Cell Wall Of Mycobacterium Tuberculosis Research Articles

Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1–4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12–80%) and specificities (14–100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

Development of a mycolic acid-graphene quantum dot probe as a potential tuberculosis biosensor.

The development of amine-functionalized graphene quantum dots (GQDs) linked to mycolic acids (MAs) as a potential fluorescent biosensor to detect tuberculosis (TB) biomarkers is described. GQDs have attractive properties: high fluorescence, excellent biocompatibility, good water solubility, and low toxicity. MAs are lipids that are found in the cell wall of Mycobacterium tuberculosis that are antigenic, however, they are soluble only in chloroform and hexane. Chloroform-soluble MAs were covalently linked to synthesized water-soluble GQDs using an amide connection to create a potential fluorescent water-soluble TB biosensor: MA-GQDs. Fluorescence results showed that GQDs had a narrow emission spectrum with the highest emission at 440 nm, while MA-GQDs had a broader spectrum with the highest emission at 470 nm, after exciting at 360 nm. The appearance of the peptide bond (amide linkage) in the Fourier-transform infrared spectrum of MA-GQDs confirmed the successful linking of MAs to GQDs. Powder X-ray diffraction exhibited an increase in the number of peaks for MA-GQDs relative to GQDs, suggesting that linking MAs to GQDs changed the crystal structure thereof. The linked MA-GQDs showed good solubility in water, high fluorescence, and visual flow through a nitrocellulose membrane. These properties are promising for biomedical fluorescence sensing applications.

Identification of Mtb GlmU Uridyltransferase Domain Inhibitors by Ligand-Based and Structure-Based Drug Design Approaches.

Targeting enzymes that play a role in the biosynthesis of the bacterial cell wall has long been a strategy for antibacterial discovery. In particular, the cell wall of Mycobacterium tuberculosis (Mtb) is a complex of three layers, one of which is Peptidoglycan, an essential component providing rigidity and strength. UDP-GlcNAc, a precursor for the synthesis of peptidoglycan, is formed by GlmU, a bi-functional enzyme. Inhibiting GlmU Uridyltransferase activity has been proven to be an effective anti-bacterial, but its similarity with human enzymes has been a deterrent to drug development. To develop Mtb selective hits, the Mtb GlmU substrate binding pocket was compared with structurally similar human enzymes to identify selectivity determining factors. Substrate binding pockets and conformational changes upon substrate binding were analyzed and MD simulations with substrates were performed to quantify crucial interactions to develop critical pharmacophore features. Thereafter, two strategies were applied to propose potent and selective bacterial GlmU Uridyltransferase domain inhibitors: (i) optimization of existing inhibitors, and (ii) identification by virtual screening. The binding modes of hits identified from virtual screening and ligand growing approaches were evaluated further for their ability to retain stable contacts within the pocket during 20 ns MD simulations. Hits that are predicted to be more potent than existing inhibitors and selective against human homologues could be of great interest for rejuvenating drug discovery efforts towards targeting the Mtb cell wall for antibacterial discovery.

Glycosyl Vinylogous Carbonates as Glycosyl Donors by Metal-Free Activation.

Synthesis of glycoconjugates employs a glycosylation reaction wherein an electrophile and a nucleophile known as a glycosyl donor and an aglycon, respectively, are involved. Glycosyl donors often contain a leaving group at the anomeric carbon that upon reaction with activator(s) results in a highly reactive electrophilic species reported as an oxycarbenium ion contact pair that will then be attacked by the aglycon. Therefore, identification of the correct glycosyl donor and activation protocol is essential for the synthesis of all glycoconjugates. Recently identified [Au]/[Ag]-catalyzed activation of ethynylcyclohexyl glycosyl carbonates is one such versatile method for the synthesis of glycosides, oligosaccharides, and glycoconjugates. In this work, stable glycosyl vinylogous carbonates were identified to undergo glycosidation in the presence of a sub-stoichiometric quantity of TfOH. The reaction is fast and suitable for donors containing both C2-ethers and C2-esters. Donors positioned with C2-ethers resulted in anomeric mixtures with greater selectivity toward 1,2-cis glycosides, whereas those with C2-esters gave 1,2-trans selective glycosides. The versatility of the method is demonstrated by conducting the glycosylation with more than 25 substrates. Furthermore, the utility of the glycosyl vinylogous carbonate donors is demonstrated with the successful synthesis of the branched pentaarabinofuranoside moiety of the Mycobacterium tuberculosis cell wall.

Detection of Mycobacterium Tuberculosis by Magnetic Nanoparticle Capture-Polymerase Chain Reaction

This work aimed to study the experiment of Mycobacterium tuberculosis (MTB) detection via magnetic nanoparticle (MNP) capture combined with polymerase chain reaction (PCR) technology. Thirty sputum samples were collected, MTB was isolated and cultured, and DNA templates were prepared. Fe3O4-polyethylene imide (PEI) MNPs were prepared, and PCR was adopted to test the 30 samples. Within the range of 0–600 °C, as the temperature rose, the quality of Fe3O4-PEI MNPs was gradually reduced. MNPs were positively charged when the pH value was 2–8. The highest capture rate of MTB with OD500 = 0.4 was 71%. The highest capture rate of MTB with OD500 = 0.6 was 58%. The highest capture rate of MTB with OD500 = 0.8 was 26%. When the temperature was heated to 900 ˚C, the thermal weight loss of MTB was about 85% if MNP wasn’t added. The thermal weight loss of MNP-captured MTB was about 45%, while that of uncaptured MTB was about 71%. In summary, polyethyleneimine could enhance the adsorption of MNP to MTB, and the capture rate gradually increased over time. The lipid of the cell wall of MTB was involved in the interaction between MTB and MNP.

Functional analysis and enzyme characterization of mannose-1-phosphate guanylyl transferase (ManB) from Mycobacterium tuberculosis

Mycobacterium tuberculosis cell wall consist variety of mannose containing glycoconjugates including lipomannan (LM) and lipoarabinomannan (LAM). These lipoglycans are involved in cell wall integrity and play role in virulence of M. tuberculosis by modulating host immune response. GDP-mannose, required for the synthesis of lipoglycans, is catalyzed by enzyme Mannose-1-phosphate guanylyl transferase (ManB). The enzyme with similar function has been studied in variety of species of prokaryotes and eukaryotes. However, biological role of ManB and its enzymatic activity remains uncharacterized in M. tuberculosis. In present study, we elucidated the role of enzyme by constructing manB knockdown strain of M. tuberculosis H37Ra. The manB knockdown decreased the cell growth and also effected the morphology of M. tuberculosis by altering the permeability of cell membrane. These findings provide the understanding on ManB function and suggesting that ManB could be the potential target for novel anti-tuberculosis drug. Furthermore, we also characterized ManB enzyme by establishing 96 well plate colorimetric assay and determined the kinetic properties including initial velocity, optimum temperature, optimum pH and other kinetic parameters. Our established assay will be helpful for further high throughput screening of potential inhibitors against ManB.

The Mur Enzymes Chink in the Armour of Mycobacterium tuberculosis cell wall.

TUBERCULOSIS: (TB) transmitted by Mycobacterium tuberculosis (Mtb) is one of the top 10 causes of death globally. Currently, the widespread occurrence of resistance toward Mtb strains is becoming a significant concern to public health. This scenario exaggerated the need for the discovery of novel targets and their inhibitors. Targeting the "Mtb cell wall peptidoglycan synthesis" is an attractive strategy to overcome drug resistance. Mur enzymes (MurA-MurF) play essential roles in the peptidoglycan synthesis by catalyzing the ligation of key amino acid residues to the stem peptide. These enzymes are unique and confined to the eubacteria and are absent in humans, representing potential targets for anti-tubercular drug discovery. Mtb Mur ligases with the same catalytic mechanism share conserved amino acid regions and structural features that can conceivably exploit for the designing of the inhibitors, which can simultaneously target more than one isoforms (MurC-MurF) of the enzyme. In light of these findings in the current review, we have discussed the recent advances in medicinal chemistry of Mtb Mur enzymes (MurA-MurF) and their inhibitors, offering attractive multi-targeted strategies to combat the problem of drug-resistant in M.tuberculosis.

Detection of Mycolic Acid Implying the Presence of Mycobacterium Tuberculosis H37Rv in Host

Tuberculosis is a widespread infectious disease that affects millions of people worldwide every year. The bacterium, responsible for causing tuberculosis is Mycobacterium tuberculosis H37Rv (Mtb) which belongs to Mycobacteriaceace family. It is a gram positive bacteria and highly aerobic in nature which enters the host by the means of inhalation. Several tests are performed for the diagnosis of tuberculosis such as chest X-rays, culture, tuberculin skin test, interferon gamma release assays and histopathology. These techniques are expensive as well as take a lot of time in confirming the presence of bacteria. A cost and time effective way to diagnose the disease could be with the help of lipids present in the cell wall of Mycobacterium tuberculosis. The cell wall skeleton is chemically composed of three covalently linked components: peptidoglycan, arabinogalactan and mycolic acids. All these components can be exposed by rupturing the cell wall by suitable means. After their exposure mycolic acids can be extracted. Mtb contains three types of mycolic acids i.e. alpha, methoxy and keto mycolic acids. These lipids consist of several functional groups which are expected to give positive result for the organic qualitative analysis. These functional groups are alcohols, carboxylic acids, ketone and ether. Each functional group has different tests which one can perform in order to confirm its presence. This review is describing several chemical tests and these can be done by using mycolic acid as sample. The chemical reactions can impart colour, show effervescence or can show precipitation for identification which in turn will confirm the presence of Mycobacterium tuberculosis in the patient.

Permeability of active ingredients of cannabis and possibility for further antituberculosis drug development

Background Cannabis is presently legalized in several countries owing to its medicinal property. The antibacterial properties of active ingredients of cannabis have been mentioned. Their usefulness in the management of tuberculosis is very interesting. Materials and methods The authors performed a bioinformatics analysis to assess the possibility of important active ingredients of cannabis. Results Based on the present analysis, it can be seen that the studied active ingredients of cannabis can effectively pass through the cell wall of Mycobacterium tuberculosis, indicating the possibility of further pharmacological actions. Conclusion The active ingredients of cannabis are the possible new targets for further antituberculosis drug development.

Chemical Space Exploration of DprE1 Inhibitors Using Chemoinformatics and Artificial Intelligence.

Tuberculosis (TB), entrained by Mycobacterium tuberculosis, continues to be an enfeebling disease, killing nearly 1.5 million people in 2019, with 2 billion people worldwide affected by latent TB. The multidrug-resistant and totally drug-resistant emerging strains further exacerbate the TB infection. The cell wall of bacteria provides critical virulence components such as cell surface proteins, regulators, signal transduction proteins, and toxins. The cell wall biosynthesis pathway of Mycobacterium tuberculosis is exhaustively studied to discover novel drug targets. Decaprenylphosphoryl-β-d-ribose-2′-epimerase (DprE1) is an important enzyme involved in the arabinogalactan biosynthetic pathway of Mycobacterium tuberculosis cell wall and is essential for both latent and persistent bacterial infection. We analyzed all known ∼1300 DprE1 inhibitors to gain deep insights into the chemogenomic space of DprE1-ligand complexes. Physicochemical descriptors of the DprE1 inhibitors showed a marked lipophilic character forming a cluster distinct from the existing TB drugs, as revealed by the principal component analysis. Similarity analysis using Murcko scaffolds and rubber band scaling revealed scarce representation of the chemical space. Further, Murcko scaffold analysis uncovered favorable and unfavorable scaffolds, where benzo and pyridine-based core scaffolds exhibit the highest biological activity, as evidenced by their MIC and IC50 values. Automatic SAR and R-group decomposition analysis resulted in the identification of substructures responsible for the inhibitory activity of the DprE1 enzyme. Further, with activity cliff analysis, we observed prominent discontinuity in the SAR of DprE1 inhibitors, where even simple structural modification in the chemical scaffold resulted in significant potency difference, presumably due to the binding orientation and interaction in the active site. Thiophene, 6-membered aromatic rings, and unsubstituted benzene ring-based toxicophores were identified in the DprE1 chemical space using an artificial intelligence approach based on inductive logic programming. This paper, hence, ushers in new insights for the design and development of potent covalent and non-covalent DprE1 inhibitors and guides hit and lead optimization for the development of non-hazardous small molecule therapeutics for Mycobacterium tuberculosis.

Synthetic molecules as DprE1 inhibitors: A patent review

ABSTRACT Introduction In recent years, the advent of multidrug-resistant tuberculosis (MDR-TB), the extensively-resistant TB (XDR-TB), and the total drug-resistant-TB (TDR-TB) have led the community to develop new antitubercular molecules. The decaprenylphosphoryl-β-D-ribose-2ʹ-epimerase-1 (DprE1) is an established target to developed new anti-TB drugs. This enzyme is required to synthesize the cell wall of Mycobacterium tuberculosis (Mtb). Area Covered This patent review focuses on the granted patents and patent applications related to the chemical entities developed as DprE1 inhibitors for TB treatment from the publication year of the BTZ-043 compound patent application (2007) till 30 September 2020. Expert Opinion The DprE1 has many advantages in the development of new antitubercular molecules, for example, its location in the periplasm of the Mtb cell wall and its absence in the human body. This indicates that the DprE1 inhibitors are selective for Mtb, and their toxic and side effects on the human body may be negligible or small. Accordingly, the use of DprE1 inhibitors may be benefic for patients with drug-resistant bacteria that require long-term medication. Four molecules are in clinical trials, which could become the drugs of the future for TB-therapy.

The utility of point-of-care urinary lipoarabinomannan testing for the diagnosis of tuberculosis in critically ill patients: a prospective observational study

BackgroundTuberculosis is a major global public health concern. Patients with tuberculosis who require critical care have a high mortality and delay in initiating antituberculous therapy is associated with increased mortality. Lipoarabinomannan (LAM) is a lipopolysaccharide found in the cell wall of Mycobacterium tuberculosis. Urinary LAM may be used as a bedside diagnostic test for tuberculosis.MethodsThe study was a single centre, prospective observational study that compared the utility of urinary LAM with conventional tuberculosis diagnostic modalities in patients with suspected tuberculosis who required intensive care admission. Urinary LAM testing was performed using the Alere Determine TB LAM Ag lateral flow assay test strips. A patient was classified as having confirmed tuberculosis if they met the following criteria: a clinical presentation compatible with tuberculosis, with either a positive TB culture, a positive GeneXpert, or a histological diagnosis of tuberculosis.ResultsFifty patients were included in the study, with 12 having confirmed tuberculosis. All patients received mechanical ventilation, and the ICU mortality was 60%. Urinary LAM had a sensitivity of 50.0% (95% CI, 21.1 to 78.9%) and a specificity of 84.2% (95% CI, 68.8 to 94.0%) for confirmed tuberculosis.ConclusionUrinary LAM allows for rapid bedside diagnosis of tuberculosis in critically ill patients. A positive urinary LAM should prompt consideration to initiate antituberculous treatment while the results of further diagnostic testing are awaited.

Mechanistic insight on the inhibition of D, D-carboxypeptidase from Mycobacterium tuberculosis by β-lactam antibiotics: an ONIOM acylation study

Mycobacterium tuberculosis cell wall is intricate and impermeable to many agents. A D, D-carboxypeptidase (DacB1) is one of the enzymes involved in the biosynthesis of cell wall peptidoglycan and catalyzes the terminal D-alanine cleavage from pentapeptide precursors. Catalytic activity and mechanism by which DacB1 functions is poorly understood. Herein, we investigated the acylation mechanism of DacB1 by β-lactams using a 6-membered ring transition state model that involves a catalytic water molecule in the reaction pathway. The full transition states (TS) optimization plus frequency were achieved using the ONIOM (B3LYP/6-31 + G(d): AMBER) method. Subsequently, the activation free energies were computed via single-point calculations on fully optimized structures using B3LYP/6-311++(d,p): AMBER and M06-2X/6-311++(d,p): AMBER with an electronic embedding scheme. The 6-membered ring transition state is an effective model to examine the inactivation of DacB1 via acylation by β-lactams antibiotics (imipenem, meropenem, and faropenem) in the presence of the catalytic water. The ΔG# values obtained suggest that the nucleophilic attack on the carbonyl carbon is the rate-limiting step with 13.62, 19.60 and 30.29 kcal mol−1 for Imi–DacB1, Mero–DacB1 and Faro–DacB1, respectively. The electrostatic potential (ESP) and natural bond orbital (NBO) analysis provided significant electronic details of the electron-rich region and charge delocalization, respectively, based on the concerted 6-membered ring transition state. The stabilization energies of charge transfer within the catalytic reaction pathway concurred with the obtained activation free energies. The outcomes of this study provide important molecular insight into the inactivation of D, D-carboxypeptidase by β-lactams. Communicated by Ramaswamy H. Sarma

Aptamer Detection of Mycobaterium tuberculosis Mannose-Capped Lipoarabinomannan in Lesion Tissues for Tuberculosis Diagnosis

Tuberculosis (TB) is the leading infectious cause of mortality worldwide. However, the diagnosis of TB, especially extrapulmonary TB (EPTB) diagnosis from lesion tissues, remains a challenge. Nucleic acid aptamers are analogous to antibodies and have advantages of easier modification, high specificity, and affinity. Mannose-capped lipoarabinomannan (ManLAM) is a unique surface lipoglycan component or constantly released from mycobacterium tuberculosis (M.tb) cell wall, which makes it a perfect candidate biomarker for TB diagnosis. Our present study aims to establish M.tb ManLAM aptamer-based immunohistochemistry (IHC) method for TB diagnosis. We performed TB diagnosis using 263 formalin-fixed paraffin-embedded tissue samples including 213 TB samples (pulmonary TB (PTB) and EPTB), and 8 samples from latent TB infection (LTBI) high risk subjects, and 42 samples from other non-TB patients with ManLAM aptamer-based IHC and routine laboratory TB diagnostic methods parallelly. The sensitivity and specificity of the ManLAM aptamer-based IHC were 86.38% and 92.86%, with much higher sensitivity than those of mycobacterial culture (9.66%) and acid-fast staining (AFS) (43.01%) and comparability to Interferon-gamma Release Assay (IGRA) (84.38%) and GeneXpert (79.31%). High agreement between ManLAM based-IHC and IGRA or GeneXpert for TB diagnosis were observed. Furthermore, ManLAM aptamer-based IHC combination with other routine TB laboratory diagnostic methods significantly increased the sensitivity up to 88.64%–97.92%. As our knowledge, this is the first report about aptamer-based IHC for disease diagnosis. Thus, ManLAM aptamer-based IHC has potentials for TB diagnosis, including PTB, and EPTB, and assists the diagnosis of LTBI with high effectiveness, feasibility, and easy production.

Easy-To-Access Quinolone Derivatives Exhibiting Antibacterial and Anti-Parasitic Activities.

The cell wall of Mycobacterium tuberculosis (Mtb) has a unique structural organisation, comprising a high lipid content mixed with polysaccharides. This makes cell wall a formidable barrier impermeable to hydrophilic agents. In addition, during host infection, Mtb resides in macrophages within avascular necrotic granulomas and cavities, which shield the bacterium from the action of most antibiotics. To overcome these protective barriers, a new class of anti-TB agents exhibiting lipophilic character have been recommended by various reports in literature. Herein, a series of lipophilic heterocyclic quinolone compounds was synthesised and evaluated in vitro against pMSp12::GFP strain of Mtb, two protozoan parasites (Plasmodium falciparum and Trypanosoma brucei brucei) and against ESKAPE pathogens. The resultant compounds exhibited varied anti-Mtb activity with MIC90 values in the range of 0.24–31 µM. Cross-screening against P. falciparum and T.b. brucei, identified several compounds with antiprotozoal activities in the range of 0.4–20 µM. Compounds were generally inactive against ESKAPE pathogens, with only compounds 8c, 8g and 13 exhibiting moderate to poor activity against S. aureus and A. baumannii.

Biophysical Insights into the Spatial Organization and Dynamics of Structurally Complex Mycobacterial Membrane Lipids

The remarkably complex cell wall of mycobacterium tuberculosis (Mtb) forms a formidable barrier to many host stresses and therapeutics. It houses distinct lipids with long hydrocarbon chains, and chemical groups such as trehalose, methyl branches and cyclopropane rings; not found in eukaryotic membranes. The current holistic picture of the Mtb cell envelope from cryo-TEM comprises of peptidoglycan (PG) covalently modified with arabinogalactan polymers that are in turn elaborated with mycolic acids (C60-C90). These mycolic acids form the inner leaflet of the mycomembrane (MM); MM also includes an outer leaflet constituted by a variety of non-covalently associated lipids such as phthiocerol dimycocerosate (PDIM), dimycolyltrehalose (TDM), sulfoglycolipids (SL), phosphatidylinositol mannosides (PIMs), lipoarabinomannan (man-LAM) and DAGs along with phospholipids. The vital role of each Mtb membrane component in forming a spatially complex barrier, and how mycobacteria uses its membrane platforms to impact infection biology remain understudied. in our current work we have characterized the outer membrane (OM) and inner membrane (IM) from Mycobacterium smegmatis by isolating and reconstituting lipids from these regions to form model membranes mimicking the natural cell envelope. Using methods such as atomic force microscopy and spectroscopy, infrared spectroscopy, time-resolved fluorescence spectroscopy, two-photon and confocal microscopy, we provide crucial insights into the lateral organization, fluidity and domain architecture of mycobacterial membrane lipids and its correlation to lipid structure. Our findings may facilitate identification of compounds with membrane perturbing activity against specific membranous regions of mycobacterial envelope. We also foresee our characterized mycobacterial model membrane system to serve as molecularly complete membrane scaffolds for anti-tubercular drug membrane screening in future.

Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle

The cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

Synthesis and evaluation of heterocycle structures as potential inhibitors of Mycobacterium tuberculosis UGM

In this study, we screen three heterocyclic structures as potential inhibitors of UDP-galactopyranose mutase (UGM), an enzyme involved in the biosynthesis of the cell wall of Mycobacterium tuberculosis. In order to understand the binding mode, docking simulations are performed on the best inhibitors. Their activity on Mycobacterium tuberculosis is also evaluated. This study made it possible to highlight an “oxazepino-indole” structure as a new inhibitor of UGM and of M. tuberculosis growth in vitro.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB2-AcpM2

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.

Group 1 CD1-restricted T cells contribute to control of systemic Staphylococcus aureus infection.

Staphylococcus aureus (SA) is the causative agent of both skin/soft tissue infections as well as invasive bloodstream infections. Though vaccines have been developed to target both humoral and T cell-mediated immune responses against SA, they have largely failed due to lack of protective efficacy. Group 1 CD1-restricted T cells recognize lipid rather than peptide antigens. Previously found to recognize lipids derived from cell wall of Mycobacterium tuberculosis (Mtb), these cells were associated with protection against Mtb infection in humans. Using a transgenic mouse model expressing human group 1 CD1 molecules (hCD1Tg), we demonstrate that group 1 CD1-restricted T cells can recognize SA-derived lipids in both immunization and infection settings. Systemic infection of hCD1Tg mice showed that SA-specific group 1 CD1-restricted T cell response peaked at 10 days post-infection, and hCD1Tg mice displayed significantly decreased kidney pathology at this time point compared with WT control mice. Immunodominant SA lipid antigens recognized by group 1 CD1-restricted T cells were comprised mainly of cardiolipin and phosphatidyl glycerol, with little contribution from lysyl-phosphatidyl glycerol which is a unique bacterial lipid not present in mammals. Group 1 CD1-restricted T cell lines specific for SA lipids also conferred protection against SA infection in the kidney after adoptive transfer. They were further able to effectively control SA replication in vitro through direct antigen presentation by group 1 CD1-expressing BMDCs. Together, our data demonstrate a previously unknown role for group 1 CD1-restricted SA lipid-specific T cells in the control of systemic MRSA infection.