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Antibiotics (Basel, Switzerland) May 2024The discovery of novel therapeutic agents, especially those targeting mycobacterial membrane protein large 3 (), has shown promise. In this study, the CRISPR...
BACKGROUND
The discovery of novel therapeutic agents, especially those targeting mycobacterial membrane protein large 3 (), has shown promise. In this study, the CRISPR interference- nuclease-deactivated Cas9 (CRISPRi-dCas9) system was utilized to suppress expression in , and its impacts on susceptibility to antimicrobial agents were evaluated.
METHODS
The repression of the gene was confirmed by RT-qPCR. The essentiality, growth curve, viability, and antimicrobial susceptibility of the knockdown strain were investigated.
RESULTS
silencing was achieved by utilizing 0.5 and 1 ng/mL anhydrotetracycline (ATc), resulting in reductions in the expression of 60.4% and 74.4%, respectively. silencing led to a significant decrease in bacterial viability when combined with one-half of the minimal inhibitory concentrations (MICs) of rifampicin, rifabutin, ceftriaxone, or isoniazid, along with 0.1 or 0.5 ng/mL ATc ( < 0.05). However, no significant difference was observed for clarithromycin or amikacin.
CONCLUSIONS
The downregulation of the gene in mycobacteria was achieved through the use of CRISPRi-dCas9, resulting in growth deficiencies and resensitization to certain antimicrobial agents. The impact was dependent upon the level of gene expression.
PubMed: 38927150
DOI: 10.3390/antibiotics13060483 -
Microbial Biotechnology Jun 2024Ethylene and ethylene oxide are widely used in the chemical industry, and ethylene is also important for its role in fruit ripening. Better sensing systems would assist...
Ethylene and ethylene oxide are widely used in the chemical industry, and ethylene is also important for its role in fruit ripening. Better sensing systems would assist risk management of these chemicals. Here, we characterise the ethylene regulatory system in Mycobacterium strain NBB4 and use these genetic parts to create a biosensor. The regulatory genes etnR1 and etnR2 and cognate promoter P were combined with a fluorescent reporter gene (fuGFP) in a Mycobacterium shuttle vector to create plasmid pUS301-EtnR12P. Cultures of M. smegmatis mc-155(pUS301-EtnR12P) gave a fluorescent signal in response to ethylene oxide with a detection limit of 0.2 μM (9 ppb). By combining the epoxide biosensor cells with another culture expressing the ethylene monooxygenase, the system was converted into an ethylene biosensor. The co-culture was capable of detecting ethylene emission from banana fruit. These are the first examples of whole-cell biosensors for epoxides or aliphatic alkenes. This work also resolves long-standing questions concerning the regulation of ethylene catabolism in bacteria.
Topics: Biosensing Techniques; Ethylenes; Ethylene Oxide; Mycobacterium; Musa; Genes, Reporter; Plasmids
PubMed: 38925606
DOI: 10.1111/1751-7915.14511 -
Archiv Der Pharmazie Jun 2024Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop...
Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop drug resistance against the most common antibiotics used to treat NTM infections. The overexpression of efflux pumps (EPs) was demonstrated to be a key mechanism of clarithromycin (CLA) resistance in NTM. Therefore, in this work, 24 compounds from an in-house library, characterized by chemical diversity, were tested as potential NTM EP inhibitors (EPIs) against Mycobacterium smegmatis mc155 and M. avium clinical isolates. Based on the acquired results, 12 novel analogs of the best derivatives 1b and 7b were designed and synthesized to improve the NTM EP inhibition activity. Among the second set of compounds, 13b emerged as the most potent NTM EPI. At a concentration of 4 µg/mL, it reduced the CLA minimum inhibitory concentration by 16-fold against the clinical isolate M. avium 2373 overexpressing EPs as primary mechanism of CLA resistance.
PubMed: 38923553
DOI: 10.1002/ardp.202400296 -
Scientific Reports Jun 2024The emergence of drug-resistant Mycobacterium tuberculosis strains is a threat to global health necessitating the discovery of novel chemotherapeutic agents. Natural...
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.
Topics: Plants, Medicinal; Microbial Sensitivity Tests; South Africa; Plant Extracts; Ghana; Mycobacterium tuberculosis; Antitubercular Agents; Mycobacterium; Mycobacterium smegmatis; Humans; Anti-Bacterial Agents
PubMed: 38918410
DOI: 10.1038/s41598-024-65369-7 -
Microbiology Spectrum Jun 2024Riboflavin (vitamin B) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin...
UNLABELLED
Riboflavin (vitamin B) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including (Mtb), can biosynthesize riboflavin . Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of or is profoundly bactericidal in Mtb; and (iv) in Msm, silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.
IMPORTANCE
The pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease.
PubMed: 38916330
DOI: 10.1128/spectrum.03207-23 -
Nature Communications Jun 2024Aerobic life is powered by membrane-bound redox enzymes that shuttle electrons to oxygen and transfer protons across a biological membrane. Structural studies suggest...
Aerobic life is powered by membrane-bound redox enzymes that shuttle electrons to oxygen and transfer protons across a biological membrane. Structural studies suggest that these energy-transducing enzymes operate as higher-order supercomplexes, but their functional role remains poorly understood and highly debated. Here we resolve the functional dynamics of the 0.7 MDa IIIIV obligate supercomplex from Mycobacterium smegmatis, a close relative of M. tuberculosis, the causative agent of tuberculosis. By combining computational, biochemical, and high-resolution (2.3 Å) cryo-electron microscopy experiments, we show how the mycobacterial supercomplex catalyses long-range charge transport from its menaquinol oxidation site to the binuclear active site for oxygen reduction. Our data reveal proton and electron pathways responsible for the charge transfer reactions, mechanistic principles of the quinone catalysis, and how unique molecular adaptations, water molecules, and lipid interactions enable the proton-coupled electron transfer (PCET) reactions. Our combined findings provide a mechanistic blueprint of mycobacterial supercomplexes and a basis for developing drugs against pathogenic bacteria.
Topics: Mycobacterium smegmatis; Electron Transport; Cryoelectron Microscopy; Oxidation-Reduction; Bacterial Proteins; Protons; Electron Transport Complex III; Oxygen; Electron Transport Complex IV; Catalytic Domain; Models, Molecular
PubMed: 38902248
DOI: 10.1038/s41467-024-49628-9 -
International Journal of Molecular... Jun 2024Mitochondria-targeted antioxidants (MTAs) have been studied quite intensively in recent years as potential therapeutic agents and vectors for the delivery of other...
Mitochondria-targeted antioxidants (MTAs) have been studied quite intensively in recent years as potential therapeutic agents and vectors for the delivery of other active substances to mitochondria and bacteria. Their most studied representatives are MitoQ and SkQ1, with its fluorescent rhodamine analog SkQR1, a decyl ester of rhodamine 19 carrying plastoquinone. In the present work, we observed a pronounced antibacterial action of SkQR1 against Gram-positive bacteria, but virtually no effect on Gram-negative bacteria. The MDR pump AcrAB-TolC, known to expel SkQ1, did not recognize and did not pump out SkQR1 and dodecyl ester of rhodamine 19 (C12R1). Rhodamine 19 butyl (C4R1) and ethyl (C2R1) esters more effectively suppressed the growth of Δ, but lost their potency with the wild-type pumping them out. The mechanism of the antibacterial action of SkQR1 may differ from that of SkQ1. The rhodamine derivatives also proved to be effective antibacterial agents against various Gram-positive species, including and . By using fluorescence correlation spectroscopy and fluorescence microscopy, SkQR1 was shown to accumulate in the bacterial membrane. Thus, the presentation of SkQR1 as a fluorescent analogue of SkQ1 and its use for visualization should be performed with caution.
Topics: Anti-Bacterial Agents; Rhodamines; Esters; Microbial Sensitivity Tests; Plastoquinone; Gram-Positive Bacteria; Escherichia coli; Mitochondria; Staphylococcus aureus; Fluorescent Dyes
PubMed: 38892325
DOI: 10.3390/ijms25116137 -
BMC Genomic Data Jun 2024The rising of antibiotic resistance has sparked a renewed interest in mycobacteriophage as alternative therapeutic strategies against mycobacterial infections. So far,...
OBJECTIVES
The rising of antibiotic resistance has sparked a renewed interest in mycobacteriophage as alternative therapeutic strategies against mycobacterial infections. So far, the vast majority of mycobacteriophages have been isolated using the model species Mycobacterium smegmatis, implying an overwhelming majority of mycobacteriophages in the environment remain uncultured, unclassified, and their specific hosts and infection strategies are still unknown. This study was undertaken to isolate and characterize novel mycobacteriophages targeting Mycobacterium septicum.
DATA DESCRIPTION
Here a novel mycobacteriophage WXIN against M. septicum was isolated from soil samples in Wuhan, China. Whole genome analysis indicates that the phage genome consists of 115,158 bp with a GC content of 61.9%. Of the 260 putative open reading frames, 46 may be associated with phage packaging, structure, lysis, lysogeny, genome modification/replication, and other functional roles. The limited genome-wide similarity, along with phylogenetic trees constructed based on viral proteome and orthologous genes show that phage WXIN represents a novel cluster distantly related to cluster J mycobacteriophages (genus Omegavirus). Overall, these results provide novel insights into the genomic properties of mycobacteriophages, highlighting the great genetic diversity of mycobacteriophages in relation to their hosts.
Topics: Genome, Viral; Mycobacteriophages; China; Phylogeny; Open Reading Frames; Mycobacterium; Soil Microbiology; Base Composition
PubMed: 38890591
DOI: 10.1186/s12863-024-01244-8 -
Tuberculosis (Edinburgh, Scotland) Jun 2024The PrrAB two-component system (TCS) is essential for Mycobacterium tuberculosis viability. Previously, it was demonstrated that PrrA binds DNA in the absence of...
The PrrAB two-component system (TCS) is essential for Mycobacterium tuberculosis viability. Previously, it was demonstrated that PrrA binds DNA in the absence of PrrB-mediated transphosphorylation and that non-cognate serine/threonine-kinases phosphorylate PrrA threonine-6 (T6). Therefore, we investigated the differential binding affinity and regulatory properties of the M. tuberculosis-derived wild-type PrrA, PrrA phosphomimetic (D58E, T6E), and PrrA phosphoablative (D58A, T6A) proteins with the prrA, dosR, and cydA genes. While we hypothesized greater DNA binding affinity and more pronounced regulation by PrrA phosphomimetic variants, recombinant, wild-type PrrA bound DNA with greatest affinity. Collectively, wild-type PrrA recombinant protein displayed the highest binding affinity to the dosR promoter (K 3.46 ± 2.09 nM), followed by the prrA promoter (K 9.00 ± 2.66 nM). To establish PrrA regulatory activity, we constructed M. smegmatis ΔprrAB::prrA strains with each of the PrrA variants and extrachromosomal prrA, dosR, and cydA promoter-mCherry reporter fusions. Our findings showed that PrrA is autoregulatory and induces dosR expression only during in vitro, hypoxic growth. Combined expression of prrAB in M. smegmatis ΔprrAB significantly induced cydA promoter-mCherry expression. Our studies advanced the understanding of PrrA function and PrrAB phosphorylation-mediated regulatory mechanisms and control of mycobacterial dosR and cydA hypoxic and low-oxygen responsive genes.
PubMed: 38885567
DOI: 10.1016/j.tube.2024.102531 -
Phenomics (Cham, Switzerland) Apr 2024ADP-ribosylation is a reversible and dynamic post-translational modification mediated by ADP-ribosyltransferases (ARTs). Poly(ADP-ribose) polymerases (PARPs) are an...
UNLABELLED
ADP-ribosylation is a reversible and dynamic post-translational modification mediated by ADP-ribosyltransferases (ARTs). Poly(ADP-ribose) polymerases (PARPs) are an important family of human ARTs. ADP-ribosylation and PARPs have crucial functions in host-pathogen interaction, especially in viral infections. However, the functions and potential molecular mechanisms of ADP-ribosylation and PARPs in infection remain unknown. In this study, bioinformatics analysis revealed significantly changed expression levels of several PARPs in tuberculosis patients compared to healthy individuals. Moreover, the expression levels of these PARPs returned to normal following tuberculosis treatment. Then, the changes in the expression levels of PARPs during infection were validated in Tohoku Hospital Pediatrics-1 (THP1)-induced differentiated macrophages infected with model strains bacillus Calmette-Guérin (BCG) and in human lung adenocarcinoma A549 cells infected with (Ms), respectively. The mRNA levels of PARP9, PARP10, PARP12, and PARP14 were most significantly increased during infection, with corresponding increases in protein levels, indicating the possible biological functions of these PARPs during infection. In addition, the biological function of host PARP9 in infection was further studied. PARP9 deficiency significantly increased the infection efficiency and intracellular proliferation ability of Ms, which was reversed by the reconstruction of PARP9. Collectively, this study updates the understanding of changes in PARP expression during infection and provides evidence supporting PARP9 as a potent suppressor for infection.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s43657-023-00112-2.
PubMed: 38884060
DOI: 10.1007/s43657-023-00112-2