Therapeutic efficacy of antimalarial drugs targeting DosRS signaling in Mycobacterium abscessus.

A search for alternative Mycobacterium abscessus treatments led to our interest in the two-component regulator DosRS, which, in Mycobacterium tuberculosis, is required for the bacterium to establish a state of nonreplicating, drug-tolerant persistence in response to a variety of host stresses. We show here that the genetic disruption of dosRS impairs the adaptation of M. abscessus to hypoxia, resulting in decreased bacterial survival after oxygen depletion, reduced tolerance to a number of antibiotics in vitro and in vivo, and the inhibition of biofilm formation. We determined that three antimalarial drugs or drug candidates, artemisinin, OZ277, and OZ439, can target DosS-mediated hypoxic signaling in M. abscessus and recapitulate the phenotypic effects of genetically disrupting dosS. OZ439 displayed bactericidal activity comparable to standard-of-care antibiotics in chronically infected mice, in addition to potentiating the activity of antibiotics used in combination. The identification of antimalarial drugs as potent inhibitors and adjunct inhibitors of M. abscessus in vivo offers repurposing opportunities that could have an immediate impact in the clinic.

Exploring the chemical space of 1,2,3-triazolyl triclosan analogs for discovery of new antileishmanial chemotherapeutic agents.

Triclosan and isoniazid are known antitubercular compounds that have proven to be also active against Leishmania parasites. On these grounds, a collection of 37 diverse 1,2,3-triazoles based on the antitubercular molecules triclosan and 5-octyl-2-phenoxyphenol (8PP) were designed in search of novel structures with leishmanicidal activity and prepared using different alkynes and azides. The 37 compounds were assayed against Leishmania donovani, the etiological agent of leishmaniasis, yielding some analogs with activity at micromolar concentrations and against M. tuberculosis H37Rv resulting in scarce active compounds with an MIC of 20 µM. To study the mechanism of action of these catechols, we analyzed the inhibition activity of the library on the M. tuberculosis enoyl-ACP reductase (ENR) InhA, obtaining poor inhibition of the enzyme. The cytotoxicity against Vero cells was also tested, resulting in none of the compounds being cytotoxic at concentrations of up to 20 µM. Derivative 5f could be considered a valuable starting point for future antileishmanial drug development. The validation of a putative leishmanial InhA orthologue as a therapeutic target needs to be further investigated.

Polysaccharide Succinylation Enhances the Intracellular Survival of Mycobacterium abscessus.

Lipoarabinomannan (LAM) and its biosynthetic precursors, phosphatidylinositol mannosides (PIMs) and lipomannan (LM) play important roles in the interactions of Mycobacterium tuberculosis with phagocytic cells and the modulation of the host immune response, but nothing is currently known of the impact of these cell envelope glycoconjugates on the physiology and pathogenicity of nontuberculous mycobacteria. We here report on the structures of Mycobacterium abscessus PIM, LM, and LAM. Intriguingly, these structures differ from those reported previously in other mycobacterial species in several respects, including the presence of a methyl substituent on one of the mannosyl residues of PIMs as well as the PIM anchor of LM and LAM, the size and branching pattern of the mannan backbone of LM and LAM, and the modification of the arabinan domain of LAM with both succinyl and acetyl substituents. Investigations into the biological significance of some of these structural oddities point to the important role of polysaccharide succinylation on the ability of M. abscessus to enter and survive inside human macrophages and epithelial cells and validate for the first time cell envelope polysaccharides as important modulators of the virulence of this emerging pathogen.

Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen.

Mechanisms of Resistance Associated with the Inhibition of the Dehydration Step of Type II Fatty Acid Synthase in Mycobacterium tuberculosis.

Isoxyl (ISO) and thiacetazone (TAC) are two antitubercular prodrugs that abolish mycolic acid biosynthesis and kill Mycobacterium tuberculosis (Mtb) through the inhibition of the essential type II fatty acid synthase (FAS-II) dehydratase HadAB. While mutations preventing ISO and TAC either from being converted to their active form or from covalently modifying their target are the most frequent spontaneous mutations associated with high-level resistance to both drugs, the molecular mechanisms underlying the high-level ISO and TAC resistance of Mtb strains harboring missense mutations in the second, nonessential, FAS-II dehydratase HadBC have remained unexplained. Using a combination of genetic, biochemical, and biophysical approaches and molecular dynamics simulation, we here show that all four reported resistance mutations in the HadC subunit of HadBC alter the stability and/or specific activity of the enzyme, allowing it in two cases (HadBCV85I and HadBCK157R) to compensate for a deficiency in HadAB in whole Mtb bacilli. The analysis of the mycolic acid profiles of Mtb strains expressing the mutated forms of HadC further points to alterations in the activity of the mycolic acid biosynthetic complex and suggests an additional contributing resistance mechanism whereby HadC mutations may reduce the accessibility of HadAB to ISO and TAC. Collectively, our results highlight the importance of developing optimized inhibitors of the dehydration step of FAS-II capable of inhibiting both dehydratases simultaneously, a goal that may be achievable given the structural resemblance of the two enzymes and their reliance on the same catalytic subunit HadB.

The MmpL3 interactome reveals a complex crosstalk between cell envelope biosynthesis and cell elongation and division in mycobacteria.

Integral membrane transporters of the Mycobacterial Membrane Protein Large (MmpL) family and their interactome play important roles in the synthesis and export of mycobacterial outer membrane lipids. Despite the current interest in the mycolic acid transporter, MmpL3, from the perspective of drug discovery, the nature and biological significance of its interactome remain largely unknown. We here report on a genome-wide screening by two-hybrid system for MmpL3 binding partners. While a surprisingly low number of proteins involved in mycolic acid biosynthesis was found to interact with MmpL3, numerous enzymes and transporters participating in the biogenesis of peptidoglycan, arabinogalactan and lipoglycans, and the cell division regulatory protein, CrgA, were identified among the hits. Surface plasmon resonance and co-immunoprecipitation independently confirmed physical interactions for three proteins in vitro and/or in vivo. Results are in line with the focal localization of MmpL3 at the poles and septum of actively-growing bacilli where the synthesis of all major constituents of the cell wall core are known to occur, and are further suggestive of a role for MmpL3 in the coordination of new cell wall deposition during cell septation and elongation. This novel aspect of the physiology of MmpL3 may contribute to the extreme vulnerability and high therapeutic potential of this transporter.

Disruption of the SucT acyltransferase in Mycobacterium smegmatis abrogates succinylation of cell envelope polysaccharides.

Similar to other prokaryotes, mycobacteria decorate their major cell envelope glycans with minor covalent substituents whose biological significance remains largely unknown. We report on the discovery of a mycobacterial enzyme, named here SucT, that adds succinyl groups to the arabinan domains of both arabinogalactan (AG) and lipoarabinomannan (LAM). Disruption of the SucT-encoding gene in Mycobacterium smegmatis abolished AG and LAM succinylation and altered the hydrophobicity and rigidity of the cell envelope of the bacilli without significantly altering AG and LAM biosynthesis. The changes in the cell surface properties of the mutant were consistent with earlier reports of transposon mutants of the closely related species Mycobacterium marinum and Mycobacterium avium harboring insertions in the orthologous gene whose ability to microaggregate and form biofilms were altered. Our findings point to an important role of SucT-mediated AG and LAM succinylation in modulating the cell surface properties of mycobacteria.

Complete auxotrophy for unsaturated fatty acids requires deletion of two sets of genes in Mycobacterium smegmatis.

The synthesis of unsaturated fatty acids in Mycobacterium smegmatis is poorly characterized. Bioinformatic analysis revealed four putative fatty acid desaturases in its genome, one of which, MSMEG_1886, is highly homologous to desA3, the only palmitoyl/stearoyl desaturase present in the Mycobacterium tuberculosis genome. A MSMEG_1886 deletion mutant was partially auxotrophic for oleic acid and viable at 37°C and 25°C, although with a long lag phase in liquid medium. Fatty acid analysis suggested that MSMEG_1886 is a palmitoyl/stearoyl desaturase, as the synthesis of palmitoleic acid was abrogated, while oleic acid contents dropped by half in the mutant. Deletion of the operon MSMEG_1741-1743 (highly homologous to a Pseudomonas aeruginosa acyl-CoA desaturase) had little effect on growth of the parental strain; however the double mutant MSMEG_1886-MSMEG_1741-1743 strictly required oleic acid for growth. The ΔMSMEG_1886-ΔMSMEG_1741 double mutant was able to grow (poorly but better than the ΔMSMEG_1886 single mutant) in solid and liquid media devoid of oleic acid, suggesting a repressor role for ΔMSMEG_1741. Fatty acid analysis of the described mutants suggested that MSMEG_1742-43 desaturates C18:0 and C24:0 fatty acids. Thus, although the M. smegmatis desA3 homologue is the major player in unsaturated fatty acid synthesis, a second set of genes is also involved.

Green Fluorescent Protein as a protein localization and topological reporter in mycobacteria.

The cell envelope-associated proteins of Mycobacterium species play critical functions in the physiology and pathogenicity of these microorganisms. Because the determination of their subcellular localization and transmembrane topology is often critical to the understanding of their function, we investigated whether the Green Fluorescent Protein (GFP) could be used as a reporter to probe protein localization and map the topology of inner membrane proteins directly in intact mycobacterial cells. To this end, two GFP-based mycobacterial reporter plasmids were engineered and their functionality validated using a variety of membrane-associated, exported and cytosolic proteins.

Design, synthesis and evaluation of indole-2-carboxamides with pan anti-mycobacterial activity.

Current treatment regimens for non-tuberculous mycobacteria (NTM) and tuberculosis (TB) generally require long duration of therapy with multiple drugs, some of which are broad spectrum antibiotics. Despite some advances in antimicrobial compounds, there remains a need in therapy for antibiotics with specific mycobacterial targets. It has been shown that MmpL3 is an essential transporter required for the translocation of mycolic acids to the mycobacterial cell envelope. Here, we synthesized a series of indole-2-carboxamides that inhibit MmpL3 and have potent pan-activity against mycobacterial species. The compounds were tested against several fast and slow-growing Mycobacterium species, including M. abscessus, M. massiliense, M. bolletii, M. chelonae, M. tuberculosis, M. avium, M. xenopi and M. smegmatis. The target of these indole-based compounds makes them selective for mycobacteria, while showing no clinically relevant bactericidal activity against S. aureus or P. aeruginosa. These compounds were tested against THP-1, a human-cell line, and showed minimal in vitro cytotoxicity and good selectivity indices. The data shown and discussed suggest that lead indole-2-carboxamides are strong contenders for further preclinical testing as NTM therapeutics.

Structure-Function Profile of MmpL3, the Essential Mycolic Acid Transporter from Mycobacterium tuberculosis.

The MmpL family of proteins translocates complex (glyco)lipids and siderophores across the cell envelope of mycobacteria and closely related Corynebacteriaceae and plays important roles in the biogenesis of the outer membrane of these organisms. Despite their significance in the physiology and virulence of Mycobacterium tuberculosis, and from the perspective of developing novel antituberculosis agents, little is known about their structure and mechanism of translocation. In this study, the essential mycobacterial mycolic acid transporter, MmpL3, and its orthologue in Corynebacterium glutamicum, CmpL1, were investigated as prototypical MmpL proteins to gain insight into the transmembrane topology, tertiary and quaternary structures, and functional regions of this transporter family. The combined genetic, biochemical, and biophysical studies indicate that MmpL3 and CmpL1 are structurally similar to Gram-negative resistance-nodulation and division efflux pumps. They harbor 12 transmembrane segments interrupted by two large soluble periplasmic domains and function as homotrimers to export long-chain (C22-C90) mycolic acids, possibly in their acetylated form, esterified to trehalose. The mapping of a number of functional residues within the middle region of the transmembrane domain of MmpL3 shows a striking overlap with mutations associated with resistance to MmpL3 inhibitors. The results suggest that structurally diverse inhibitors of MmpL3 all target the proton translocation path of the transporter and that multiresistance to these inhibitors is enabled by conformational changes in MmpL3.

Assembling of the Mycobacterium tuberculosis Cell Wall Core.

The unique cell wall of mycobacteria is essential to their viability and the target of many clinically used anti-tuberculosis drugs and inhibitors under development. Despite intensive efforts to identify the ligase(s) responsible for the covalent attachment of the two major heteropolysaccharides of the mycobacterial cell wall, arabinogalactan (AG) and peptidoglycan (PG), the enzyme or enzymes responsible have remained elusive. We here report on the identification of the two enzymes of Mycobacterium tuberculosis, CpsA1 (Rv3267) and CpsA2 (Rv3484), responsible for this function. CpsA1 and CpsA2 belong to the widespread LytR-Cps2A-Psr (LCP) family of enzymes that has been shown to catalyze a variety of glycopolymer transfer reactions in Gram-positive bacteria, including the attachment of wall teichoic acids to PG. Although individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtained, the combined inactivation of both genes appears to be lethal. In the closely related microorganism Corynebacterium glutamicum, the ortholog of cpsA1 is the only gene involved in this function, and its conditional knockdown leads to dramatic changes in the cell wall composition and morphology of the bacteria due to extensive shedding of cell wall material in the culture medium as a result of defective attachment of AG to PG. This work marks an important step in our understanding of the biogenesis of the unique cell envelope of mycobacteria and opens new opportunities for drug development.

Biosynthesis and translocation of unsulfated acyltrehaloses in Mycobacterium tuberculosis.

A number of species-specific polymethyl-branched fatty acid-containing trehalose esters populate the outer membrane of Mycobacterium tuberculosis. Among them, 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) not only play a structural role in the cell envelope but also contribute to the ability of M. tuberculosis to multiply and persist in the infected host, promoting the intracellular survival of the bacterium and modulating host immune responses. The nature of the machinery, topology, and sequential order of the reactions leading to the biosynthesis, assembly, and export of these complex glycolipids to the cell surface are the object of the present study. Our genetic and biochemical evidence corroborates a model wherein the biosynthesis and translocation of DAT and PAT to the periplasmic space are coupled and topologically split across the plasma membrane. The formation of DAT occurs on the cytosolic face of the plasma membrane through the action of PapA3, FadD21, and Pks3/4; that of PAT occurs on the periplasmic face via transesterification reactions between DAT substrates catalyzed by the acyltransferase Chp2 (Rv1184c). The integral membrane transporter MmpL10 is essential for DAT to reach the cell surface, and its presence in the membrane is required for Chp2 to be active. Disruption of mmpL10 or chp2 leads to an important build-up of DAT inside the cells and to the formation of a novel form of unsulfated acyltrehalose esterified with polymethyl-branched fatty acids normally found in sulfolipids that is translocated to the cell surface.

The cell envelope glycoconjugates of Mycobacterium tuberculosis.

Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of the most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last 10 years in the discovery and development of novel inhibitors targeting their biogenesis.