ID: mdl-34949834

ABSTRACT

During our screening for antibiotics against Mycobacterium avium complex (MAC) with a mass spectrometry network-based indexing approach, a new compound named kimidinomycin was isolated from the culture broth of Streptomyces sp. KKTA-0263 by solvent extraction, HP20 column chromatography, and preparative HPLC. From the structural elucidation, the compound possesses a 38-membered macrolide structure with an N-methylguanidyl group at the terminal side chain. The compound exhibited antimycobacterial activity against M. avium, M. intracellulare, M. smegmatis, and M. bovis BCG with respective MIC values of 12.5, 0.78, 12.5, and 25.0 µg ml-1.


Subject(s)
Antibiotics, Antitubercular , Mycobacterium avium Complex , Streptomyces , Animals , Cricetinae , Humans , Antibiotics, Antitubercular/biosynthesis , Antibiotics, Antitubercular/pharmacology , Antibiotics, Antitubercular/toxicity , CHO Cells , Chromatography, High Pressure Liquid , Cricetulus , Fermentation , HeLa Cells , Macrolides/chemistry , Microbial Sensitivity Tests , Mycobacterium avium Complex/drug effects , Mycobacterium avium-intracellulare Infection , Mycobacterium bovis/drug effects , Mycobacterium smegmatis/drug effects , Streptomyces/metabolism
15.
Nucleic Acids Res ; 49(22): 12805-12819, 2021 12 16.
Article in English | MEDLINE | ID: mdl-34871411

ABSTRACT

DNA repair systems allow microbes to survive in diverse environments that compromise chromosomal integrity. Pathogens such as Mycobacterium tuberculosis must contend with the genotoxic host environment, which generates the mutations that underlie antibiotic resistance. Mycobacteria encode the widely distributed SOS pathway, governed by the LexA repressor, but also encode PafBC, a positive regulator of the transcriptional DNA damage response (DDR). Although the transcriptional outputs of these systems have been characterized, their full functional division of labor in survival and mutagenesis is unknown. Here, we specifically ablate the PafBC or SOS pathways, alone and in combination, and test their relative contributions to repair. We find that SOS and PafBC have both distinct and overlapping roles that depend on the type of DNA damage. Most notably, we find that quinolone antibiotics and replication fork perturbation are inducers of the PafBC pathway, and that chromosomal mutagenesis is codependent on PafBC and SOS, through shared regulation of the DnaE2/ImuA/B mutasome. These studies define the complex transcriptional regulatory network of the DDR in mycobacteria and provide new insight into the regulatory mechanisms controlling the genesis of antibiotic resistance in M. tuberculosis.


Subject(s)
Bacterial Proteins/genetics , DNA Repair/genetics , Mutagenesis , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , SOS Response, Genetics/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Ciprofloxacin/pharmacology , DNA Damage , Gene Expression Profiling/methods , Gene Expression Regulation, Bacterial/drug effects , Gene Regulatory Networks/drug effects , Gene Regulatory Networks/genetics , Microbial Viability/drug effects , Microbial Viability/genetics , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Species Specificity
16.
Proc Natl Acad Sci U S A ; 118(47)2021 11 23.
Article in English | MEDLINE | ID: mdl-34782468

ABSTRACT

The structure has been determined by electron cryomicroscopy of the adenosine triphosphate (ATP) synthase from Mycobacterium smegmatis This analysis confirms features in a prior description of the structure of the enzyme, but it also describes other highly significant attributes not recognized before that are crucial for understanding the mechanism and regulation of the mycobacterial enzyme. First, we resolved not only the three main states in the catalytic cycle described before but also eight substates that portray structural and mechanistic changes occurring during a 360° catalytic cycle. Second, a mechanism of auto-inhibition of ATP hydrolysis involves not only the engagement of the C-terminal region of an α-subunit in a loop in the γ-subunit, as proposed before, but also a "fail-safe" mechanism involving the b'-subunit in the peripheral stalk that enhances engagement. A third unreported characteristic is that the fused bδ-subunit contains a duplicated domain in its N-terminal region where the two copies of the domain participate in similar modes of attachment of the two of three N-terminal regions of the α-subunits. The auto-inhibitory plus the associated "fail-safe" mechanisms and the modes of attachment of the α-subunits provide targets for development of innovative antitubercular drugs. The structure also provides support for an observation made in the bovine ATP synthase that the transmembrane proton-motive force that provides the energy to drive the rotary mechanism is delivered directly and tangentially to the rotor via a Grotthuss water chain in a polar L-shaped tunnel.


Subject(s)
Adenosine Triphosphate/metabolism , Mitochondrial Proton-Translocating ATPases/chemistry , Mitochondrial Proton-Translocating ATPases/drug effects , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/enzymology , Tuberculosis/drug therapy , Animals , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Bacterial Proteins , Cattle , Cryoelectron Microscopy , Hydrolysis , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Models, Molecular , Protein Conformation , Protein Subunits/chemistry , Proteins/chemistry , Proton-Motive Force , Tuberculosis/microbiology , ATPase Inhibitory Protein
17.
Microbiology (Reading) ; 167(10)2021 10.
Article in English | MEDLINE | ID: mdl-34665112

ABSTRACT

MSMEG_2295 is a TetR family protein encoded by the first gene of a Mycobacterium smegmatis (Msm) operon that expresses the gene for DinB2 (MSMEG_2294), a translesion DNA repair enzyme. We have carried out investigations to understand its function by performing DNA binding studies and gene knockout experiments. We found that the protein binds to a conserved inverted repeat sequence located upstream of the dinB2 operon and several other genes. Using a knockout of MSMEG_2295, we show that MSMEG_2295 controls the expression of at least five genes, the products of which could potentially influence carbohydrate and fatty acid metabolism as well as antibiotic and oxidative stress resistance. We have demonstrated that MSMEG_2295 is a repressor by performing complementation analysis. Knocking out of MSMEG_2295 had a significant impact on pyruvate metabolism. Pyruvate dehydrogenase activity was virtually undetectable in ΔMSMEG_2295, although in the complemented strain, it was high. We also show that knocking out of MSMEG_2295 causes resistance to H2O2, reversed in the complemented strain. We have further found that the mycobacterial growth inhibitor plumbagin, a compound of plant origin, acts as an inducer of MSMEG_2295 regulated genes. We, therefore, establish that MSMEG_2295 functions by exerting its role as a repressor of multiple Msm genes and that by doing so, it plays a vital role in controlling pyruvate metabolism and response to oxidative stress.


Subject(s)
Bacterial Proteins/metabolism , Mycobacterium smegmatis/metabolism , Repressor Proteins/metabolism , Bacterial Proteins/genetics , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial/drug effects , Mutation , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Naphthoquinones/pharmacology , Operator Regions, Genetic , Operon/genetics , Promoter Regions, Genetic , Pyruvic Acid/metabolism , Repressor Proteins/genetics , Superoxides/metabolism
18.
Bioorg Chem ; 117: 105422, 2021 12.
Article in English | MEDLINE | ID: mdl-34700110

ABSTRACT

The emergence of bacterial resistance has triggered a multitude of efforts to develop new antibacterial agents. There are many compounds in literature that were reported as potent antibacterial agents, however, they lacked the required safety to mammalian cells or no clear picture about their toxicity profile was presented. Inspired by discovered hit from our in-house library and by previously reported 2,4-diaminosubstituted quinazolines, we describe the design and synthesis of novel 2,4-disubstituted-thioquinazolines (3-13 and 36), 2-thio-4-amino substituted quinazolines (14-33) and 6-substituted 2,4-diamonsubstituted quinazolines (37-39). The synthesized compounds showed potent antibacterial activity against a panel of Gram-positive, efflux deficient E.coli and Mycobacterium smegmatis. The panel also involved resistant strains including methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis and vancomycin-resistant Enterococcus faecium, in addition to Mycobacterium smegmatis. The newly synthesized compounds revealed MIC values against the tested strains ranging from 1 to 64 µg/mL with a good safety profile. Most of the 2-thio-4-amino substituted-quinazolines showed significant antimycobacterial activity with the variations at position 2 and 4 offering additional antibacterial activity against the different strains. Compared to previously reported 2,4-diaminosubstituted quinazolines, the bioisosteric replacement of the 2-amino with sulfur offered a successful approach to keep the high antibacterial potency while substantially improving safety profile as indicated by the reduced activity on different cell lines and a lack of hemolytic activity.


Subject(s)
Anti-Bacterial Agents/pharmacology , Quinazolines/pharmacology , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , CHO Cells , Cell Survival/drug effects , Cricetulus , Dose-Response Relationship, Drug , Enterococcus faecalis/drug effects , Hep G2 Cells , Humans , Microbial Sensitivity Tests , Molecular Structure , Mycobacterium smegmatis/drug effects , Quinazolines/chemical synthesis , Quinazolines/chemistry , Staphylococcus aureus/drug effects , Streptococcus pneumoniae/drug effects , Structure-Activity Relationship
19.
Biomed Pharmacother ; 144: 112264, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34624680

ABSTRACT

In Sudanese traditional medicine, decoctions, macerations, and tonics of the stem and root of Combretum hartmannianum are used for the treatment of persistent cough, a symptom that could be related to tuberculosis (TB). To verify these traditional uses, extracts from the stem wood, stem bark, and roots of C. hartmannianum were screened for their growth inhibitory effects against Mycobacterium smegmatis ATCC 14468. Methanol Soxhlet and ethyl acetate extracts of the root gave the strongest effects (MIC 312.5 and 625 µg/ml, respectively). HPLC-UV/DAD and UHPLC/QTOF-MS analysis of the ethyl acetate extract of the root led to the detection of 54 compounds, of which most were polyphenols and many characterized for the first time in C. hartmannianum. Among the major compounds were terflavin B and its two isomers, castalagin, corilagin, tellimagrandin I and its derivative, (S)-flavogallonic acid dilactone, punicalagin, and methyl-ellagic acid xylopyranoside. In addition, di-, tri- and tetra-galloyl glucose, combregenin, terminolic acid, cordifoliside D, luteolin, and quercetin-3-O-galactoside-7-O-rhamnoside-(2→1)-O-ß-D-arabinopyranoside were characterized. Luteolin gave better growth inhibition against M. smegmatis (MIC 250 µg/ml) than corilagin, ellagic acid, and gallic acid (MIC 500-1000 µg/ml). Our study justifies the use of C. hartmannianum in Sudanese folk medicine against prolonged cough that could be related to TB infection. This study demonstrates that C. hartmannianum should be explored further for new anti-TB drug scaffolds and antibiotic adjuvants.


Subject(s)
Anti-Bacterial Agents/pharmacology , Combretum , Flavonoids/pharmacology , Glycosides/pharmacology , Hydrolyzable Tannins/pharmacology , Mycobacterium smegmatis/drug effects , Pentacyclic Triterpenes/pharmacology , Phytochemicals/pharmacology , Plant Extracts/pharmacology , Anti-Bacterial Agents/isolation & purification , Combretum/chemistry , Ethnopharmacology , Flavonoids/isolation & purification , Glycosides/isolation & purification , Humans , Hydrolyzable Tannins/isolation & purification , Microbial Sensitivity Tests , Mycobacterium smegmatis/growth & development , Pentacyclic Triterpenes/isolation & purification , Phytochemicals/isolation & purification , Plant Extracts/isolation & purification , Sudan
20.
Elife ; 102021 09 30.
Article in English | MEDLINE | ID: mdl-34590581

ABSTRACT

The imidazopyridine telacebec, also known as Q203, is one of only a few new classes of compounds in more than 50 years with demonstrated antituberculosis activity in humans. Telacebec inhibits the mycobacterial respiratory supercomplex composed of complexes III and IV (CIII2CIV2). In mycobacterial electron transport chains, CIII2CIV2 replaces canonical CIII and CIV, transferring electrons from the intermediate carrier menaquinol to the final acceptor, molecular oxygen, while simultaneously transferring protons across the inner membrane to power ATP synthesis. We show that telacebec inhibits the menaquinol:oxygen oxidoreductase activity of purified Mycobacterium smegmatis CIII2CIV2 at concentrations similar to those needed to inhibit electron transfer in mycobacterial membranes and Mycobacterium tuberculosis growth in culture. We then used electron cryomicroscopy (cryoEM) to determine structures of CIII2CIV2 both in the presence and absence of telacebec. The structures suggest that telacebec prevents menaquinol oxidation by blocking two different menaquinol binding modes to prevent CIII2CIV2 activity.


Subject(s)
Antitubercular Agents/pharmacology , Imidazoles/pharmacology , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , Piperidines/pharmacology , Pyridines/pharmacology , Electron Transport , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/metabolism , Oxidation-Reduction
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