Publications by authors named "Gurdyal S Besra"

380 Publications

CD1a selectively captures endogenous cellular lipids that broadly block T cell response.

J Exp Med 2021 Jul 7;218(7). Epub 2021 May 7.

Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.

We optimized lipidomics methods to broadly detect endogenous lipids bound to cellular CD1a proteins. Whereas membrane phospholipids dominate in cells, CD1a preferentially captured sphingolipids, especially a C42, doubly unsaturated sphingomyelin (42:2 SM). The natural 42:2 SM but not the more common 34:1 SM blocked CD1a tetramer binding to T cells in all human subjects tested. Thus, cellular CD1a selectively captures a particular endogenous lipid that broadly blocks its binding to TCRs. Crystal structures show that the short cellular SMs stabilized a triad of surface residues to remain flush with CD1a, but the longer lipids forced the phosphocholine group to ride above the display platform to hinder TCR approach. Whereas nearly all models emphasize antigen-mediated T cell activation, we propose that the CD1a system has intrinsic autoreactivity and is negatively regulated by natural endogenous inhibitors selectively bound in its cleft. Further, the detailed chemical structures of natural blockers could guide future design of therapeutic blockers of CD1a response.
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http://dx.doi.org/10.1084/jem.20202699DOI Listing
July 2021

Discovery of Novel Thiophene-arylamide Derivatives as DprE1 Inhibitors with Potent Antimycobacterial Activities.

J Med Chem 2021 Apr 14. Epub 2021 Apr 14.

Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation & Chinese Academy of Medical Sciences Key Laboratory of Anti-DR TB Innovative Drug Research, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, P. R. China.

In this study, we report the design and synthesis of a series of novel thiophene-arylamide compounds derived from the noncovalent decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1) inhibitor TCA1 through a structure-based scaffold hopping strategy. Systematic optimization of the two side chains flanking the thiophene core led to new lead compounds bearing a thiophene-arylamide scaffold with potent antimycobacterial activity and low cytotoxicity. Compounds , , , and exhibited potent activity against both drug-susceptible (minimum inhibitory concentration (MIC) = 0.02-0.12 μg/mL) and drug-resistant (MIC = 0.031-0.24 μg/mL) tuberculosis strains while retaining potent DprE1 inhibition (half maximal inhibitory concentration (IC) = 0.2-0.9 μg/mL) and good intracellular antimycobacterial activity. In addition, these compounds showed good hepatocyte stability and low inhibition of the human ether-à-go-go related gene (hERG) channel. The representative compound with acceptable pharmacokinetic property demonstrated significant bactericidal activity in an acute mouse model of tuberculosis. Moreover, the molecular docking study of template compound provides new insight into the discovery of novel antitubercular agents targeting DprE1.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00263DOI Listing
April 2021

Generation of Liposomes to Study the Effect of Lipids on HIV-1 - and -Infections.

Int J Mol Sci 2021 Feb 16;22(4). Epub 2021 Feb 16.

Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 7BE, UK.

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and () co-infection is an early precipitate to AIDS. We aimed to determine whether strains differentially modulate cellular susceptibility to HIV-1 infection (- and -infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 H37Rv, CDC1551 and lineage 2 HN878, EU127) and non-pathogenic ( BCG and ) strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 - and -infection of TZM-bl cells using single-cycle infectious virus particles. glycolipids did not affect HIV-1 direct infection however, -infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from , H37Rv and EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 -infection. These findings indicate that variant strains of have differential effect on HIV-1 -infection with the potential to influence HIV-1 disease course in co-infected individuals.
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http://dx.doi.org/10.3390/ijms22041945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920488PMC
February 2021

Synthesis and recycling of the mycobacterial cell envelope.

Curr Opin Microbiol 2021 Apr 18;60:58-65. Epub 2021 Feb 18.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. Electronic address:

Mycobacterium tuberculosis (Mtb), the causative agent of the disease tuberculosis, is a recognised global health concern. The efficacy of the current treatment regime is under threat due to the emergence of antibiotic resistance, directing an urgent requirement for the discovery of new anti-tubercular agents and drug targets. The mycobacterial cell wall is a well-validated drug target for Mtb and is composed of three adaptive macromolecular structures, peptidoglycan, arabinogalactan and mycolic acids, an array of complex lipids and carbohydrates. The majority of the enzymes involved in cell wall synthesis have been established, whilst studies directed towards the mechanisms of remodelling and recycling have been neglected. This review briefly describes mycobacterial cell wall synthesis, and focuses on aspects of remodelling and recycling, thus highlighting opportunities for future research.
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http://dx.doi.org/10.1016/j.mib.2021.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035080PMC
April 2021

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T Cell Engagers Generates Anti-Tumor Immunity While Avoiding Anergy.

Cancer Res 2021 Jan 22. Epub 2021 Jan 22.

Microbiology & Immunology, Albert Einstein College of Medicine

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong anti-tumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogs of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T cell responses. The anti-tumor effects of iNKT cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT anergy and exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2219DOI Listing
January 2021

Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in .

ACS Infect Dis 2021 01 15;7(1):141-152. Epub 2020 Dec 15.

Department of Microbiology and Immunology, Weill Cornell Medicine, New York, New York 10065, United States.

MmpL3, an essential mycolate transporter in the inner membrane of (), has been identified as a target of multiple, chemically diverse antitubercular drugs. However, several of these molecules seem to have secondary targets and inhibit bacterial growth by more than one mechanism. Here, we describe a cell-based assay that utilizes two-way regulation of MmpL3 expression to readily identify MmpL3-specific inhibitors. We successfully used this assay to identify a novel guanidine-based MmpL3 inhibitor from a library of 220 compounds that inhibit growth of by largely unknown mechanisms. We furthermore identified inhibitors of cytochrome - oxidase as one class of off-target hits in whole-cell screens for MmpL3 inhibitors and report a novel sulfanylacetamide as a potential QcrB inhibitor.
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http://dx.doi.org/10.1021/acsinfecdis.0c00675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802072PMC
January 2021

Chemical insights into the search for MAIT cells activators.

Mol Immunol 2021 01 6;129:114-120. Epub 2020 Dec 6.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom. Electronic address:

Mucosal-associated invariant T cells (MAIT cells) represent a potential therapeutic target as they can tune or enhance immune responses. They recognise and become activated by antigens, presented by the monomorphic MHC-I related molecule, MR1. To assess the significance of MAIT cells in human diseases, a better understanding of the MAIT cell-MR1-antigen interaction is imperative. Easy access to MR1 ligands and MAIT cells activators can help achieve this. In this review, we summarise current literature that has identified the natural ligands and drug-like molecules that activate MAIT cells and provide insight into their key molecular interactions with MR1 and MAIT T cell receptors (TCRs). We focus on the progress made in synthesizing and isolating 5-amino-6-d-ribitylaminouracil (5-A-RU), a key precursor in the synthesis of the known natural ligands, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil(5-OP-RU) and 5-(2-oxoethylideneamino)-6-d-ribitylaminouracil (5-OE-RU), and also on the stabilisation and optimisation of the latter compounds.
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http://dx.doi.org/10.1016/j.molimm.2020.11.017DOI Listing
January 2021

Tuberculosis diagnostics: overcoming ancient challenges with modern solutions.

Emerg Top Life Sci 2020 12;4(4):423-436

School of Chemical Engineering, Advanced Nanomaterials Structures and Applications Laboratories, College of Engineering and Physical Sciences, University of Birmingham, Birmingham B15 2TT, U.K.

Rapid, sensitive, accurate and portable diagnostics are a mainstay of modern medicine. Tuberculosis is a disease that has been with us since time immemorial and, despite the fact that it can be treated and cured, it still remains the world's biggest infectious killer, taking the lives of millions annually. There have been important developments in the diagnostic devices for tuberculosis however, these are often prone to error, expensive, lack the necessary sensitivity or accuracy and, crucially, not sufficiently portable and thus not applicable in the remote, rural areas, where they are most needed. Modern solutions have been emerging in the past decade, seeking to overcome many of the inhibiting issues in this field by utilising recent advances in molecular biology, genetics and sequencing or even completely 'reinventing the wheel', by developing novel and unprecedented diagnostic techniques. In this mini review, the issues and challenges arising from the historical methods of diagnosing tuberculosis are discussed, followed by outlaying their particular lack of appropriateness for regions of the world where tuberculosis still remains endemic. Subsequently, more recent developments of new methods and technological advancements as 'modern weapons' in the battle to defeat this disease and associated challenges are reviewed, and finally an outlook is presented, highlighting the future of the modern solutions under development, which are envisioned to lay the platform for improvements in delivering timely intervention, reduce immense expense and burden on healthcare systems worldwide, while saving millions of lives and eventually, may enable the eradication of this ancient disease.
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http://dx.doi.org/10.1042/ETLS20200335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733669PMC
December 2020

Development of a novel secondary phenotypic screen to identify hits within the mycobacterial protein synthesis pipeline.

FASEB Bioadv 2020 Oct 20;2(10):600-612. Epub 2020 Aug 20.

Institute of Microbiology and Infection School of Biosciences University of Birmingham Birmingham UK.

Background: Whole-cell phenotypic screening is the driving force behind modern anti-tubercular drug discovery efforts. Focus has shifted from screening for bactericidal scaffolds to screens incorporating target deconvolution. Target-based screening aims to direct drug discovery toward known effective targets and avoid investing resources into unproductive lines of enquiry. The protein synthesis pipeline, including RNA polymerase and the ribosome, is a clinically proven target in . Screening for new hits of this effective target pathway is an invaluable tool in the drug discovery arsenal.

Methods: Using . H37Rv augmented with anhydrotetracycline-inducible expression of mCherry, a phenotypic screen was developed for the identification of protein synthesis inhibitors in a medium throughput screening format.

Results: The assay was validated using known inhibitors of protein synthesis to show a dose-dependent reduction in mCherry fluorescence. This was expanded to a proprietary screen of hypothetical protein synthesis hits and modified to include quantitative viability measurement of cells using resazurin.

Conclusion: Following the success of the proprietary screen, a larger scale screen of the GlaxoSmithKline anti-tubercular library containing 2799 compounds was conducted. Combined single shot and dose-response screening yielded 18 hits, 0.64% of all screened compounds.
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http://dx.doi.org/10.1096/fba.2020-00022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566049PMC
October 2020

Antibiotics and resistance: the two-sided coin of the mycobacterial cell wall.

Cell Surf 2020 Dec 2;6:100044. Epub 2020 Sep 2.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

, the bacterium responsible for tuberculosis, is the global leading cause of mortality from an infectious agent. Part of this success relies on the unique cell wall, which consists of a thick waxy coat with tightly packed layers of complexed sugars, lipids and peptides. This coat provides a protective hydrophobic barrier to antibiotics and the host's defences, while enabling the bacterium to spread efficiently through sputum to infect and survive within the macrophages of new hosts. However, part of this success comes at a cost, with many of the current first- and second-line drugs targeting the enzymes involved in cell wall biosynthesis. The flip side of this coin is that resistance to these drugs develops either in the target enzymes or the activation pathways of the drugs, paving the way for new resistant clinical strains. This review provides a synopsis of the structure and synthesis of the cell wall and the major current drugs and targets, along with any mechanisms of resistance.
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http://dx.doi.org/10.1016/j.tcsw.2020.100044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502851PMC
December 2020

Evasion of MAIT cell recognition by the African Typhimurium ST313 pathovar that causes invasive disease.

Proc Natl Acad Sci U S A 2020 08 11;117(34):20717-20728. Epub 2020 Aug 11.

Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal strains activate MAIT cells. However, Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of Typhimurium ST313 lineage 2.
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http://dx.doi.org/10.1073/pnas.2007472117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456131PMC
August 2020

Anti-tubercular derivatives of rhein require activation by the monoglyceride lipase Rv0183.

Cell Surf 2020 Dec 21;6:100040. Epub 2020 Apr 21.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

The emergence and perseverance of drug resistant strains of () ensures that drug discovery efforts remain at the forefront of tuberculosis research. There are numerous different approaches that can be employed to lead to the discovery of anti-tubercular agents. In this work, we endeavored to optimize the anthraquinone chemical scaffold of a known drug, rhein, converting it from a compound with negligible activity against , to a series of compounds with potent activity. Two compounds exhibited low toxicity and good liver microsome stability and were further progressed in attempts to identify the biological target. Whole genome sequencing of resistant isolates revealed inactivating mutations in a monoglyceride lipase. Over-expression trials and an enzyme assay confirmed that the designed compounds are prodrugs, activated by the monoglyceride lipase. We propose that rhein is the active moiety of the novel compounds, which requires chemical modifications to enable access to the cell through the extensive cell wall structure. This work demonstrates that re-engineering of existing antimicrobial agents is a valid method in the development of new anti-tubercular compounds.
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http://dx.doi.org/10.1016/j.tcsw.2020.100040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389528PMC
December 2020

The thick waxy coat of mycobacteria, a protective layer against antibiotics and the host's immune system.

Biochem J 2020 05;477(10):1983-2006

School of Biosciences, Institute of Microbiology and Infection, University of Birmingham, Edgbaston, B15 2TT Birmingham, U.K.

Tuberculosis, caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease, with a mortality rate of over a million people per year. This pathogen's remarkable resilience and infectivity is largely due to its unique waxy cell envelope, 40% of which comprises complex lipids. Therefore, an understanding of the structure and function of the cell wall lipids is of huge indirect clinical significance. This review provides a synopsis of the cell envelope and the major lipids contained within, including structure, biosynthesis and roles in pathogenesis.
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http://dx.doi.org/10.1042/BCJ20200194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261415PMC
May 2020

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM.

Protein Cell 2020 07 3;11(7):505-517. Epub 2020 May 3.

State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and College of Pharmacy, Nankai University, Tianjin, 300353, China.

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.
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http://dx.doi.org/10.1007/s13238-020-00726-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305291PMC
July 2020

Ligand-dependent downregulation of MR1 cell surface expression.

Proc Natl Acad Sci U S A 2020 05 27;117(19):10465-10475. Epub 2020 Apr 27.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom.

The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A'-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.
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http://dx.doi.org/10.1073/pnas.2003136117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229755PMC
May 2020

Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol.

Science 2020 06 23;368(6496):1211-1219. Epub 2020 Apr 23.

Shanghai Institute for Advanced Immunochemical Studies, iHuman Institute, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo-electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.
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http://dx.doi.org/10.1126/science.aba9102DOI Listing
June 2020

A novel mutation alters the stability of PapA2 resulting in the complete abrogation of sulfolipids in clinical mycobacterial strains.

FASEB Bioadv 2019 May 10;1(5):306-319. Epub 2019 Apr 10.

Cardio Respiratory Disease Biology CSIR-Institute of Genomics and Integrative Biology New Delhi India.

The analysis of whole genomes has revealed specific geographical distribution of (Mtb) strains across the globe suggestive of unique niche dependent adaptive mechanisms. We provide an important correlation of a genome-based mutation to a molecular phenotype across two predominant clinical Mtb lineages of the Indian subcontinent. We have identified a distinct lineage specific mutation-G247C, translating into an alanine-proline conversion in the A2 gene of Indo-oceanic lineage 1 (L1) Mtb strains, and restoration of cell wall sulfolipids by simple genetic complementation of 2 from lineage 3 (L3) or from H37Rv (lineage 4-L4) attributed the loss of this glycolipid to this specific mutation in Indo-Oceanic L1 Mtb. The investigation of structure of Mtb PapA2 revealed a distinct nonribosomal peptide synthetase (NRPS) C domain conformation with an unconventional presence of a zinc binding motif. Surprisingly, the A83P mutation did not map to either the catalytic center in the N-terminal subdomain or any of the substrate-binding region of the protein. On the contrary, the inherent ability of mutant PapA2 to form insoluble aggregates and molecular simulations with the wild-type/mutant (Wt/mut) PapA2 purports an important role for the surface associated 83rd residue in protein conformation. This study demonstrates the importance of a critical structural residue in the papA2 protein of Mtb and helps establish a link between observed genomic alteration and its molecular consequence in the successful human pathogen Mtb. We demonstrate the effect of a unique SNP in gene of Indo-oceanic (Mtb) strains leading to the loss of sulfolipid from these strains. By X-ray crystallographic analysis and molecular dynamics (MD) simulations, we show the importance of this residue in the global PapA2 structure. The presence of a Zn atom has not been reported before for this class of proteins. Here, we provide an important link between genomic alteration and its molecular consequence in Mtb highlighting one of the many adaptive mechanisms that have contributed to its success as a human pathogen. A high degree of identity with PapA1, 3, or 4 would help in interpreting the structure of these PapA proteins and other acyl transferases of other biological systems.
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http://dx.doi.org/10.1096/fba.2018-00039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996325PMC
May 2019

Targeting mitochondrial dysfunction in MAIT cells limits IL-17 production in obesity.

Cell Mol Immunol 2020 11 27;17(11):1193-1195. Epub 2020 Feb 27.

Human Health Institute, Department of Biology, Maynooth University, Maynooth, County Kildare, Ireland.

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http://dx.doi.org/10.1038/s41423-020-0375-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784973PMC
November 2020

Tissue-specific shaping of the TCR repertoire and antigen specificity of iNKT cells.

Elife 2019 12 16;8. Epub 2019 Dec 16.

The Peter Gorer Department of Immunobiology, King's College London, London, United Kingdom.

Tissue homeostasis is critically dependent on the function of tissue-resident lymphocytes, including lipid-reactive invariant natural killer T (iNKT) cells. Yet, if and how the tissue environment shapes the antigen specificity of iNKT cells remains unknown. By analysing iNKT cells from lymphoid tissues of mice and humans we demonstrate that their T cell receptor (TCR) repertoire is highly diverse and is distinct for cells from various tissues resulting in differential lipid-antigen recognition. Within peripheral tissues iNKT cell recent thymic emigrants exhibit a different TCR repertoire than mature cells, suggesting that the iNKT population is shaped after arrival to the periphery. Consistent with this, iNKT cells from different organs show distinct basal activation, proliferation and clonal expansion. Moreover, the iNKT cell TCR repertoire changes following immunisation and is shaped by age and environmental changes. Thus, post-thymic modification of the TCR-repertoire underpins the distinct antigen specificity for iNKT cells in peripheral tissues.
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http://dx.doi.org/10.7554/eLife.51663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930077PMC
December 2019

Mucosal-Associated Invariant T Cells Display Diminished Effector Capacity in Oesophageal Adenocarcinoma.

Front Immunol 2019 10;10:1580. Epub 2019 Jul 10.

Department of Surgery, Trinity Translational Medicine Institute, St. James's Hospital, Dublin, Ireland.

Oesophageal adenocarcinoma (OAC) is an aggressive malignancy with poor prognosis, and incidence is increasing rapidly in the Western world. Mucosal-associated invariant T (MAIT) cells recognize bacterial metabolites and kill infected cells, yet their role in OAC is unknown. We aimed to elucidate the role of MAIT cells during cancer development by characterizing the frequency, phenotype, and function of MAIT cells in human blood and tissues, from OAC and its pre-malignant inflammatory condition Barrett's oesophagus (BO). Blood and tissues were phenotyped by flow cytometry and conditioned media from explanted tissue was used to model the effects of the tumor microenvironment on MAIT cell function. Associations were assessed between MAIT cell frequency, circulating inflammatory markers, and clinical parameters to elucidate the role of MAIT cells in inflammation driven cancer. MAIT cells were decreased in BO and OAC blood compared to healthy controls, but were increased in oesophageal tissues, compared to BO-adjacent tissue, and remained detectable after neo-adjuvant treatment. MAIT cells in tumors expressed CD8, PD-1, and NKG2A but lower NKG2D than BO cohorts. MAIT cells produced less IFN-γ and TNF-α in the presence of tumor-conditioned media. OAC cell line viability was reduced upon exposure to expanded MAIT cells. Serum levels of chemokine IP-10 were inversely correlated with MAIT cell frequency in both tumors and blood. MAIT cells were higher in the tumors of node-negative patients, but were not significantly associated with other clinical parameters. This study demonstrates that OAC tumors are infiltrated by MAIT cells, a type of CD8 T cell featuring immune checkpoint expression and cytotoxic potential. These findings may have implications for immunotherapy and immune scoring approaches.
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http://dx.doi.org/10.3389/fimmu.2019.01580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635552PMC
October 2020

The hydrolase LpqI primes mycobacterial peptidoglycan recycling.

Nat Commun 2019 06 14;10(1):2647. Epub 2019 Jun 14.

Institute of Microbiology and Infection, School of Biological Sciences, University of Birmingham, Birmingham, B15 2TT, UK.

Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ β-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.
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http://dx.doi.org/10.1038/s41467-019-10586-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572805PMC
June 2019

Identification and characterization of a novel anti-inflammatory lipid isolated from Mycobacterium vaccae, a soil-derived bacterium with immunoregulatory and stress resilience properties.

Psychopharmacology (Berl) 2019 May 22;236(5):1653-1670. Epub 2019 May 22.

Department of Integrative Physiology, Center for Neuroscience, and Center for Microbial Exploration, University of Colorado Boulder, Boulder, CO, 80309, USA.

Rationale: Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known.

Objectives: Our objective was to identify and characterize novel anti-inflammatory molecules from M. vaccae NCTC 11659.

Methods: We have purified and identified a unique anti-inflammatory triglyceride, 1,2,3-tri [Z-10-hexadecenoyl] glycerol, from M. vaccae and evaluated its effects in freshly isolated murine peritoneal macrophages.

Results: The free fatty acid form of 1,2,3-tri [Z-10-hexadecenoyl] glycerol, 10(Z)-hexadecenoic acid, decreased lipopolysaccharide-stimulated secretion of the proinflammatory cytokine IL-6 ex vivo. Meanwhile, next-generation RNA sequencing revealed that pretreatment with 10(Z)-hexadecenoic acid upregulated genes associated with peroxisome proliferator-activated receptor alpha (PPARα) signaling in lipopolysaccharide-stimulated macrophages, in association with a broad transcriptional repression of inflammatory markers. We confirmed using luciferase-based transfection assays that 10(Z)-hexadecenoic acid activated PPARα signaling, but not PPARγ, PPARδ, or retinoic acid receptor (RAR) α signaling. The effects of 10(Z)-hexadecenoic acid on lipopolysaccharide-stimulated secretion of IL-6 were prevented by PPARα antagonists and absent in PPARα-deficient mice.

Conclusion: Future studies should evaluate the effects of 10(Z)-hexadecenoic acid on stress-induced exaggeration of peripheral inflammatory signaling, central neuroinflammatory signaling, and anxiety- and fear-related defensive behavioral responses.
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http://dx.doi.org/10.1007/s00213-019-05253-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626661PMC
May 2019

Obesity Reduces mTORC1 Activity in Mucosal-Associated Invariant T Cells, Driving Defective Metabolic and Functional Responses.

J Immunol 2019 06 10;202(12):3404-3411. Epub 2019 May 10.

Obesity Immunology Group, Education and Research Centre, St. Vincent's University Hospital, University College Dublin, Dublin 4, Ireland;

Obesity underpins the development of numerous chronic diseases, such as type II diabetes mellitus. It is well established that obesity negatively alters immune cell frequencies and functions. Mucosal-associated invariant T (MAIT) cells are a population of innate T cells, which we have previously reported are dysregulated in obesity, with altered circulating and adipose tissue frequencies and a reduction in their IFN-γ production, which is a critical effector function of MAIT cells in host defense. Hence, there is increased urgency to characterize the key molecular mechanisms that drive MAIT cell effector functions and to identify those which are impaired in the obesity setting. In this study, we found that MAIT cells significantly upregulate their rates of glycolysis upon activation in an mTORC1-dependent manner, and this is essential for MAIT cell IFN-γ production. Furthermore, we show that mTORC1 activation is dependent on amino acid transport via SLC7A5. In obese patients, using RNA sequencing, Seahorse analysis, and a series of in vitro experiments, we demonstrate that MAIT cells isolated from obese adults display defective glycolytic metabolism, mTORC1 signaling, and SLC7A5 aa transport. Collectively, our data detail the intrinsic metabolic pathways controlling MAIT cell cytokine production and highlight mTORC1 as an important metabolic regulator that is impaired in obesity, leading to altered MAIT cell responses.
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http://dx.doi.org/10.4049/jimmunol.1801600DOI Listing
June 2019

Promotion or Suppression of Murine Intestinal Polyp Development by iNKT Cell Directed Immunotherapy.

Front Immunol 2019 1;10:352. Epub 2019 Mar 1.

Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.

The glycosphingolipid α-galactosylceramide (α-GalCer) is a well-described immune activator with strong anti-tumor properties in animal models. It is presented on CD1d and acts by stimulating the invariant, type I, natural killer T (iNKT) lymphocytes to rapidly secrete TH1 and TH2 associated cytokines. This in turn promotes activation of a diversity of immune cells including natural killer (NK) cells with anti-tumor functions. Prior to tumor development, iNKT cells can also perform tumor surveillance and naturally protect from emergence of cancer. In contrast, we have recently demonstrated that iNKT cells naturally promote polyps in the spontaneous murine adenomatous polyposis coli (Apc) model for colon cancer, associated with suppressed TH1 immunity and enhanced immunoregulation. Here we investigated whether iNKT cell directed immunotherapy could subvert the polyp promoting function of iNKT cells and reduce polyp growth in this model. We treated mice with α-GalCer, or synthetic derivatives of this ligand (C-glycoside and C20:2) that have enhanced immunoregulatory properties. Treatment with iNKT cell ligands led to increased iNKT cell division, but reduced iNKT cell frequencies, lower NK1.1 expression and elevation of PD-1. mice that had been treated either long-term (5-15 weeks of age), or short-term (12-15 weeks of age) with α-GalCer demonstrated a significant decrease in polyp burden. Surprisingly, long-term treatment with the TH1 biasing ligand C-glycoside did not have significant effects on polyps, while long-term treatment with the TH2 biasing ligand C20:2 enhanced polyp growth. In stark contrast, short-term treatment with C20:2 led to reduction in polyp numbers and size. Reduced polyp burden after long-term treatment was associated with increased expression of genes indicating a pro-inflammatory polyp microenvironment. Polyp-reducing short-term treatment led to CD8 T cell activation specifically in polyps, and decreased tumor infiltrating and splenic macrophages, and a switch toward a pro-inflammatory phenotype. Thus, iNKT cell directed therapy could subvert the natural polyp enhancing function of iNKT cells, overcome immunosuppression, and reduce polyps. However, different iNKT cell activating ligands had opposite effects, and the timing of treatment had a major influence on outcomes.
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http://dx.doi.org/10.3389/fimmu.2019.00352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405695PMC
May 2020

Crystal structure of the TreS:Pep2 complex, initiating α-glucan synthesis in the GlgE pathway of mycobacteria.

J Biol Chem 2019 05 15;294(18):7348-7359. Epub 2019 Mar 15.

From the Institute of Microbiology & Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

A growing body of evidence implicates the mycobacterial capsule, the outermost layer of the mycobacterial cell envelope, in modulation of the host immune response and virulence of mycobacteria. Mycobacteria synthesize the dominant capsule component, α(1→4)-linked glucan, via three interconnected and potentially redundant metabolic pathways. Here, we report the crystal structure of the TreS:Pep2 complex, containing trehalose synthase (TreS) and maltokinase (Pep2), which converts trehalose to maltose 1-phosphate as part of the TreS:Pep2-GlgE pathway. The structure, at 3.6 Å resolution, revealed that a diamond-shaped TreS tetramer forms the core of the complex and that pairs of Pep2 monomers bind to opposite apices of the tetramer in a 4 + 4 configuration. However, for the orthologues, results from isothermal titration calorimetry and analytical ultracentrifugation experiments indicated that the prevalent stoichiometry in solution is 4 TreS + 2 Pep2 protomers. The observed discrepancy between the crystallized complex and the behavior in the solution state may be explained by the relatively weak affinity of Pep2 for TreS ( 3.5 μm at mildly acidic pH) and crystal packing favoring the 4 + 4 complex. Proximity of the ATP-binding site in Pep2 to the complex interface provides a rational basis for rate enhancement of Pep2 upon binding to TreS, but the complex structure appears to rule out substrate channeling between the active sites of TreS and Pep2. Our findings provide a structural model for the trehalose synthase:maltokinase complex in that offers critical insights into capsule assembly.
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http://dx.doi.org/10.1074/jbc.RA118.004297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509496PMC
May 2019

Utilisation of the Prestwick Chemical Library to identify drugs that inhibit the growth of mycobacteria.

PLoS One 2019 12;14(3):e0213713. Epub 2019 Mar 12.

Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

Tuberculosis (TB) is an infectious bacterial disease that kills approximately 1.3 million people every year. Despite global efforts to reduce both the incidence and mortality associated with TB, the emergence of drug resistant strains has slowed any progress made towards combating the spread of this deadly disease. The current TB drug regimen is inadequate, takes months to complete and poses significant challenges when administering to patients suffering from drug resistant TB. New treatments that are faster, simpler and more affordable are urgently required. Arguably, a good strategy to discover new drugs is to start with an old drug. Here, we have screened a library of 1200 FDA approved drugs from the Prestwick Chemical library using a GFP microplate assay. Drugs were screened against GFP expressing strains of Mycobacterium smegmatis and Mycobacterium bovis BCG as surrogates for Mycobacterium tuberculosis, the causative agent of TB in humans. We identified several classes of drugs that displayed antimycobacterial activity against both M. smegmatis and BCG, however each organism also displayed some selectivity towards certain drug classes. Variant analysis of whole genomes sequenced for resistant mutants raised to florfenicol, vanoxerine and pentamidine highlight new pathways that could be exploited in drug repurposing programmes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213713PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414029PMC
December 2019

Development of a whole-cell high-throughput phenotypic screen to identify inhibitors of mycobacterial amino acid biosynthesis.

FASEB Bioadv 2019 Apr 22;1(4):246-254. Epub 2019 Jan 22.

School of Biosciences University of Birmingham Birmingham UK.

Anti-tubercular drug discovery continues to be dominated by whole-cell high-throughput screening campaigns, enabling the rapid discovery of new inhibitory chemical scaffolds. Target-based screening is a popular approach to direct inhibitor discovery with a specified mode of action, eliminating the discovery of anti-tubercular agents against unsuitable targets. Herein, a screening method has been developed using BCG to identify inhibitors of amino acid biosynthesis. The methodology was initially optimized using the known branched-chain amino acid biosynthetic inhibitors metsulfuron-methyl (MSM) and sulfometuron-methyl (SMM), and subsequently, whole genome sequencing of resistant mutants and the use of over-expressor strains confirming their mode of action. The GlaxoSmithKline compound library of small molecule inhibitors with known activity against was then used to validate the screen. In this paper, we have shown that media supplementation with amino acids can rescue BCG from known amino acid synthesis inhibitors, MSM and SMM, in a pathway specific manner. The therapeutic potential of amino acid biosynthesis inhibitors emphasizes the importance of this innovative screen, enabling the discovery of compounds targeting a multitude of related essential biochemical pathways, without limiting drug discovery toward a single target.
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http://dx.doi.org/10.1096/fba.2018-00048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996392PMC
April 2019

Crystal structure of Mycobacterium tuberculosis FadB2 implicated in mycobacterial β-oxidation.

Acta Crystallogr D Struct Biol 2019 Jan 8;75(Pt 1):101-108. Epub 2019 Jan 8.

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, England.

The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long-lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty-acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in β-oxidation (∼100 genes). In a previous study, the enzymology of the M. tuberculosis L-3-hydroxyacyl-CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand-free form is reported at 2.1 Å resolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann-fold motif in the N-terminal domain, while the helical C-terminal domain mediates dimer formation. Comparison with the CoA- and NAD-bound human orthologue mitochondrial hydroxyacyl-CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi-catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self-association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA-FadB complex as it lacks the N-terminal hydratase domain of FadB. Instead, FadB2 may functionally (or physically) associate with the enoyl-CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein-network database.
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http://dx.doi.org/10.1107/S2059798318017242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333283PMC
January 2019

Deciphering the molecular basis of mycobacteria and lipoglycan recognition by the C-type lectin Dectin-2.

Sci Rep 2018 11 15;8(1):16840. Epub 2018 Nov 15.

Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Paul Sabatier, 31077, Toulouse, France.

Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus, Candida albicans, Schistosoma mansonii, and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 → 2)-linked mannosides forming the caps. Here, using isogenic M. tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M. tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl-myo-inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 → 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola, suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.
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http://dx.doi.org/10.1038/s41598-018-35393-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237770PMC
November 2018