Coarse-Graining the Recognition of a Glycolipid by the C-Type Lectin Mincle Receptor.

Macrophage inducible Ca2+-dependent lectin (Mincle) receptor recognizes Mycobacterium tuberculosis glycolipids to trigger an immune response. This host membrane receptor is thus a key player in the modulation of the immune response to infection by M. tuberculosis and has emerged as a promising target for the development of new vaccines against tuberculosis. The recent development of the Martini 3 force field for coarse-grained (CG) molecular modeling allows the study of interactions of soluble proteins with small ligands which was not typically modeled well with the previous Martini 2 model. Here, we present a refined approach detailing a protocol for modeling interactions between a glycolipid and its receptor at a CG level using the Martini 3 force field. Using this approach, we studied Mincle and identified critical parameters governing ligand recognition, such as loop flexibility and the regulation of hydrophobic groove formation by calcium ions. In addition, we assessed ligand affinity using free energy perturbation calculations. Our results offer mechanistic insight into the interactions between Mincle and glycolipids, providing a basis for the rational design of molecules targeting this type of membrane receptors.

Inhibitor binding and disruption of coupled motions in MmpL3 protein: Unraveling the mechanism of trehalose monomycolate transport.

Mycobacterial membrane protein Large 3 (MmpL3) of Mycobacterium tuberculosis (Mtb) is crucial for the translocation of trehalose monomycolate (TMM) across the inner bacterial cell membrane, making it a promising target for anti-tuberculosis (TB) drug development. While several structural, microbiological, and in vitro studies have provided significant insights, the precise mechanisms underlying TMM transport by MmpL3 and its inhibition remain incompletely understood at the atomic level. In this study, molecular dynamic (MD) simulations for the apo form and seven inhibitor-bound forms of Mtb MmpL3 were carried out to obtain a thorough comprehension of the protein's dynamics and function. MD simulations revealed that the seven inhibitors in this work stably bind to the central channel of the transmembrane domain and primarily forming hydrogen bonds with ASP251, ASP640, or both residues. Through dynamical cross-correlation matrix and principal component analysis analyses, several types of coupled motions between different domains were observed in the apo state, and distinct conformational states were identified using Markov state model analysis. These coupled motions and varied conformational states likely contribute to the transport of TMM. However, simulations of inhibitor-bound MmpL3 showed an enlargement of the proton channel, potentially disrupting coupled motions. This indicates that inhibitors may impair MmpL3's transport function by directly blocking the proton channel, thereby hindering coordinated domain movements and indirectly affecting TMM translocation.

Identification of fluoroquinolone-resistant Mycobacterium tuberculosis through high-level data fusion of Raman and laser-induced breakdown spectroscopy.

Accurate and rapid diagnosis of drug susceptibility of Mycobacterium tuberculosis is crucial for the successful treatment of tuberculosis, a persistent global public health threat. To shorten diagnosis times and enhance accuracy, this study introduces a fusion model combining laser-induced breakdown spectroscopy (LIBS) and Raman spectroscopy. This model offers a rapid and accurate method for diagnosing drug-resistance. LIBS and Raman spectroscopy provide complementary information, enabling accurate identification of drug resistance in tuberculosis. Although individual use of LIBS or Raman spectroscopy achieved approximately 90% accuracy in identifying drug resistance, the fusion model significantly improved identification accuracy to 98.3%. Given the fast measurement capabilities of both techniques, this fusion approach is expected to markedly decrease the time required for diagnosis.

Benzohydroxamate and nitrobenzohydroxamate affect membrane order: Correlations between spectroscopic and molecular dynamics to approach tuberculosis.

This work correlates the effects of benzohydroxamate (BH) and nitrobenzohydroxamate (NBH) anions in two membrane models which may be used for anti-tuberculosis (anti-TB) spectroscopic studies and/or computational studies. Firstly, the BH and NBH influence in the physico-chemical properties of soy asolectin (ASO)-based large multilamellar vesicles (MLVs) were evaluated by spectroscopic and calorimetric studies. In parallel, the BH and NBH interaction with a Mycobacterium tuberculosis (Mtb) inner membrane model, composed of phosphatidyl-myo-inositol-dimannoside (PIM2), was investigated by molecular dynamics (MD) simulations. Spectroscopic data showed a localization of BH close to the lipid phosphate group, while NBH was found close to the choline region. The BH ordered the ASO choline, phosphate and carbonyl regions and disrupted the acyl methylenes, reducing the membrane packing of the lipid hydrophobic region. On the other hand, NBH showed an ordering effect in all the lipid groups (polar, interface and hydrophobic ones). By MD studies, it was found that NBH enhanced the stability of the PIM2 membrane more than BH, while also being positioned closer to its mannosyl oxygens. As in ASO MLVs, BH was localized close to the PIM2 phosphate group and disrupted its acyl chains. However, higher values of lateral diffusion were observed for NBH than BH. Despite this, BH and NBH increased the membrane thickness by 35 %, which suggests a global ordering effect of both drugs. Findings of this work reinforce the accordance and complementarity between MLVs based on ASO and the PIM2 MD model results to study the drug effects in Mtb membrane properties.

Orthogonal Deprotection of Photolabile Protecting Groups and Its Application in Oligosaccharide Synthesis.

We herein report for the first time the inter- and intramolecular orthogonal cleavage of two ortho-nitrobenzyl (NB) analogues. It is shown that the nitroveratryl (NV) group can be photolyzed with high priority when NV and ortho-nitrobenzyl carbonate (oNBC) are used together as the protecting groups of glycans. Notably, the photolytic products could be used directly in the subsequent glycosylation without further purification. With the above-mentioned orthogonal photolabile protecting group strategy in hand, a Mycobacterium tuberculosis tetrasaccharide and a derivative of glucosyl glycerol were rapidly prepared.

Preliminary X-ray diffraction and ligand-binding analyses of the N-terminal domain of hypothetical protein Rv1421 from Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis can reside and persist in deep tissues; latent tuberculosis can evade immune detection and has a unique mechanism to convert it into active disease through reactivation. M. tuberculosis Rv1421 (MtRv1421) is a hypothetical protein that has been proposed to be involved in nucleotide binding-related metabolism in cell-growth and cell-division processes. However, due to a lack of structural information, the detailed function of MtRv1421 remains unclear. In this study, a truncated N-terminal domain (NTD) of MtRv1421, which contains a Walker A/B-like motif, was purified and crystallized using PEG 400 as a precipitant. The crystal of MtRv1421-NTD diffracted to a resolution of 1.7 Šand was considered to belong to either the C-centered monoclinic space group C2 or the I-centered orthorhombic space group I222, with unit-cell parameters a = 124.01, b = 58.55, c = 84.87 Å, ß = 133.12° or a = 58.53, b = 84.86, c = 90.52 Å, respectively. The asymmetric units of the C2 or I222 crystals contained two or one monomers, respectively. In terms of the binding ability of MtRv1421-NTD to various ligands, uridine diphosphate (UDP) and UDP-N-acetylglucosamine significantly increased the melting temperature of MtRv1421-NTD, which indicates structural stabilization through the binding of these ligands. Altogether, the results reveal that a UDP moiety may be required for the interaction of MtRv1421-NTD as a nucleotide-binding protein with its ligand.

Evolution of fungal tuberculosis necrotizing toxin (TNT) domain-containing enzymes reveals divergent adaptations to enhance NAD cleavage.

Tuberculosis necrotizing toxin (TNT) is a protein domain discovered on the outer membrane of Mycobacterium tuberculosis (Mtb), and the fungal pathogen Aspergillus fumigatus. TNT domains have pure NAD(P) hydrolytic activity, setting them apart from other NAD-cleaving domains such as ADP-ribosyl cyclase and Toll/interleukin-1 receptor homology (TIR) domains which form a wider set of products. Importantly, the Mtb TNT domain has been shown to be involved in immune evasion via depletion of the intracellular NAD pool of macrophages. Therefore, an intriguing hypothesis is that TNT domains act as "NAD killers" in host cells facilitating pathogenesis. Here, we explore the phylogenetic distribution of TNT domains and detect their presence solely in bacteria and fungi. Within fungi, we discerned six TNT clades. In addition, X-ray crystallography and AlphaFold2 modeling unveiled clade-specific strategies to promote homodimer stabilization of the fungal enzymes, namely, Ca2+ binding, disulfide bonds, or hydrogen bonds. We show that dimer stabilization is a requirement for NADase activity and that the group-specific strategies affect the active site conformation, thereby modulating enzyme activity. Together, these findings reveal the evolutionary lineage of fungal TNT enzymes, corroborating the hypothesis of them being pure extracellular NAD (eNAD) cleavers, with possible involvement in microbial warfare and host immune evasion.

Enhancing the sequence coverage of nanodiamond-extracted early secretory proteins from the Mycobacterium tuberculosis complex.

The unambiguous identification of protein species requires high sequence coverage. In this study, we successfully improved the sequence coverage of early secretory 10 kDa cell filtrate protein (CFP-10) and 6 kDa early secretory antigenic target (ESAT-6) proteins from the Mycobacterium tuberculosis complex (MTC) in broth culture media with the use of the 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Conventional matrices, α-cyano-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), were also used for comparison. After nanodiamond (ND) extraction, the sequence coverage of the CFP-10 protein was 87% when CHCA and DHB matrices were used, and the ESAT-6 protein was not detected. On the other hand, the sequence coverage for ND-extracted CFP-10 and ESAT-6 could reach 94% and 100%, respectively, when the Cl-CCA matrix was used and with the removal of interference from bovine serum albumin (BSA) protein and α-crystallin (ACR) protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was also adopted to analyze the protein mass spectra. A total of 6 prominent ion signals were observed, including ESAT-6 protein peaks at mass-to-charge ratios (m/z) of ∼7931, ∼7974, ∼9768, and ∼9813 and CFP-10 protein peaks at m/z of ∼10 100 and ∼10 660. The ESAT-6 ion signals were always detected concurrently with CFP-10 ion signals, but CFP-10 ion signals could be detected alone without the ESAT-6 ion signals. Furthermore, the newly found ESAT-6 peaks were also confirmed using a Mag-Beads-Protein G kit with an ESAT-6 antibody to capture the ESAT-6 protein, which was also consistent with the sequence coverage analysis.

Unusual triflic acid-promoted oligomerization of arabinofuranosides during glycosylation.

We discovered an unusual triflic acid-promoted oligomerization of arabinofuranosides during glycosylation of the primary hydroxy group of α-(1 â†’ 5)-linked tetraarabinofuranoside bearing 4-(2-chloroethoxy)phenyl aglycone with α-(1 â†’ 5), ß-(1 â†’ 2)-linked tetraarabinofuranoside containing N-phenyltrifluoroacetimidoyl leaving group, which led to octa-, dodeca- and hexadecaarabinofuranosides. The possible mechanism of triflic acid-promoted oligomerization was proposed. The choice of promoter was found to be a critical factor for the discovered oligomerization of arabinofuranosides. The obtained octa-, dodeca- and hexadecaarabinofuranosides may serve as useful blocks in the synthesis of oligosaccharide fragments of polysaccharides of Mycobacterium tuberculosis.

Mechanism of Peroxynitrite Interaction with Ferric M. tuberculosis Nitrobindin: A Computational Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO-) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO- is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO- and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO- and ONOOH. Our results indicate that ONOO- and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt-Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO- exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.

Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

Asymmetric Total Synthesis and Structural Revision of DAT2, an Antigenic Glycolipid from Mycobacterium tuberculosis.

DAT2 is a member of the diacyl trehalose family (DAT) of antigenic glycolipids located in the mycomembrane of Mycobacterium tuberculosis (Mtb). Recently it was shown that the molecular structure of DAT2 had been incorrectly assigned, but the correct structure remained elusive. Herein, the correct molecular structure of DAT2 and its methyl-branched acyl substituent mycolipanolic acid is determined. For this, four different stereoisomers of mycolipanolic acid were prepared in a stereoselective and unified manner, and incorporated into DAT2. A rigorous comparison of the four isomers to the DAT isolated from Mtb H37Rv by NMR, HPLC, GC, and mass spectrometry allowed a structural revision of mycolipanolic acid and DAT2. Activation of the macrophage inducible Ca2+-dependent lectin receptor (Mincle) with all four stereoisomers shows that the natural stereochemistry of mycolipanolic acid / DAT2 provides the strongest activation, which indicates its high antigenicity and potential application in serodiagnostics and vaccine adjuvants.

Chemical remodeling of the mycomembrane with chain-truncated lipids sensitizes mycobacteria to rifampicin.

The outer mycomembrane of Mycobacterium tuberculosis and related pathogens is a robust permeability barrier that protects against antibiotic treatment. Here, we demonstrate that synthetic analogues of the mycomembrane biosynthetic precursor trehalose monomycolate bearing truncated lipid chains increase permeability of Mycobacterium smegmatis cells and sensitize them to treatment with the first-line anti-tubercular drug rifampicin. The reported strategy may be useful for enhancing entry of drugs and other molecules to mycobacterial cells, and represents a new way to study mycomembrane structure and function.

ATP-independent substrate recruitment to proteasomal degradation in mycobacteria.

Mycobacteria and other actinobacteria possess proteasomal degradation pathways in addition to the common bacterial compartmentalizing protease systems. Proteasomal degradation plays a crucial role in the survival of these bacteria in adverse environments. The mycobacterial proteasome interacts with several ring-shaped activators, including the bacterial proteasome activator (Bpa), which enables energy-independent degradation of heat shock repressor HspR. However, the mechanism of substrate selection and processing by the Bpa-proteasome complex remains unclear. In this study, we present evidence that disorder in substrates is required but not sufficient for recruitment to Bpa-mediated proteasomal degradation. We demonstrate that Bpa binds to the folded N-terminal helix-turn-helix domain of HspR, whereas the unstructured C-terminal tail of the substrate acts as a sequence-specific threading handle to promote efficient proteasomal degradation. In addition, we establish that the heat shock chaperone DnaK, which interacts with and co-regulates HspR, stabilizes HspR against Bpa-mediated proteasomal degradation. By phenotypical characterization of Mycobacterium smegmatis parent and bpa deletion mutant strains, we show that Bpa-dependent proteasomal degradation supports the survival of the bacterium under stress conditions by degrading HspR that regulates vital chaperones.

Structure of an endogenous mycobacterial MCE lipid transporter.

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.

Ion mobility mass spectrometry for the study of mycobacterial mycolic acids.

Lipids are highly structurally diverse molecules involved in a wide variety of biological processes. The involvement of lipids is even more pronounced in mycobacteria, including the human pathogen Mycobacterium tuberculosis, which produces a highly complex and diverse set of lipids in the cell envelope. These lipids include mycolic acids, which are among the longest fatty acids in nature and can contain up to 90 carbon atoms. Mycolic acids are ubiquitously found in mycobacteria and are alpha branched and beta hydroxylated lipids. Discrete modifications, such as alpha, alpha', epoxy, methoxy, keto, and carboxy, characterize mycolic acids at the species level. Here, we used high precision ion mobility-mass spectrometry to build a database including 206 mass-resolved collision cross sections (CCSs) of mycolic acids originating from the strict human pathogen M. tuberculosis, the opportunistic strains M. abscessus, M. marinum and M. avium, and the nonpathogenic strain M. smegmatis. Primary differences between the mycolic acid profiles could be observed between mycobacterial species. Acyl tail length and modifications were the primary structural descriptors determining CCS magnitude. As a resource for researchers, this work provides a detailed catalogue of the mass-resolved collision cross sections for mycolic acids along with a workflow to generate and analyse the dataset generated.

Mycobacterial type VII secretion systems.

Mycobacteria, such as the pathogen M. tuberculosis, utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates' co-dependencies for transport allow for the first time more detailed and testable models for secretion.

Immune Targeting of Mycobacteria through Cell Surface Glycan Engineering.

Mycobacteria and other organisms in the order Mycobacteriales cause a range of significant human diseases, including tuberculosis, leprosy, diphtheria, Buruli ulcer, and non-tuberculous mycobacterial (NTM) disease. However, the intrinsic drug tolerance engendered by the mycobacterial cell envelope undermines conventional antibiotic treatment and contributes to acquired drug resistance. Motivated by the need to augment antibiotics with novel therapeutic approaches, we developed a strategy to specifically decorate mycobacterial cell surface glycans with antibody-recruiting molecules (ARMs), which flag bacteria for binding to human-endogenous antibodies that enhance macrophage effector functions. Mycobacterium-specific ARMs consisting of a trehalose targeting moiety and a dinitrophenyl hapten (Tre-DNPs) were synthesized and shown to specifically incorporate into outer-membrane glycolipids of Mycobacterium smegmatis via trehalose metabolism, enabling recruitment of anti-DNP antibodies to the mycobacterial cell surface. Phagocytosis of Tre-DNP-modified M. smegmatis by macrophages was significantly enhanced in the presence of anti-DNP antibodies, demonstrating proof-of-concept that our strategy can augment the host immune response. Because the metabolic pathways responsible for cell surface incorporation of Tre-DNPs are conserved in all Mycobacteriales organisms but absent from other bacteria and humans, the reported tools may be enlisted to interrogate host-pathogen interactions and develop immune-targeting strategies for diverse mycobacterial pathogens.

Natural Metabolite Ursolic Acid as an Inhibitor of Dormancy Regulator DosR of Mycobacterium tuberculosis: Evidence from Molecular Docking, Molecular Dynamics Simulation and Free Energy Analysis.

BACKGROUND: DosR is a transcriptional regulator of Mycobacterium tuberculosis (MTB), governing the expression of a set of nearly 50 genes that is often referred to as 'dormancy regulon'. The inhibition of DosR expression by an appropriate inhibitor may be a crucial step against MTB. OBJECTIVE: We targeted the DosR with natural metabolites, ursolic acid (UA) and carvacrol (CV), using in silico approaches. METHODS: The molecular docking, molecular dynamics (MD) simulation for 200 ns, calculation of binding energies by MM-GBSA method, and ADMET calculation were performed to evaluate the inhibitory potential of natural metabolites ursolic acid (UA) and carvacrol (CV) against DosR of MTB. RESULTS: Our study demonstrated that UA displayed significant compatibility with DosR during the 200 ns timeframe of MD simulation. The thermodynamic binding energies by MM-GBSA also suggested UA conformational stability within the binding pocket. The SwissADME, pkCSM, and OSIRIS DataWarrior showed a drug-likeness profile of UA, where Lipinski profile was satisfied with one violation (MogP > 4.15) with no toxicities, no mutagenicity, no reproductive effect, and no irritant nature. CONCLUSION: The present study suggests that UA has the potency to inhibit the DosR expression and warrants further investigation on harnessing its clinical potential.

Automated Glycan Assembly of Mycobacterial Hexaarabinofuranoside and Docosasaccharide Arabinan (Araf23 ) Motifs found on Mycobacterium tuberculosis.

Mycobacteria are covered in a thick layer of different polysaccharides that helps to avert the innate immune response. Lipoarabinomannan (LAM) and arabinogalactan (AG) are ubiquitously contained in these envelopes, and rapid access to defined oligo- and polysaccharides is essential to elucidate their structural and biological roles. Arabinofuranose (Araf) residues in LAM and AG are connected either via α-1,2-trans linkages that are synthetically straightforward to install or the more challenging ß-(1,2-cis) linkages. Herein, it was demonstrated that automated glycan assembly (AGA) can be used to quickly prepare 1,2-cis-ß-Araf as illustrated by the assembly of a highly branched arabinan hexasaccharide and a docosasaccharide arabinan (Araf23 ) motif.