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.

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.

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.

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.

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.

Conformational Dynamics and Stability of Bilayers Formed by Mycolic Acids from the Mycobacterium tuberculosis Outer Membrane.

Bilayers of mycolic acids (MAs) form the outer membrane of Mycobacterium tuberculosis that has high strength and extremely low permeability for external molecules (including antibiotics). For the first time, we were able to study them using the all-atom long-term molecular dynamic simulations (from 300 ns up to 1.2 µs) in order to investigate the conformational changes and most favorable structures of the mycobacterial membranes. The structure and properties of the membranes are crucially dependent on the initial packing of the α-mycolic acid (AMA) molecules, as well as on the presence of the secondary membrane components, keto- and methoxy mycolic acids (KMAs and MMAs). In the case of AMA-based membranes, the most labile conformation is W while other types of conformations (sU as well as sZ, eU, and eZ) are much more stable. In the multicomponent membranes, the presence of the KMA and MMA components (in the W conformation) additionally stabilizes both the W and eU conformations of AMA. The membrane in which AMA prevails in the eU conformation is much thicker and, at the same time, much denser. Such a packing of the MA molecules promotes the formation of a significantly stronger outer mycobacterial membrane that should be much more resistant to the threatening external factors.

A Metabolic-Tag-Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria.

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.

Structure-based drug design of novel M. tuberculosis InhA inhibitors based on fragment molecular orbital calculations.

2-trans enoyl-acyl carrier protein reductase (InhA) is a promising target for developing novel chemotherapy agents for tuberculosis, and their inhibitory effects on InhA activity were widely investigated by the physicochemical experiments. However, the reason for the wide range of their inhibitory effects induced by similar agents was not explained by only the difference in their chemical structures. In our previous molecular simulations, a series of heteroaryl benzamide derivatives were selected as candidate inhibitors against InhA, and their binding properties with InhA were investigated to propose novel derivatives with higher binding affinity to InhA. In the present study, we extended the simulations for a series of 4-hydroxy-2-pyridone derivatives to search widely for more potent inhibitors against InhA. Using ab initio fragment molecular orbital (FMO) calculations, we elucidated the specific interactions between InhA residues and the derivatives at an electronic level and highlighted key interactions between InhA and the derivatives. The FMO results clearly indicated that the most potent inhibitor has strong hydrogen bonds with the backbones of Tyr158, Thr196, and NADH of InhA. This finding may provide informative structural concepts for designing novel 4-hydroxy-2-pyridone derivatives with higher binding affinity to InhA. Our previous and present molecular simulations could provide important guidelines for the rational design of more potent InhA inhibitors.

Tunable Strategy for the Asymmetric Synthesis of Sulfoglycolipids from Mycobacterium tuberculosis To Elucidate the Structure and Immunomodulatory Property Relationships.

We developed a versatile asymmetric strategy to synthesize different classes of sulfoglycolipids (SGLs) from Mycobacterium tuberculosis. The strategy features the use of asymmetrically protected trehaloses, which were acquired from the glycosylation of TMS α-glucosyl acceptors with benzylidene-protected thioglucosyl donors. The positions of the protecting groups at the donors and acceptors can be fine-tuned to obtain different protecting-group patterns, which is crucial for regioselective acylation and sulfation. In addition, a chemoenzymatic strategy was established to prepare the polymethylated fatty acid building blocks. The strategy employs inexpensive lipase as a desymmetrization agent in the preparation of the starting substrate and readily available chiral oxazolidinone as a chirality-controlling agent in the construction of the polymethylated fatty acids. A subsequent investigation on the immunomodulatory properties of each class of SGLs showed how the structures of SGLs impact the host innate immunity response.

Synthesis of the Mycobacterium tuberculosis Canetti Lipooligosaccharide II Nonasaccharide.

A route for preparing lipooligosaccharide (LOS) glycans from Mycobacterium tuberculosis Canetti was developed and applied to the most complex of these structures, LOS II. The synthesis of the target nonasaccharide was achieved via a convergent [3+3+3] approach. Key features of the strategy include the stereoselective synthesis of an asymmetrically substituted trehalose moiety from two protected glucose residues and several chemoselective glycosylations involving thioglycoside donors.

The essential M. tuberculosis Clp protease is functionally asymmetric in vivo.

The Clp protease system is a promising, noncanonical drug target against Mycobacterium tuberculosis (Mtb). Unlike in Escherichia coli, the Mtb Clp protease consists of two distinct proteolytic subunits, ClpP1 and ClpP2, which hydrolyze substrates delivered by the chaperones ClpX and ClpC1. While biochemical approaches uncovered unique aspects of Mtb Clp enzymology, its essentiality complicates in vivo studies. To address this gap, we leveraged new genetic tools to mechanistically interrogate the in vivo essentiality of the Mtb Clp protease. While validating some aspects of the biochemical model, we unexpectedly found that only the proteolytic activity of ClpP1, but not of ClpP2, is essential for substrate degradation and Mtb growth and infection. Our observations not only support a revised model of Mtb Clp biology, where ClpP2 scaffolds chaperone binding while ClpP1 provides the essential proteolytic activity of the complex; they also have important implications for the ongoing development of inhibitors toward this emerging therapeutic target.

A modular and reconfigurable open-channel gated device for the electrokinetic extraction of cell-free DNA assays.

The high-efficiency separation and extraction of short fragments of cell-free DNA (cfDNA) remain challenging due to their low abundance and short lengths. This study presents a method for separating short cfDNA fragments, with lengths ranging from about 100 to 200 base pairs, from liquid human plasma samples into separable and extractable bands as solid agarose gel slabs. To achieve this, a novel millimeter-scale fluidic device is used for sample handling, transient isotachophoresis, and extraction. The device features open-to-atmosphere liquid chambers that define and manually actuated (i.e., movable) agarose-made gate valve structures. The agarose gates then define discrete zones for buffers, sample injection, DNA pre-concentration via isotachophoresis, size-based gel separation, and DNA-band extraction. As a demonstration of its efficacy, the device is applied to the enrichment and purification of M. tuberculosis genomic DNA fragments spiked in human plasma samples. This purified cfDNA is analyzed using the quantitative polymerase chain reaction (qPCR) of the IS6110 repetitive sequence in the M. tuberculosis genome. The data from this study demonstrates that high sensitivity can be achieved in cfDNA detection, as shown by the comparison with a typical solid-phase extraction method and buffer spiked with cfDNA. Evidence is presented that suggests plasma peptides generated by treatment of the sample with proteinase K acts as endogenous spacer molecules, which improve the resolution and purification of DNA relative to the marker dye and other contaminants that decrease the signal level in qPCR.

Tailor made: New insights into lipoarabinomannan structure may improve TB diagnosis.

Detecting the mycobacterial glycolipid lipoarabinomannan (LAM) in urine by anti-LAM antibodies fills a gap in the diagnostic armamentarium of much needed simple rapid tests for tuberculosis, but lacks high sensitivity in all patient groups. A better understanding of LAM structure from clinically relevant strains may allow improvements in diagnostic performance. De et al. have recently determined the structures of LAM from three epidemiologically important lineages of Mycobacterium tuberculosis and probed their interaction with an anti-LAM monoclonal antibody. Their results not only identify a series of tailoring modifications that impact antibody binding but also provide a roadmap for improving U-LAM-based diagnostics.

Determination of tuberculosis-related volatile organic biomarker methyl nicotinate in vapor using fluorescent assay based on quantum dots and cobalt-containing porphyrin nanosheets.

Methyl nicotinate (MN) is a representative and typical volatile organic marker of Mycobacterium tuberculosis, and the specific detection of MN in human breath facilitates non-invasive, rapid, and accurate epidemic screening of tuberculosis infection. Herein, we constructed a fluorescent assay consisted of CdTe quantum dots (QD) and cobalt-metalized tetrakis(4-carboxyphenyl) porphyrin (CoTCPP) nanosheets to determine methyl nicotinate (MN) in vapor samples. Red-emission QD (λex=370 nm, λem=658 nm) acts as signal switches whose fluorescence signals can be effectively quenched by CoTCPP nanosheets but restored in the presence of MN. The strategy relied on the distinct binding affinity of cobalt ion and MN. MN restored the fluorescence of QD quenched by CoTCPP in a concentration-dependent manner, which exhibited a well-linear relationship in the range 1-100 µM, and a limit of detection of 0.59 µM. The proposed platform showed sensitivity and selectivity to detect MN in vapor samples with satisfactory RSD below 3.33%. The method is cheap, simple, and relatively rapid (detected within 4 min), which suggests a potential in tuberculosis diagnosis in resource- and professional-lacked areas.

Crystal structure of the cytokinin-producing enzyme "lonely guy" (LOG) from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is an extremely successful intracellular pathogen that cause a large number of death worldwide. It is interesting that this non-phytopathogen can synthesize cytokinin by "lonely guy" (LOG) protein. The cytokinin biosynthesis pathway in Mtb is not clear. Here we determined the crystal structure of LOG from Mtb (MtLOG) at a high resolution of 1.8 Å. MtLOG exists as dimer which belongs to type-I LOG and shows a typical α-ß Rossmann fold. Like other LOGs, MtLOG also contains a conserved "PGGXGTXXE" motif that contributes to the formation of an active site. For the first time, we found that the MtLOG binds to Mg2+ in the negative potential pocket. According to the docking result, we found that Arg78, Arg98 and Tyr162 should be the key amino acid involved in substrate binding. Our findings provide a structural basis for cytokinin study in Mtb and will play an important role in design and development of enzyme inhibitors.

The Structural Basis of Mycobacterium tuberculosis RpoB Drug-Resistant Clinical Mutations on Rifampicin Drug Binding.

Tuberculosis (TB), caused by the Mycobacterium tuberculosis infection, continues to be a leading cause of morbidity and mortality in developing countries. Resistance to the first-line anti-TB drugs, isoniazid (INH) and rifampicin (RIF), is a major drawback to effective TB treatment. Genetic mutations in the ß-subunit of the DNA-directed RNA polymerase (rpoB) are reported to be a major reason of RIF resistance. However, the structural basis and mechanisms of these resistant mutations are insufficiently understood. In the present study, thirty drug-resistant mutants of rpoB were initially modeled and screened against RIF via a comparative molecular docking analysis with the wild-type (WT) model. These analyses prioritized six mutants (Asp441Val, Ser456Trp, Ser456Gln, Arg454Gln, His451Gly, and His451Pro) that showed adverse binding affinities, molecular interactions, and RIF binding hinderance properties, with respect to the WT. These mutant models were subsequently analyzed by molecular dynamics (MD) simulations. One-hundred nanosecond all-atom MD simulations, binding free energy calculations, and a dynamic residue network analysis (DRN) were employed to exhaustively assess the impact of mutations on RIF binding dynamics. Considering the global structural motions and protein-ligand binding affinities, the Asp441Val, Ser456Gln, and His454Pro mutations generally yielded detrimental effects on RIF binding. Locally, we found that the electrostatic contributions to binding, particularly by Arg454 and Glu487, might be adjusted to counteract resistance. The DRN analysis revealed that all mutations mostly distorted the communication values of the critical hubs and may, therefore, confer conformational changes in rpoB to perturb RIF binding. In principle, the approach combined fundamental molecular modeling tools for robust "global" and "local" level analyses of structural dynamics, making it well suited for investigating other similar drug resistance cases.

Discovery of Mycobacterium tuberculosis Rv3364c-Derived Small Molecules as Potential Therapeutic Agents to Target SNX9 for Sepsis.

The serine protease inhibitor Rv3364c of Mycobacterium tuberculosis (MTB) is highly expressed in cells during MTB exposure. In this study, we showed that the 12WLVSKF17 motif of Rv3364c interacts with the BAR domain of SNX9 and inhibits endosome trafficking to interact with p47phox, thereby suppressing TLR4 inflammatory signaling in macrophages. Derived from the structure of this Rv3364c peptide motif, 2,4-diamino-6-(4-tert-butylphenyl)-1,3,5-trazine, DATPT as a 12WLVSKF17 peptide-mimetic small molecule has been identified. DATPT can block the SNX9-p47phox interaction in the endosome and suppress reactive oxygen species and inflammatory cytokine production; it demonstrated significant therapeutic effects in a mouse model of cecal ligation and puncture-induced sepsis. DATPT has considerably improved potency, with an IC50 500-fold (in vitro) or 2000-fold (in vivo) lower than that of the 12WLVSKF17 peptide. Furthermore, DATPT shows potent antibacterial activities by reduction in ATP production and leakage of intracellular ATP out of bacteria. These results provide evidence for peptide-derived small molecule DATPT with anti-inflammatory and antibacterial functions for the treatment of sepsis.