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Nature Communications Oct 2023Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and...
Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein LprG are involved in transport of triacylglycerides (TAGs) that seal the mycomembrane. Here, we report a 2.7 Å structure of a mycobacterial Rv1410 homologue, which adopts an outward-facing conformation and exhibits unusual transmembrane helix 11 and 12 extensions that protrude ~20 Å into the periplasm. A small, very hydrophobic cavity suitable for lipid transport is constricted by a functionally important ion-lock likely involved in proton coupling. Combining mutational analyses and MD simulations, we propose that TAGs are extracted from the core of the inner membrane into the central cavity via lateral clefts present in the inward-facing conformation. The functional role of the periplasmic helix extensions is to channel the extracted TAG into the lipid binding pocket of LprG.
Topics: Membrane Transport Proteins; Mycobacterium tuberculosis; Biological Transport; Membranes; Lipids; Protein Conformation
PubMed: 37833269
DOI: 10.1038/s41467-023-42073-0 -
International Journal of Infectious... Feb 2020High accuracy diagnostic screening tests for tuberculosis (TB) are required to improve the diagnosis of both active TB and latent Mycobacterium tuberculosis (MTB)...
OBJECTIVES
High accuracy diagnostic screening tests for tuberculosis (TB) are required to improve the diagnosis of both active TB and latent Mycobacterium tuberculosis (MTB) infection (LTBI). The novel IGRA LIOFeron®TB/LTBI assay was tested and its accuracy was compared to the QuantiFERON®-TB Gold Plus assay.
METHODS
A total of 389 subjects were enrolled in two cohorts and classified as healthy, active TB or LTBI persons. The blood of all the patients was tested with LIOFeron®TB/LTBI assay, containing MTB alanine dehydrogenase, able to differentiate active TB from LTBI diagnosis. The results obtained with both IGRAs, performed on the same 250 samples, were finally compared.
RESULTS
The two assays demonstrated an excellent concordance of their results with patients' diagnosis of MTB infection. ROC analysis for QuantiFERON®-TB Gold Plus showed sensitivity and specificity respectively of 98% and 97% in diagnosing active TB patients and 85% and 94% in diagnosing LTBI subjects. LIOFeron®TB/LTBI assay showed sensitivity and specificity respectively of 90% and 98% in diagnosing active TB patients and 94% and 97% in diagnosing LTBI subjects.
CONCLUSIONS
The two IGRAs displayed the same high accuracy in diagnosing MTB infection/TB disease, and LIOFeron®TB/LTBI assay demonstrated higher sensitivity than QuantiFERON®-TB Gold Plus test in LTBI detection.
Topics: Adult; Aged; Cohort Studies; Diagnostic Tests, Routine; Female; Humans; Latent Tuberculosis; Male; Middle Aged; Mycobacterium tuberculosis; ROC Curve; Sensitivity and Specificity; T-Lymphocytes
PubMed: 31877486
DOI: 10.1016/j.ijid.2019.12.012 -
Proceedings of the National Academy of... Apr 2022Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been...
Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug–drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemical–genetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo–relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemical–genetic–environmental interactions that can be used to optimize drug–drug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.
Topics: Antitubercular Agents; Carbon; Cell Wall; Drug Interactions; Gene-Environment Interaction; Humans; Mycobacterium tuberculosis
PubMed: 35380903
DOI: 10.1073/pnas.2201632119 -
Electrophoresis Oct 2019Tuberculosis is highly persistent and displays phenotypic resistance to high concentrations of antimicrobials. Recent reports exhibited that Mycobacterium tuberculosis... (Comparative Study)
Comparative Study
Tuberculosis is highly persistent and displays phenotypic resistance to high concentrations of antimicrobials. Recent reports exhibited that Mycobacterium tuberculosis biofilm was implicated to its pathogenicity and drug resistance. In this study, there were 47 kinds of differential proteins in the biofilm of M. tuberculosis H37Rv cells compared with the planktonic bacteria, and 37 proteins were nonredundant and identified by proteomics approach, such as 2DE and LC-MS/MS. Moreover, six kinds of proteins were identified as HspX, which were conservative and highly expressed in biofilm. Note that 47 differential proteins were divided into seven categories, such as cell wall and cell processes, conserved hypotheticals, intermediary metabolism and respiration, and so on by TUBERCULIST. The Gene Ontology classification results showed that the largest protein group involved in metabolism, binding proteins, and catalytic function accounts for 30% and 57% of all identified proteins, respectively. Moreover, the protein interaction network analyzed by STRING showed that the minority proteins such as RpoA, SucC, Cbs, Tuf, DnaK, and GroeL in the interaction network have high network connectivity. These results implied that the proteins involved in metabolic process and catalytic function and the minority proteins mentioned above may play an important role in M. tuberculosis biofilm formation. To our knowledge, this is the first report about differential proteins between biofilm and planktonic M. tuberculosis, which provided the potential antigens for vaccines and target proteins for anti-mycobacterial drugs.
Topics: Bacterial Proteins; Biofilms; Mycobacterium tuberculosis; Protein Interaction Maps; Proteome; Proteomics
PubMed: 31141184
DOI: 10.1002/elps.201900030 -
Scientific Reports May 2024Ethiopia is one of the countries with a high tuberculosis (TB) burden, yet little is known about the spatial distribution of Mycobacterium tuberculosis (Mtb) lineages....
Ethiopia is one of the countries with a high tuberculosis (TB) burden, yet little is known about the spatial distribution of Mycobacterium tuberculosis (Mtb) lineages. This study identifies the spoligotyping of 1735 archived Mtb isolates from the National Drug Resistance Survey, collected between November 2011 and June 2013, to investigate Mtb population structure and spatial distribution. Spoligotype International Types (SITs) and lineages were retrieved from online databases. The distribution of lineages was evaluated using Fisher's exact test and logistic regression models. The Global Moran's Index and Getis-Ord Gi statistic were utilized to identify hotspot areas. Our results showed that spoligotypes could be interpreted and led to 4 lineages and 283 spoligotype patterns in 91% of the isolates, including 4% of those with multidrug/rifampicin resistance (MDR/RR) TB. The identified Mtb lineages were lineage 1 (1.8%), lineage 3 (25.9%), lineage 4 (70.6%) and lineage 7 (1.6%). The proportion of lineages 3 and 4 varied by regions, with lineage 3 being significantly greater than lineage 4 in reports from Gambella (AOR = 4.37, P < 0.001) and Tigray (AOR = 3.44, P = 0.001) and lineage 4 being significantly higher in Southern Nations Nationalities and Peoples Region (AOR = 1.97, P = 0.026) than lineage 3. Hotspots for lineage 1 were located in eastern Ethiopia, while a lineage 7 hotspot was identified in northern and western Ethiopia. The five prevalent spoligotypes, which were SIT149, SIT53, SIT25, SIT37 and SIT26 account for 42.8% of all isolates under investigation, while SIT149, SIT53 and SIT21 account for 52-57.8% of drug-resistant TB cases. TB and drug resistant TB are mainly caused by lineages 3 and 4, and significant proportions of the prevalent spoligotypes also influence drug-resistant TB and the total TB burden. Regional variations in lineages may result from both local and cross-border spread.
Topics: Ethiopia; Mycobacterium tuberculosis; Humans; Female; Male; Adult; Middle Aged; Adolescent; Young Adult; Tuberculosis, Multidrug-Resistant; Tuberculosis; Bacterial Typing Techniques
PubMed: 38714745
DOI: 10.1038/s41598-024-59435-3 -
Tuberculosis (Edinburgh, Scotland) Sep 2020Ocular tuberculosis (OTB) encompasses all forms of intra- and extra-ocular inflammation associated with Mycobacterium tuberculosis (Mtb) infection. However, the organism... (Review)
Review
Ocular tuberculosis (OTB) encompasses all forms of intra- and extra-ocular inflammation associated with Mycobacterium tuberculosis (Mtb) infection. However, the organism is rarely found in ocular fluid samples of diseased eyes, rendering the pathomechanisms of the disease unclear. This confounds clinical decision-making in diagnosis and treatment of OTB. Here, we critically review existing human and animal data related to ocular inflammation and TB pathogenesis to unravel likely pathomechanisms of OTB. Broadly there appear to be two fundamental mechanisms that may underlie the development of TB-associated ocular inflammation: a. inflammatory response to live/replicating Mtb in the eye, and b. immune mediated ocular inflammation induced by non-viable Mtb or its components in the eye. This distinction is significant as in direct Mtb-driven mechanisms, diagnosis and treatment would be aimed at detection of Mtb-infection and its elimination; while indirect mechanisms would primarily require anti-inflammatory therapy with adjunctive anti-TB therapy. Further, we discuss how that most clinical phenotypes of OTB likely represent a combination of both mechanisms, with one being predominant than the other.
Topics: Animals; Anti-Inflammatory Agents; Antitubercular Agents; Autoimmunity; Eye; Host-Pathogen Interactions; Humans; Mycobacterium tuberculosis; Tuberculosis, Ocular; Uveitis
PubMed: 33010848
DOI: 10.1016/j.tube.2020.101961 -
Methods in Molecular Biology (Clifton,... 2021The utility of fluorescent proteins in bacterial research has long been appreciated, with extensive use in the Mycobacterium tuberculosis field. In more recent years, a...
The utility of fluorescent proteins in bacterial research has long been appreciated, with extensive use in the Mycobacterium tuberculosis field. In more recent years, a new generation of fluorescent tools has been developed for use in M. tuberculosis research. These new fluorescent reporters exploit the immense genetic and transcriptional knowledge now available, and enable the use of the bacteria as direct reporters of the local environment during infection, as well as provide insight into bacterial replication status in situ. Here we describe methods for the construction of such fluorescent reporter M. tuberculosis strains, and their use in combination with confocal microscopy and flow cytometry approaches for single bacterium-level analyses of M. tuberculosis physiology and M. tuberculosis-host interactions.
Topics: Animals; Flow Cytometry; Host-Pathogen Interactions; Luminescent Proteins; Lung; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Mycobacterium tuberculosis; Tuberculosis, Pulmonary
PubMed: 34235663
DOI: 10.1007/978-1-0716-1460-0_17 -
Epidemiology and Infection Jan 2022Whole-genome sequencing (WGS) has shown tremendous potential in rapid diagnosis of drug-resistant tuberculosis (TB). In the current study, we performed WGS on...
Whole-genome sequencing (WGS) has shown tremendous potential in rapid diagnosis of drug-resistant tuberculosis (TB). In the current study, we performed WGS on drug-resistant Mycobacterium tuberculosis isolates obtained from Shanghai (n = 137) and Russia (n = 78). We aimed to characterise the underlying and high-frequency novel drug-resistance-conferring mutations, and also create valuable combinations of resistance mutations with high predictive sensitivity to predict multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB) phenotype using a bootstrap method. Most strains belonged to L2.2, L4.2, L4.4, L4.5 and L4.8 lineages. We found that WGS could predict 82.07% of phenotypically drug-resistant domestic strains. The prediction sensitivity for rifampicin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (STR), ofloxacin (OFL), amikacin (AMK) and capreomycin (CAP) was 79.71%, 86.30%, 76.47%, 88.37%, 83.33%, 70.00% and 70.00%, respectively. The mutation combination with the highest sensitivity for MDR prediction was rpoB S450L + rpoB H445A/P + katG S315T + inhA I21T + inhA S94A, with a sensitivity of 92.17% (0.8615, 0.9646), and the mutation combination with highest sensitivity for XDR prediction was rpoB S450L + katG S315T + gyrA D94G + rrs A1401G, with a sensitivity of 92.86% (0.8158, 0.9796). The molecular information presented here will be of particular value for the rapid clinical detection of MDR- and XDR-TB isolates through laboratory diagnosis.
Topics: Antitubercular Agents; Bacterial Proteins; China; Drug Resistance, Multiple, Bacterial; Genome, Bacterial; Humans; Microbial Sensitivity Tests; Mutation; Mycobacterium tuberculosis; Phylogeny; Russia; Sensitivity and Specificity; Tuberculosis, Multidrug-Resistant; Whole Genome Sequencing
PubMed: 35086603
DOI: 10.1017/S095026882100279X -
ACS Infectious Diseases Aug 2019MTBVAC is a live attenuated vaccine constructed by genetic deletions in the and virulence genes. The MTBVAC vaccine is currently in phase 2 clinical trials with... (Comparative Study)
Comparative Study
MTBVAC is a live attenuated vaccine constructed by genetic deletions in the and virulence genes. The MTBVAC vaccine is currently in phase 2 clinical trials with newborns and adults in South Africa, one of the countries with the highest incidence. Although MTBVAC has been extensively characterized by genomics, transcriptomics, lipidomics, and proteomics, its metabolomic profile is yet unknown. Accordingly, in this study we aim to identify differential metabolites between and MTBVAC. To this end, an untargeted metabolomics approach based on liquid chromatography coupled to high-resolution mass spectrometry was implemented in order to explore the main metabolic differences between and MTBVAC. As an outcome, we identified a set of 34 metabolites involved in diverse bacterial biosynthetic pathways. A consistent increase in the phosphatidylinositol species was observed in the vaccine candidate relative to its parental strain. This phenotype resulted in an increased production of phosphatidylinositol mannosides, a novel PhoP-regulated phenotype in the most widespread lineages of . This study represents a step ahead in our understanding of the MTBVAC vaccine, and some of the differential metabolites identified in this work might be used as potential vaccination biomarkers.
Topics: Bacterial Proteins; Biosynthetic Pathways; Chromatography, Liquid; Mass Spectrometry; Metabolomics; Mycobacterium tuberculosis; Phosphatidylinositols; Tuberculosis Vaccines
PubMed: 31099236
DOI: 10.1021/acsinfecdis.9b00008 -
Infection, Genetics and Evolution :... Aug 2019Tuberculosis remains a devastating disease to Mankind, ranking as the ninth cause of death worldwide. Eliminating tuberculosis as proven much more difficult than once... (Review)
Review
Tuberculosis remains a devastating disease to Mankind, ranking as the ninth cause of death worldwide. Eliminating tuberculosis as proven much more difficult than once anticipated. In addition to the delay in diagnosis and drug resistance problems that compromise the efficacy of treatment, the enormous reservoir of latently infected individuals continuously feeds the epidemics. However, targeting latency with prophylactic antibiotic administration is not possible at the populational level. Together, these issues call for a better understanding of latency, as well as for a more precise identification of individuals at high risk of reactivation. For this, recent paradigm changing evidence need to be taken into account, most notably, the existence of a tuberculosis spectrum; the genetic diversity of both humans and tuberculosis-causing bacteria; and the changes in the human population that interfere with tuberculosis. Here we discuss latency in the light of these variables and how that understanding can move forward tuberculosis research and elimination.
Topics: Animals; Biomarkers; Humans; Latent Tuberculosis; Lung; MicroRNAs; Mycobacterium tuberculosis; Positron Emission Tomography Computed Tomography; Post-Exposure Prophylaxis; Receptors, IgG; T-Lymphocyte Subsets; Transcriptome; Tuberculoma
PubMed: 30576838
DOI: 10.1016/j.meegid.2018.12.019