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Infection, Genetics and Evolution :... Nov 2023Spoligotyping is one of the molecular typing methods widely used for exploring the genetic variety of Mycobacterium tuberculosis. The aim of this study was to compare...
Spoligotyping is one of the molecular typing methods widely used for exploring the genetic variety of Mycobacterium tuberculosis. The aim of this study was to compare the spoligoprofiles of M. tuberculosis clinical isolates, obtained using in vitro and in silico approaches. The study included 230 M. tuberculosis isolates, recovered from Poland and Lithuania between 2018 and 2021. Spoligotyping in vitro was performed with a commercially available kit. Whole genome sequencing (WGS) was done with Illumina NovaSeq 6000 sequencer. Spoligotype International Types (SITs) were assigned according to the SITVIT2 database or using three different in silico tools, and based on WGS data, namely SpoTyping, SpolPred, and lorikeet. Upon in vitro spoligotyping, the isolates produced 65 different spoligotypes. Spoligotypes inferred from the WGS data were congruent with in vitro generated patterns in 81.7% (188/230) for lorikeet and 81.3% (187/230) for SpolPred and SpoTyping. Spacers 18 and 31 produced the highest ratio of discrepant results between in vitro and in silico approaches, with their signals discordantly assigned for 15 (6.5%) and 9 (3.9%) isolates, respectively. All three in silico approaches used were similarly efficient for M. tuberculosis spoligotype prediction. However, only SpoTyping could predict spoligotypes without a need for manual curation. Thus, we consider it as the most accurate tool. Its use is further advocated by the shortest time of analysis. A relatively high (ca. 20%) discordance between in vitro and in silico spoligotyping results was observed. While we discourage comparing conventional spoligotyping with in silico equivalents, we advise the use of the latter, as it improves the accuracy of spoligopatterns, and thus depicts the relatedness between the isolates more reliably.
Topics: Humans; Mycobacterium tuberculosis; Bacterial Typing Techniques; Molecular Typing; Whole Genome Sequencing; Tuberculosis; Genotype
PubMed: 37757901
DOI: 10.1016/j.meegid.2023.105508 -
International Journal of Biological... Jul 2023Advancement in the area of anti-tubercular drug development has been full-fledged, yet, a very less number of drug molecules have reached phase II clinical trials, and... (Review)
Review
Advancement in the area of anti-tubercular drug development has been full-fledged, yet, a very less number of drug molecules have reached phase II clinical trials, and therefore "End-TB" is still a global challenge. Inhibitors to specific metabolic pathways of Mycobacterium tuberculosis (Mtb) gain importance in strategizing anti-tuberculosis drug discovery. The lead compounds that target DNA replication, protein synthesis, cell wall biosynthesis, bacterial virulence and energy metabolism are emerging as potential chemotherapeutic options against Mtb growth and survival within the host. In recent times, the in silico approaches have become most promising tools in the identification of suitable inhibitors for specific protein targets of Mtb. An update in the fundamental understanding of these inhibitors and the mechanism of interaction may bring hope to future perspectives in novel drug development and delivery approaches. This review provides a collective impression of the small molecules with potential antimycobacterial activities and their target pathways in Mtb such as cell wall biosynthesis, DNA replication, transcription and translation, efflux pumps, antivirulence pathways and general metabolism. The mechanism of interaction of specific inhibitor with their respective protein targets has been discussed. The comprehensive knowledge of such an impactful area of research would essentially reflect in the discovery of novel drug molecules and effective delivery approaches. This narrative review encompasses the knowledge of emerging targets and promising chemical inhibitors that could potentially translate in to the anti-TB-drug discovery.
Topics: Mycobacterium tuberculosis; Antitubercular Agents; Drug Discovery; DNA Replication
PubMed: 37244342
DOI: 10.1016/j.ijbiomac.2023.125022 -
Nature Mar 2024Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs...
Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis (TB), an infectious disease that is responsible for major health and economic costs worldwide. Mtb encounters diverse environments during its life cycle and responds to these changes largely by reprogramming its transcriptional output. However, the mechanisms of Mtb transcription and how they are regulated remain poorly understood. Here we use a sequencing method that simultaneously determines both termini of individual RNA molecules in bacterial cells to profile the Mtb transcriptome at high resolution. Unexpectedly, we find that most Mtb transcripts are incomplete, with their 5' ends aligned at transcription start sites and 3' ends located 200-500 nucleotides downstream. We show that these short RNAs are mainly associated with paused RNA polymerases (RNAPs) rather than being products of premature termination. We further show that the high propensity of Mtb RNAP to pause early in transcription relies on the binding of the σ-factor. Finally, we show that a translating ribosome promotes transcription elongation, revealing a potential role for transcription-translation coupling in controlling Mtb gene expression. In sum, our findings depict a mycobacterial transcriptome that prominently features incomplete transcripts resulting from RNAP pausing. We propose that the pausing phase constitutes an important transcriptional checkpoint in Mtb that allows the bacterium to adapt to environmental changes and could be exploited for TB therapeutics.
Topics: DNA-Directed RNA Polymerases; Mycobacterium tuberculosis; RNA, Bacterial; Transcriptome; Tuberculosis; RNA, Messenger; Gene Expression Regulation, Bacterial; Transcription Initiation Site; Sigma Factor; Ribosomes; Protein Biosynthesis
PubMed: 38418874
DOI: 10.1038/s41586-024-07105-9 -
induces host autophagic ferritin degradation for enhanced iron bioavailability and bacterial growth.Autophagy Apr 2024Mtb, ; TB, tuberculosis; NCOA4, nuclear receptor coactivator 4; p38, p38 protein kinase; AKT1, AKT serine/threonine kinase 1; TRIM21, tripartite motif containing 21;...
Mtb, ; TB, tuberculosis; NCOA4, nuclear receptor coactivator 4; p38, p38 protein kinase; AKT1, AKT serine/threonine kinase 1; TRIM21, tripartite motif containing 21; FTH1, ferritin heavy chain 1; FTL, ferritin light chain; HERC2, HECT and RLD domain containing E3 ubiquitin protein ligase 2.
Topics: Mycobacterium tuberculosis; Ferritins; Autophagy; Humans; Iron; Biological Availability; Tuberculosis; Animals
PubMed: 37198940
DOI: 10.1080/15548627.2023.2213983 -
ELife Aug 2023A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in during anti-tuberculosis (TB) chemotherapy. However, the...
A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.
Topics: Bacterial Proteins; Mycobacterium tuberculosis; Mutagenesis; DNA Repair; Antitubercular Agents
PubMed: 37530405
DOI: 10.7554/eLife.75628 -
Infection and Immunity Oct 2023Hematopoietic stem and progenitor cells (HSPCs) play a vital role in the host response to infection through the rapid and robust production of mature immune cells. These...
Hematopoietic stem and progenitor cells (HSPCs) play a vital role in the host response to infection through the rapid and robust production of mature immune cells. These HSPC responses can be influenced, directly and indirectly, by pathogens as well. Infection with () can drive lymphopoiesis through modulation of type I interferon (IFN) signaling. We have previously found that the presence of a drug resistance (DR)-conferring mutation in drives altered host-pathogen interactions and heightened type I IFN production . But the impacts of this DR mutation on host responses to infection, particularly the hematopoietic compartment, remain unexplored. Using a mouse model, we show that, while drug-sensitive infection induces expansion of HSPC subsets and a skew toward lymphopoiesis, DR infection fails to induce an expansion of these subsets and an accumulation of mature granulocytes in the bone marrow. Using single-cell RNA sequencing, we show that the HSCs from DR -infected mice fail to upregulate pathways related to cytokine signaling across all profiled HSC subsets. Collectively, our studies report a novel finding of a chronic infection that fails to induce a potent hematopoietic response that can be further investigated to understand pathogen-host interaction at the level of hematopoiesis.
Topics: Humans; Bone Marrow; Tuberculosis; Hematopoietic Stem Cells; Mycobacterium tuberculosis; Hematopoiesis; Bacterial Infections; Bone Marrow Cells
PubMed: 37754680
DOI: 10.1128/iai.00201-23 -
The Journal of Cell Biology Dec 2023Peroxisomes are organelles involved in many metabolic processes including lipid metabolism, reactive oxygen species (ROS) turnover, and antimicrobial immune responses....
Peroxisomes are organelles involved in many metabolic processes including lipid metabolism, reactive oxygen species (ROS) turnover, and antimicrobial immune responses. However, the cellular mechanisms by which peroxisomes contribute to bacterial elimination in macrophages remain elusive. Here, we investigated peroxisome function in iPSC-derived human macrophages (iPSDM) during infection with Mycobacterium tuberculosis (Mtb). We discovered that Mtb-triggered peroxisome biogenesis requires the ESX-1 type 7 secretion system, critical for cytosolic access. iPSDM lacking peroxisomes were permissive to Mtb wild-type (WT) replication but were able to restrict an Mtb mutant missing functional ESX-1, suggesting a role for peroxisomes in the control of cytosolic but not phagosomal Mtb. Using genetically encoded localization-dependent ROS probes, we found peroxisomes increased ROS levels during Mtb WT infection. Thus, human macrophages respond to the infection by increasing peroxisomes that generate ROS primarily to restrict cytosolic Mtb. Our data uncover a peroxisome-controlled, ROS-mediated mechanism that contributes to the restriction of cytosolic bacteria.
Topics: Humans; Cytosol; Macrophages; Mycobacterium tuberculosis; Peroxisomes; Reactive Oxygen Species; Type VII Secretion Systems
PubMed: 37737955
DOI: 10.1083/jcb.202303066 -
Proceedings of the National Academy of... Jul 2023Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process...
Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC ( complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC ( < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.
Topics: Mycobacterium tuberculosis; Phase Variation; Genomics; Adaptation, Physiological; Virulence; Phylogeny; Genome, Bacterial
PubMed: 37399390
DOI: 10.1073/pnas.2301394120 -
International Journal of... 2023
Topics: Humans; Mycobacterium tuberculosis; Microscopy; Tuberculosis, Multidrug-Resistant; Antitubercular Agents; Microbial Sensitivity Tests; Drug Resistance, Multiple, Bacterial
PubMed: 38149529
DOI: 10.4103/ijmy.ijmy_196_23 -
JCI Insight Oct 2023Host cytosolic sensing of Mycobacterium tuberculosis (M. tuberculosis) RNA by the RIG-I-like receptor (RLR) family perturbs innate immune control within macrophages;...
Host cytosolic sensing of Mycobacterium tuberculosis (M. tuberculosis) RNA by the RIG-I-like receptor (RLR) family perturbs innate immune control within macrophages; however, a distinct role of MDA5, a member of the RLR family, in M. tuberculosis pathogenesis has yet to be fully elucidated. To further define the role of MDA5 in M. tuberculosis pathogenesis, we evaluated M. tuberculosis intracellular growth and innate immune responses in WT and Mda5-/- macrophages. Transfection of M. tuberculosis RNA strongly induced proinflammatory cytokine production in WT macrophages, which was abrogated in Mda5-/- macrophages. M. tuberculosis infection in macrophages induced MDA5 protein expression, accompanied by an increase in MDA5 activation as assessed by multimer formation. IFN-γ-primed Mda5-/- macrophages effectively contained intracellular M. tuberculosis proliferation to a markedly greater degree than WT macrophages. Further comparisons of WT versus Mda5-/- macrophages revealed that during M. tuberculosis infection MDA5 contributed to IL-1β production and inflammasome activation and that loss of MDA5 led to a substantial increase in autophagy. In the mouse TB model, loss of MDA5 conferred host survival benefits with a concomitant reduction in M. tuberculosis bacillary burden. These data reveal that loss of MDA5 is host protective during M. tuberculosis infection in vitro and in vivo, suggesting that M. tuberculosis exploits MDA5 to subvert immune containment.
Topics: Animals; Mice; Immunity, Innate; Macrophages; Mycobacterium tuberculosis; RNA; Tuberculosis
PubMed: 37725440
DOI: 10.1172/jci.insight.166242