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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 -
Microbiology Resource Announcements Jun 2024, a member of the family and species , is an F1 cluster phage that infects mc²155. The Maravista genome has 61.3% GC content, is 60,140 bp in length, and encodes 104...
, a member of the family and species , is an F1 cluster phage that infects mc²155. The Maravista genome has 61.3% GC content, is 60,140 bp in length, and encodes 104 putative genes. Maravista encodes two putative glycosyltransferases, suggesting glycosylation of its capsid protein.
PubMed: 38860805
DOI: 10.1128/mra.00502-24 -
Microbiology Spectrum Jun 2024Iron scavenging is required for full virulence of mycobacterial pathogens. During infection, the host immune response restricts mycobacterial access to iron, which is...
Iron scavenging is required for full virulence of mycobacterial pathogens. During infection, the host immune response restricts mycobacterial access to iron, which is essential for bacterial respiration and DNA synthesis. The iron-dependent regulator (IdeR) responds to changes in iron accessibility by repressing iron-uptake genes when iron is available. In contrast, iron-uptake gene transcription is induced when iron is depleted. The gene is essential in and is required for bacterial growth. To further study how iron regulates transcription, wee developed an iron responsive reporter system that relies on an IdeR-regulated promoter to drive Cre and mediated recombination in . Recombination leads to the expression of an antibiotic resistance gene so that mutations that activate the IdeR-regulated promoter can be selected. A transposon library in the background of this reporter system was exposed to media containing iron and hemin, and this resulted in the selection of mutants in the antioxidant mycothiol synthesis pathway. We validated that inactivation of the mycothiol synthesis gene results in increased recombination and increased IdeR-regulated promoter activity in the reporter system. Further, we show that vitamin C, which has been shown to oxidize iron through the Fenton reaction, can decrease promoter activity in the mutant. We conclude that the intracellular redox state balanced by mycothiol can alter IdeR activity in the presence of iron.IMPORTANCE is a tractable organism to study mycobacterial gene regulation. We used to construct a novel recombination-based reporter system that allows for the selection of mutations that deregulate a promoter of interest. Transposon mutagenesis and insertion sequencing (TnSeq) in the recombination reporter strain identified genes that impact iron regulated promoter activity in mycobacteria. We found that the mycothiol synthesis gene is required for IdeR mediated transcriptional regulation by maintaining intracellular redox balance. By affecting the oxidative state of the intracellular environment, mycothiol can modulate iron-dependent transcriptional activity. Taken more broadly, this novel reporter system can be used in combination with transposon mutagenesis to identify genes that are required by to overcome temporary or local changes in iron availability during infection.
PubMed: 38860795
DOI: 10.1128/spectrum.00487-24 -
Diagnostic Microbiology and Infectious... May 2024We present a patient who suffered an agricultural rollover trauma and developed a fracture-associated tissue infection caused by Mycobacterium smegmatis. Since cases are...
We present a patient who suffered an agricultural rollover trauma and developed a fracture-associated tissue infection caused by Mycobacterium smegmatis. Since cases are rare, treatment of infections with M. smegmatis requires an interprofessional approach and the combination of surgery and adjunctive antimicrobial treatment.
PubMed: 38850688
DOI: 10.1016/j.diagmicrobio.2024.116379 -
MLife Mar 2024Insertion sequences (ISs) exist widely in bacterial genomes, but their roles in the evolution of bacterial antiphage defense remain to be clarified. Here, we report...
Insertion sequences (ISs) exist widely in bacterial genomes, but their roles in the evolution of bacterial antiphage defense remain to be clarified. Here, we report that, under the pressure of phage infection, the IS transposition of into the gene can occur at high frequencies, which endows the mutant mycobacterium with a broad-spectrum antiphage ability. Lsr2 functions as a negative regulator and directly silences expression of a gene island composed of 11 lipid metabolism-related genes. The complete or partial loss of the gene island leads to a significant decrease of bacteriophage adsorption to the mycobacterium, thus defending against phage infection. Strikingly, a phage that has evolved mutations in two tail-filament genes can re-escape from the inactivation-triggered host defense. This study uncovered a new signaling pathway for activating antimycobacteriophage immunity by IS transposition and provided insight into the natural evolution of bacterial antiphage defense.
PubMed: 38827510
DOI: 10.1002/mlf2.12106 -
Journal of Biomedical Research May 2024Tumor vaccines are a promising avenue in cancer immunotherapy. Despite the progress in targeting specific immune epitopes, tumor cells lacking them can evade treatment....
Tumor vaccines are a promising avenue in cancer immunotherapy. Despite the progress in targeting specific immune epitopes, tumor cells lacking them can evade treatment. Here, we aimed to construct an efficient tumor vaccine Vac-SM, utilizing shikonin (SKN) to induce immunogenic cell death (ICD) and ( ) as an immune adjuvant to enhance tumor vaccine efficacy. SKN demonstrated a dose-dependent and time-dependent cytotoxic effect on the tumor cell line as seen using the CCK-8 assay and induced ICD in tumor cells by detecting the expression of relevant indicators respectively. Compared to that in the control groups, Vac-SM injection in mouse subcutaneous metastatic tumors significantly inhibited tumor growth and distant tumor growth and improved survival rates. effectively induced bone marrow-derived dendritic cells (DC) maturation and activation and tumor-draining lymph nodes showed increased maturation of DC and a higher proportion of effector memory T-cell subsets with Vac-SM treatment, based on flow cytometry analysis results.Collectively, Vac-SM vaccine effectively induces ICD, improves antigen presentation by DC, activates a specific systemic antitumor T-cell immune response, exhibits favorable safety profile, and holds promise for clinical translation for local tumor immunotherapy.
PubMed: 38807377
DOI: 10.7555/JBR.38.20240049 -
Frontiers in Microbiology 2024() genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis...
() genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using comparative genome analysis, we identified one of the genes, Rv1509, as a signature protein exclusively present in . To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in () constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, and studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in pathogenesis.
PubMed: 38803374
DOI: 10.3389/fmicb.2024.1344857