Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis.

BACKGROUND: Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure-function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis. RESULTS: We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. CONCLUSIONS: The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.

Effect of Mycolic Acids on Host Immunity and Lipid Metabolism.

Mycolic acids constitute pivotal constituents within the cell wall structure of Mycobacterium tuberculosis. Due to their structural diversity, the composition of mycolic acids exhibits substantial variations among different strains, endowing them with the distinctive label of being the 'signature' feature of mycobacterial species. Within Mycobacterium tuberculosis, the primary classes of mycolic acids include α-, keto-, and methoxy-mycolic acids. While these mycolic acids are predominantly esterified to the cell wall components (such as arabinogalactan, alginate, or glucose) of Mycobacterium tuberculosis, a fraction of free mycolic acids are secreted during in vitro growth of the bacterium. Remarkably, different types of mycolic acids possess varying capabilities to induce foamy macro-phages and trigger immune responses. Additionally, mycolic acids play a regulatory role in the lipid metabolism of host cells, thereby exerting influence over the progression of tuberculosis. Consequently, the multifaceted properties of mycolic acids shape the immune evasion strategy employed by Mycobacterium tuberculosis. A comprehensive understanding of mycolic acids is of paramount significance in the pursuit of developing tuberculosis therapeutics and unraveling the intricacies of its pathogenic mechanisms.

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 stable home for an equine pathogen: valid publication of the binomial Prescottella equi gen. nov., comb. nov., and reclassification of four rhodococcal species into the genus Prescottella.

Opinion 106 of the Judicial Commission has clarified the nomenclature of the taxon variously named Rhodococcus equi, 'Prescottella equi' and Rhodococcus hoagii. As a consequence, we present here the genus name Prescottella and that of its nomenclatural type species, Prescottella equi comb. nov., for valid publication and propose the reclassification of four rhodococcal species as novel combinations in the genus, namely Prescottella agglutinans Guo et al. 2015 comb. nov., Prescottella defluvii Kämpfer et al. 2014 comb. nov., Prescottella soli Li et al. 2015 comb. nov. and Prescottella subtropica Lee et al. 2019 comb. nov. In addition, we note that a clinical isolate, strain 86-07 (=W8901), likely represents an additional species within the genus Prescottella. Nearly a century after the original description of the type strain of the type species as Corynebacterium equi, we provide a stable home for Prescottella equi and its relatives.

Synthesis and characterisation of quantum dots coupled to mycolic acids as a water-soluble fluorescent probe for potential lateral flow detection of antibodies and diagnosis of tuberculosis.

This work explores the potential use of cadmium-based quantum dots (QDs) coupled to mycolic acids (MAs) as a fluorescent probe to detect anti-MA antibodies which are biomarkers for tuberculosis (TB). The use of free MAs as antigens for the serodiagnosis of TB is known but has not been developed into a point of care test. This study focuses on the synthesis, solubility, and lateral flow of QDs coupled to MAs. Water-soluble CdSe/ZnS QDs capped with l-cysteine were synthesised and covalently coupled to MAs via amide linkages to form a water-soluble fluorescent probe: MA-CdSe/ZnS QDs. The MA-CdSe/ZnS QDs showed broad absorption bands and coupling, confirmed by the presence of amide bonds in the Fourier-transform infrared (FTIR) spectrum, resulting in a blue shift in fluorescence. Powder X-ray diffraction (XRD) revealed a shift and increase in the number of peaks for MA-CdSe/ZnS QDs relative to the L-cys-CdSe/ZnS QDs, suggesting that coupling changed the crystal structure. The average particle size of MA-CdSe/ZnS QDs was ~3.0 nm. Visual paper-based lateral flow of MA-CdSe/ZnS QDs was achieved on strips of nitrocellulose membrane with both water and membrane blocking solution eluents. The highly fluorescent MA-CdSe/ZnS QDs showed good water solubility and lateral flow, which are important properties for fluorescence sensing applications.

Interactions between the Re-Emerging Pathogen Corynebacterium diphtheriae and Host Cells.

Corynebacterium diphtheriae, the etiological agent of diphtheria, is a re-emerging pathogen, responsible for several thousand deaths per year. In addition to diphtheria, systemic infections, often by non-toxigenic strains, are increasingly observed. This indicates that besides the well-studied and highly potent diphtheria toxin, various other virulence factors may influence the progression of the infection. This review focuses on the known components of C. diphtheriae responsible for adhesion, invasion, inflammation, and cell death, as well as on the cellular signaling pathways activated upon infection.

Biological and Biochemical Evaluation of Isatin-Isoniazid Hybrids as Bactericidal Candidates against Mycobacterium tuberculosis.

Tuberculosis remains a leading cause of mortality among infectious diseases worldwide, prompting the need to discover new drugs to fight this disease. We report here the design, synthesis, and antimycobacterial activity of isatin-mono/bis-isoniazid hybrids. Most of the compounds exhibited very high activity against Mycobacterium tuberculosis, with MICs in the range of 0.195 to 0.39 µg/ml, and exerted a more potent bactericidal effect than the standard antitubercular drug isoniazid (INH). Importantly, these compounds were found to be well tolerated at high doses (>200 µg/ml) on Vero kidney cells, leading to high selectivity indices. Two of the most promising hybrids were evaluated for activity in THP-1 macrophages infected with M. tuberculosis, among which compound 11e was found to be slightly more effective than INH. Overexpression of InhA along with cross-resistance determination of the most potent compounds, selection of resistant mutants, and biochemical analysis, allowed us to decipher their mode of action. These compounds effectively inhibited mycolic acid biosynthesis and required KatG to exert their biological effects. Collectively, this suggests that the synthesized isatin-INH hybrids are promising antitubercular molecules for further evaluation in preclinical settings.

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

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.

Mycobacterial Epoxide Hydrolase EphD Is Inhibited by Urea and Thiourea Derivatives.

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.

The crystal structure of the mycobacterial trehalose monomycolate transport factor A, TtfA, reveals an atypical fold.

Trehalose monomycolate (TMM) represents an essential element of the mycobacterial envelope. While synthesized in the cytoplasm, TMM is transported across the inner membrane by MmpL3 but, little is known regarding the MmpL3 partners involved in this process. Recently, the TMM transport factor A (TtfA) was found to form a complex with MmpL3 and to participate in TMM transport, although its biological role remains to be established. Herein, we report the crystal structure of the Mycobacterium smegmatis TtfA core domain. The phylogenetic distribution of TtfA homologues in non-mycolate containing bacteria suggests that TtfA may exert additional functions.

Lack of Specificity of Phenotypic Screens for Inhibitors of the Mycobacterium tuberculosis FAS-II System.

Phenotypic screening of inhibitors of the essential Mycobacterium tuberculosis FAS-II dehydratase HadAB led to the identification of GSK3011724A, a compound previously reported to inhibit the condensation step of FAS-II. Whole-cell-based and cell-free assays confirmed the lack of activity of GSK3011724A against the dehydratase despite evidence of cross-resistance between GSK3011724A and HadAB inhibitors. The nature of the resistance mechanisms is suggestive of alterations in the FAS-II interactome reducing access of GSK3011724A to KasA.

Assembly of the Mycobacterial Cell Wall.

Mycobacterium tuberculosis remains one of the most successful bacterial pathogens, claiming over 1.3 million lives worldwide in 2013. The emergence of multidrug-resistant and extensively drug-resistant isolates has prompted the need for new drugs and drug targets. M. tuberculosis possesses an unusual cell wall dominated by lipids and carbohydrates that provides a permeability barrier against hydrophilic drugs and is crucial for its survival and virulence. This large macromolecular structure, termed the mycolyl-arabinogalactan-peptidoglycan complex, and the phosphatidyl-myo-inositol-based lipoglycans are key features of the mycobacterial cell wall. Assembly of these cell wall components is an attractive target for the development of chemotherapeutics against tuberculosis. Herein, we focus on recent biochemical and molecular insights into these complex molecules of M. tuberculosis cell wall.

Enhancing the bioconversion of phytosterols to steroidal intermediates by the deficiency of kasB in the cell wall synthesis of Mycobacterium neoaurum.

BACKGROUND: The bioconversion of phytosterols into high value-added steroidal intermediates, including the 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall in M. neoaurum strain strikingly enhanced the cell permeability for the high production of steroids. RESULTS: The deletion of the nonessential kasB, encoding a ß-ketoacyl-acyl carrier protein synthase, led to a disturbed proportion of mycolic acids (MAs), which is one of the most important components in the cell wall of Mycobacterium neoaurum ATCC 25795. The determination of cell permeability displayed about two times improvement in the kasB-deficient strain than that of the wild type M. neoaurum. Thus, the deficiency of kasB in the 9-OHAD-producing strain resulted in a significant increase of 137.7% in the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). Ultimately, the 9-OHAD productivity in an industrial used resting cell system was reached 0.1135 g/L/h (10.9 g/L 9-OHAD from 20 g/L phytosterol) and the conversion time was shortened by 33%. In addition, a similar self-enhancement effect (34.5%) was realized in the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain. CONCLUSIONS: The modification of kasB resulted in a meaningful change in the cell wall mycolic acids. Deletion of the kasB gene remarkably improved the cell permeability, leading to a self-enhancement of the steroidal intermediate conversion. The results showed a high efficiency and feasibility of this construction strategy.

Application of Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID) to Total Lipid Quantitation in Mycolic-Acid Synthesizing Rhodococcus and Williamsia Species.

In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis.

Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria.

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl ß-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.

Synthesis of a Di-Mycoloyl Tri-Arabinofuranosyl Glycerol Fragment of the Mycobacterial Cell Wall, Based on Synthetic Mycolic Acids.

Fragments of mycobacterial cell walls such as arabinoglycerol mycolate and dimycoloyl diarabinoglycerol, comprising complex mixtures of mycolic acids, have immunostimulatory and antigenic properties. A related di-mycoloyl tri-arabinofuranosyl glycerol fragment has been isolated from cell wall hydrolysates. An effective stereoselective synthesis of tri-arabinofuranosyl glycerol, followed by coupling with stereochemically defined mycolic acids of different structural classes, to provide unique di-mycoloyl tri-arabinofuranosyl glycerols is now described.

Deletion of MSMEG_1350 in Mycobacterium smegmatis causes loss of epoxy-mycolic acids, fitness alteration at low temperature and resistance to a set of mycobacteriophages.

Mycobacterium smegmatis is intrinsically resistant to thiacetazone, an anti-tubercular thiourea; however we report here that it causes a mild inhibition in growth in liquid medium. Since mycolic acid biosynthesis was affected, we cloned and expressed Mycobacterium smegmatis mycolic acid methyltransferases, postulated as targets for thiacetazone in other mycobacterial species. During this analysis we identified MSMEG_1350 as the methyltransferase involved in epoxy mycolic acid synthesis since its deletion led to their total loss. Phenotypic characterization of the mutant strain showed colony morphology alterations at all temperatures, reduced growth and a slightly increased susceptibility to SDS, lipophilic and large hydrophilic drugs at 20 °C with little effect at 37 °C. No changes were detected between parental and mutant strains in biofilm formation, sliding motility or sedimentation rate. Intriguingly, we found that several mycobacteriophages severely decreased their ability to form plaques in the mutant strain. Taken together our results prove that, in spite of being a minor component of the mycolic acid pool, epoxy-mycolates are required for a proper assembly and functioning of the cell envelope. Further studies are warranted to decipher the role of epoxy-mycolates in the M. smegmatis cell envelope.

Structural and mechanistic comparison of the Cyclopropane Mycolic Acid Synthases (CMAS) protein family of Mycobacterium tuberculosis.

Tuberculosis (TB) is a chronic disease caused by the bacillus Mycobacterium tuberculosis(Mtb) and remains a leading cause of mortality worldwide. The bacteria has an external wall which protects it from being killed, and the enzymes involved in the biosynthesis of the cell wall components have been proposed as promising targets for future drug development efforts. Cyclopropane Mycolic Acid Synthases (CMAS) constitute a group of ten homologous enzymes which belong to the mycolic acid biosynthesis pathway. These enzymes have S-adenosyl-l-methionine (SAM) dependent methyltransferase activity with a peculiarity, each one of them has strong substrate selectivity and reaction specificity, being able to produce among other things cyclopropanes or methyl-alcohol groups from the lipid olefin group. How each CMAS processes its substrate and how the specificity and selectivity are encoded in the protein sequence and structure, is still unclear. In this work, by using a combination of modeling tools, including comparative modeling, docking, all-atom MD and QM/MM methodologies we studied in detail the reaction mechanism of cmaA2, mmaA4, and mmaA1 CMAS and described the molecular determinants that lead to different products. We have modeled the protein-substrate complex structure and determined the free energy pathway for the reaction. The combination of modeling tools at different levels of complexity allows having a complete picture of the CMAS structure-activity relationship.

Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is recognized as a global health emergency as promoted by the World Health Organization. Over 1 million deaths per year, along with the emergence of multi- and extensively-drug resistant strains of Mtb, have triggered intensive research into the pathogenicity and biochemistry of this microorganism, guiding the development of anti-TB chemotherapeutic agents. The essential mycobacterial cell wall, sharing some common features with all bacteria, represents an apparent 'Achilles heel' that has been targeted by TB chemotherapy since the advent of TB treatment. This complex structure composed of three distinct layers, peptidoglycan, arabinogalactan and mycolic acids, is vital in supporting cell growth, virulence and providing a barrier to antibiotics. The fundamental nature of cell wall synthesis and assembly has rendered the mycobacterial cell wall as the most widely exploited target of anti-TB drugs. This review provides an overview of the biosynthesis of the prominent cell wall components, highlighting the inhibitory mechanisms of existing clinical drugs and illustrating the potential of other unexploited enzymes as future drug targets.

Synergistic Interactions of MmpL3 Inhibitors with Antitubercular Compounds In Vitro.

A number of inhibitors of the essential Mycobacterium tuberculosis mycolic acid transporter, MmpL3, are currently under development as potential novel antituberculosis agents. Using the checkerboard method to study the interaction profiles of various antituberculosis drugs or experimental compounds with two different chemotypes inhibiting this transporter (indolcarboxamides and adamantyl ureas), we showed that MmpL3 inhibitors act synergistically with rifampin, bedaquiline, clofazimine, and ß-lactams.