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We investigated the mechanism of action of an arylsulfonamide with whole-cell activity against Mycobacterium tuberculosis . We newly synthesized the molecule and confirmed it had activity against both extracellular and intracellular bacilli. The molecule had some activity against HepG2 cells but maintained some selectivity. Bacterial cytological profiling suggested that the mechanism of action was via disruption of cell wall synthesis, with similarities to an inhibitor of the mycolic acid exporter MmpL3. The compound induced expression from the IniB promoter and caused a boost in ATP production but did not induce reactive oxygen species. A mutation in MmpL3 (S591I) led to low-level resistance. Taken together, these data confirm the molecule targets cell wall biosynthesis with MmpL3 as the most probable target., Competing Interests: The authors declare no conflict of interest.

Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis , an essential inner membrane transporter of the resistance-nodulation-division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro , inside macrophages, and in M. tuberculosis- infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections.IMPORTANCEMmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition., Competing Interests: The authors declare no conflict of interest.

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis , the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved σD envelope stress response in porin export and identified MarP as the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the σD pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the σD regulon in protein export to the MM., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hart et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Worldwide, tuberculosis is the second leading infectious killer and multidrug resistance severely hampers disease control. Mycolic acids are a unique category of lipids that are essential for viability, virulence, and persistence of the causative agent, Mycobacterium tuberculosis (Mtb). Therefore, enzymes involved in mycolic acid biosynthesis represent an important class of drug targets. We previously showed that the (3R)-hydroxyacyl-ACP dehydratase (HAD) protein HadD is dedicated mainly to the production of ketomycolic acids and plays a determinant role in Mtb biofilm formation and virulence. Here, we discovered that HAD activity requires the formation of a tight heterotetramer between HadD and HadB, a HAD unit encoded by a distinct chromosomal region. Using biochemical, structural, and cell-based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. Based on HadBD Mtb crystal structure and substrate-bound models, we identified determinants of the ultra-long-chain lipid substrate specificity and revealed details of structure-function relationship. HadBD Mtb unique function is partly due to a wider opening and a higher flexibility of the substrate-binding crevice in HadD, as well as the drastically truncated central α-helix of HadD hotdog fold, a feature described for the first time in a HAD enzyme. Taken together, our study shows that HadBD Mtb , and not HadD alone, is the biologically relevant functional unit. These results have important implications for designing innovative antivirulence molecules to fight tuberculosis, as they suggest that the target to consider is not an isolated subunit, but the whole HadBD complex., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Background: Mycobacterium tuberculosis is a bacterium that has historically had a substantial impact on human health. Despite advances in understanding and management of tuberculosis (TB), the disease remains a crucial problem that necessitates ongoing work to discover effective drugs, minimize transmission, and improve global health outcomes., Methods: The purpose of this study is to use molecular docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses to explore the molecular interactions of different proteins that are involved in mycolic acid biosynthesis (HadAB, InhA, KasA, FabD, and beta-ketoacyl-acyl carrier protein synthase III) of M. tuberculosis with Demospongiae metabolites. The docking findings were evaluated using the glide gscore, and the top 10 compounds docked against each protein receptor were chosen. Furthermore, the selected compounds underwent ADMET analysis, indicating that they have the potential for therapeutic development., Results: Among the selected compounds, makaluvamine G showed the highest binding affinity against HadAB, psammaplysin E showed highest binding affinity against InhA, pseudotheonamide D showed the highest binding affinity against KasA protein, dinordehydrobatzelladine B showed the highest binding affinity against FabD, and nagelamide X showed the highest binding affinity against beta-ketoacyl-acyl carrier protein synthase III. Additionally, molecular mechanics generalized born surface area (MM-GBSA) binding free energy and molecular dynamics simulations were used to support the docking investigations., Conclusion: The results of the study suggest that these compounds may eventually be used to treat TB. However, computer validations were included in this study, and more in vitro research is required to turn these prospective inhibitors into clinical drugs., Competing Interests: None

Lipids are prominent components of the mycobacterial cell wall, and they play critical roles not only in maintaining biofilm formation but also in resisting environmental stress, including drug resistance. However, information regarding the mechanism mediating mycobacterial lipid synthesis remains limited. PatA is a membrane-associated acyltransferase and synthesizes phosphatidyl- myo -inositol mannosides (PIMs) in mycobacteria. Here, we found that PatA could regulate the synthesis of lipids (except mycolic acids) to maintain biofilm formation and environmental stress resistance in Mycolicibacterium smegmatis. Interestingly, the deletion of patA significantly enhanced isoniazid (INH) resistance in M. smegmatis, although it reduced bacterial biofilm formation. This might be due to the fact that the patA deletion promoted the synthesis of mycolic acids through an unknown synthesis pathway other than the reported fatty acid synthase (FAS) pathway, which could effectively counteract the inhibition by INH of mycolic acid synthesis in mycobacteria. Furthermore, the amino acid sequences and physiological functions of PatA were highly conserved in mycobacteria. Therefore, we found a mycolic acid synthesis pathway regulated by PatA in mycobacteria. In addition, PatA also affected biofilm formation and environmental stress resistance by regulating the synthesis of lipids (except mycolic acids) in mycobacteria. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, leads to a large number of human deaths every year. This is so serious, which is due mainly to the drug resistance of mycobacteria. INH kills M. tuberculosis by inhibiting the synthesis of mycolic acids, which are synthesized by the FAS pathway. However, whether there is another mycolic acid synthesis pathway is unknown. In this study, we found a PatA-mediated mycolic acid synthesis pathway that led to INH resistance of in patA -deleted mutant. In addition, we first report the regulatory effect of PatA on mycobacterial biofilm formation, which could affect the bacterial response to environmental stress. Our findings represent a new model for regulating biofilm formation by mycobacteria. More importantly, the discovery of the PatA-mediated mycolic acid synthesis pathway indicates that the study of mycobacterial lipids has entered a new stage, and the enzymes might be new targets of antituberculosis drugs., Competing Interests: The authors declare no conflict of interest.

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid-rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we establish that the translocation of non-essential trehalose polyphleates (TPP) occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MmpL3 and MmpL10, respectively involved in the export of mycolic acids and TPP, in growing cells and their colocalization with Wag31, a protein playing a critical role in regulating peptidoglycan biosynthesis in mycobacteria. We found that MmpL3, like Wag31, displays polar localization and preferential accumulation at the old pole whereas MmpL10 is more homogenously distributed in the plasma membrane and slightly accumulates at the new pole. These results led us to propose a model in which insertion of TPP and mycolic acids into the mycomembrane is spatially uncoupled., (© 2023. The Author(s).)

The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Corynebacterium glutamicum has been widely utilized for the industrial production of various amino acids. Trehalose is a prerequisite for the biosynthesis of mycolates which are structurally important constituents of the cell envelope in C. glutamicum. In this study, C. glutamicum mutant ΔSYA, which is unable to synthesize trehalose was constructed by deleting genes treS, treY and otsA in the three pathways of trehalose biosynthesis. In the fermentation medium, ΔSYA grew as well as the control C. glutamicum ATCC13869, synthesized similar levels of glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids to ATCC13869, but produced 12.5 times more L-glutamate than ATCC13869. Transcriptional levels of the genes relevant to L-arginine biosynthesis, encoding ODHC and relevant to the biosynthesis of sulfur-containing amino acids were down-regulated in ΔSYA. In minimal medium with different concentrations of glucose, ΔSYA grew worse than ATCC13869 but excreted more L-glutamate. When grown in minimal medium, phospholipids are the major lipid in ΔSYA, while glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids are the major lipid in ATCC13869. The transcriptional levels of mscCG in ΔSYA was significantly up-regulated when grown in minimal medium., Competing Interests: Disclosures The authors have no conflicts of interest., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Bacteria of the order Corynebacteriales including pathogens such as Mycobacterium tuberculosis and Corynebacterium diphtheriae are characterized by their complex, multi-layered envelope. In addition to a peptidoglycan layer, these organisms possess an additional polysaccharide layer made of arabinogalactan and an outer membrane layer composed predominantly of long-chain fatty acids called mycolic acids. This so-called mycolata envelope structure is both a potent barrier against antibiotic entry into cells and a target of several antibacterial therapeutics. A better understanding of the mechanisms underlying mycolata envelope assembly therefore promises to reveal new ways of disrupting this unique structure for the development of antibiotics and antibiotic potentiators. Because they engage with receptors on the cell surface during infection, bacteriophages have long been used as tools to uncover important aspects of host envelope assembly. However, surprisingly little is known about the interactions between Corynebacteriales phages and their hosts. We therefore made use of the phages Cog and CL31 that infect Corynebacterium glutamicum ( Cglu ), a model member of the Corynebacteriales, to discover host factors important for phage infection. A high-density transposon library of Cglu was challenged with these phages followed by transposon sequencing to identify resistance loci. The analysis identified an important role for mycomembrane proteins in phage infection as well as components of the arabinogalactan and mycolic acid synthesis pathways. Importantly, the approach also implicated a new gene ( cgp_0396 ) in the process of arabinogalactan modification and identified a conserved new factor (AhfA, Cpg_0475) required for mycolic acid synthesis in Cglu ., Competing Interests: AM, TB No competing interests declared, (© 2022, McKitterick and Bernhardt.)

New anti-tubercular agents are urgently needed to address the emerging threat of drug resistance to human tuberculosis. Here, we have used structure-assisted methods to develop compounds that target mycobacterial membrane protein large 3 (MmpL3). MmpL3 is essential for the transport of mycolic acids, an important cell-wall component of mycobacteria. We prepared compounds that potently inhibit the growth of Mycobacterium tuberculosis (Mtb) and other mycobacteria in cell culture. The cryoelectron microscopy (cryo-EM) structure of mycobacterial MmpL3 in complex with one of these compounds (ST004) was determined using lipid nanodiscs at an overall resolution of 3.36 Å. The structure reveals the binding mode of ST004 to MmpL3, with the S4 and S5 subsites of the inhibitor-binding pocket in the proton translocation channel playing vital roles. These data are a promising starting point for the development of anti-tuberculosis drugs that target MmpL3., Competing Interests: Declaration of interests ShanghaiTech University has applied for a PCT patent that covers all the compounds presented here. All authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality and involvement in various aspects of mycobacterial pathogenesis, the synthesis of mycolic acids-and the identification of the enzymes involved-is a valuable target for drug development. Although most of the core pathway is comparable between species, subtle structure differences lead to different structures delineating the mycolic acid repertoire of tuberculous and some nontuberculous mycobacteria. We here report the characterization of an α'-mycolic acid-deficient Mycobacterium smegmatis mutant obtained by chemical mutagenesis. Whole-genome sequencing and bioinformatic analysis identified a premature stop codon in MSMEG_4301, encoding an acyl-CoA synthetase. Orthologs of MSMEG_4301 are present in all mycobacterial species containing α'-mycolic acids. Deletion of the Mycobacterium abscessus ortholog MAB_1915 abrogated synthesis of α'-mycolic acids; likewise, deletion of MSMEG_4301 in an otherwise wild-type M. smegmatis background also caused loss of these short mycolates. IMPORTANCE Mycobacterium abscessus is a nontuberculous mycobacterium responsible for an increasing number of hard-to-treat infections due to the impervious nature of its cell envelope, a natural barrier to several antibiotics. Mycolic acids are key components of that envelope; thus, their synthesis is a valuable target for drug development. Our results identify the first enzyme involved in α'-mycolic acids, a short-chain member of mycolic acids, loss of which greatly affects growth of this opportunistic pathogen.

Comprehending the molecular strategies employed by Mycobacterium tuberculosis (Mtb) in FAS-II regulation is of paramount significance for curbing tuberculosis progression. Mtb employs two sets of dehydratases, namely HadAB and HadBC (β-hydroxyacyl acyl carrier protein dehydratase), for the regulation of the fatty acid synthase (FAS-II) pathway. We utilized a sequence similarity network to discern the basis for the presence of two copies of the dehydratase gene in Mtb. This analysis groups HadC and HadA in different clusters, which could be attributed to the variability in their physiological role with respect to the acyl chain uptake. Our study reveals structural details pertaining to the crystal structure of the last remaining enzyme of the FAS-II pathway. It also provides insights into the highly flexible hot-dog helix and substrate regulatory loop. Additionally, mutational studies assisted in establishing the role of the C-terminal end in HadC of HadBC in the regulation of acyl carrier protein from Mtb-mediated interactions. Complemented with surface plasmon resonance and molecular dynamics simulation studies, the present study provides the first evidence of the molecular mechanisms involved in the differential binding affinity of the acyl carrier protein from Mtb towards both mtbHadAB and mtbHadBC., (© 2022 Federation of European Biochemical Societies.)

Transporters belonging to the Resistance-Nodulation-cell Division (RND) superfamily of proteins such as Mycobacterium tuberculosis MmpL3 and its analogs are the focus of intense investigations due to their importance in the physiology of Corynebacterium-Mycobacterium-Nocardia species and antimycobacterial drug discovery. These transporters deliver trehalose monomycolates, the precursors of major lipids of the outer membrane, to the periplasm by a proton motive force-dependent mechanism. In this study, we successfully purified, from native membranes, the full-length and the C-terminal truncated M. tuberculosis MmpL3 and Corynebacterium glutamicum CmpL1 proteins and reconstituted them into proteoliposomes. We also generated a series of substrate mimics and inhibitors specific to these transporters, analyzed their activities in the reconstituted proteoliposomes, and carried out molecular dynamics simulations of the model MmpL3 transporter at different pH. We found that all reconstituted proteins facilitate proton translocation across a phospholipid bilayer, but MmpL3 and CmpL1 differ dramatically in their responses to pH and interactions with substrate mimics and indole-2-carboxamide inhibitors. Our results further suggest that some inhibitors abolish the transport activity of MmpL3 and CmpL1 by inhibition of proton translocation.

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., (© 2022. The Author(s).)

Background: Bacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3 secretion system is required by mycobacteria for iron homeostasis. The ESX-3 operon encodes for one cytoplasmic component (EccA 3 ) and five membrane components (EccB3 - EccE3 and MycP 3 ). In this study we sought to identify the sub-cellular location of EccA 3 of the ESX-3 secretion system in mycobacteria., Results: Fluorescently tagged EccA 3 localized to a single pole in the majority of Mycobacterium smegmatis cells and time-lapse fluorescent microscopy identified this pole as the growing pole. Deletion of ESX-3 did not prevent polar localization of fluorescently tagged EccA 3 , suggesting that EccA 3 unipolar localization is independent of other ESX-3 components. Affinity purification - mass spectrometry was used to identify EccA 3 associated proteins which may contribute to the localization of EccA 3 at the growing pole. EccA 3 co-purified with fatty acid metabolism proteins (FAS, FadA3, KasA and KasB), mycolic acid synthesis proteins (UmaA, CmaA1), cell division proteins (FtsE and FtsZ), and cell shape and cell cycle proteins (MurS, CwsA and Wag31). Secretion system related proteins Ffh, SecA1, EccA1, and EspI were also identified., Conclusions: Time-lapse microscopy demonstrated that EccA3 is located at the growing pole in M. smegmatis. The co-purification of EccA 3 with proteins known to be required for polar growth, mycolic acid synthesis, the Sec secretion system (SecA1), and the signal recognition particle pathway (Ffh) also suggests that EccA 3 is located at the site of active cell growth., (© 2022. The Author(s).)

Co-culture is an efficient strategy for natural product discovery. We have used mycolic acid-containing bacteria (MACB) Tsukamurella pumonis TP-B0596 to induce secondary metabolism by actinomycetes and have found several natural products. We also observed that MACB attached to the mycelium of Streptomyces lividans forming coaggregates during combined-culture. This stimulated interest in the interactions among actinomycetes and MACB, and we found that soil isolated cultures contained a mixture of actinomycetes and MACB. Our previously observed interactions were the result of selective screening and combination of bacteria in the lab, which warranted investigation of the existence of these interactions in the natural soil environment. Therefore, in this paper, we report the interaction between a co-isolated natural pair of actinomycetes and MACB in terms of morphology and metabolic changes. A natural pair of actinomycetes and MACB co-aggregated in liquid culture and showed metabolic changes. Interestingly, co-aggregated actinomycetes and MACB were re-isolated from soil with no obvious morphological colony differences from the colony of a single strain. The results demonstrate that there is a stochastic chance of picking colonies containing co-aggregated actinomycetes and MACB, which suggests that the pair can exist in co-aggregate form in the soil environment and interact with each other., (© 2022. The Author(s).)

Mycolic acids are key components of the complex cell envelope of Corynebacteriales . These fatty acids, conjugated to trehalose or to arabinogalactan form the backbone of the mycomembrane. While mycolic acids are essential to the survival of some species, such as Mycobacterium tuberculosis , their absence is not lethal for Corynebacterium glutamicum, which has been extensively used as a model to depict their biosynthesis. Mycolic acids are first synthesized on the cytoplasmic side of the inner membrane and transferred onto trehalose to give trehalose monomycolate (TMM). TMM is subsequently transported to the periplasm by dedicated transporters and used by mycoloyltransferase enzymes to synthesize all the other mycolate-containing compounds. Using a random transposition mutagenesis, we recently identified a new uncharacterized protein (Cg1246) involved in mycolic acid metabolism. Cg1246 belongs to the DUF402 protein family that contains some previously characterized nucleoside phosphatases. In this study, we performed a functional and structural characterization of Cg1246. We showed that absence of the protein led to a significant reduction in the pool of TMM in C. glutamicum , resulting in a decrease in all other mycolate-containing compounds. We found that, in vitro , Cg1246 has phosphatase activity on organic pyrophosphate substrates but is most likely not a nucleoside phosphatase. Using a computational approach, we identified important residues for phosphatase activity and constructed the corresponding variants in C. glutamicum . Surprisingly complementation with these non-functional proteins fully restored the defect in TMM of the Δ cg1246 mutant strain, suggesting that in vivo , the phosphatase activity is not involved in mycolic acid biosynthesis.

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2 , controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR -null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of β-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1 / desA2 is relieved upon binding of saturated acyl-CoAs of chain length C 16 to C 24 , while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Some nontuberculous mycobacteria (NTM) are considered opportunistic pathogens. Nevertheless, NTM infections are increasing worldwide, becoming a major public health threat. Furthermore, there is no current specific drugs to treat these infections, and the recommended regimens generally lack efficacy, emphasizing the need for novel antibacterial compounds. In this paper, we focused on the essential mycolic acids transporter MmpL3, which is a well-characterized target of several antimycobacterial agents, to identify new compounds active against Mycobacterium abscessus ( Mab ). From the crystal structure of MmpL3 in complex with known inhibitors, through an in silico approach, we developed a pharmacophore that was used as a three-dimensional filter to identify new putative MmpL3 ligands within databases of known drugs. Among the prioritized compounds, mefloquine showed appreciable activity against Mab (MIC = 16 μg/mL). The compound was confirmed to interfere with mycolic acids biosynthesis, and proved to also be active against other NTMs, including drug-resistant clinical isolates. Importantly, mefloquine is a well-known antimalarial agent, opening the possibility of repurposing an already approved drug, which is a useful strategy to reduce the time and cost of disclosing novel drug candidates.

Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

The Mycobacterium tuberculosis cell envelope is a critical interface between the host and pathogen and provides a protective barrier against the immune response and antibiotics. Cell envelope lipids are also mycobacterial virulence factors that influence the host immune response. The mycobacterial membrane protein large (MmpL) proteins transport cell envelope lipids and siderophores that are important for the basic physiology and pathogenesis of M. tuberculosis. We recently identified MmpL11 as a conserved transporter of mycolic acid-containing lipids including monomeromycolyl diacylglycerol (MMDAG), mycolate wax ester (MWE), and long-chain triacylglycerols (LC-TAGs). These lipids contribute to biofilm formation in M. tuberculosis and M. smegmatis, and non-replicating persistence in M. tuberculosis. In this report, we identified domains and residues that are essential for MmpL11 TB lipid transporter activity. Specifically, we show that the D1 periplasmic loop and a conserved tyrosine are essential for the MmpL11 function. Intriguingly, we found that MmpL11 levels are regulated by the phosphorylation of threonine in the cytoplasmic C-terminal domain, providing the first direct evidence of the phospho-regulation of MmpL11 transporter activity in M. tuberculosis and M. smegmatis. Our results offer further insight into the function of MmpL transporters and regulation of mycobacterial cell envelope biogenesis., (© 2020 John Wiley & Sons Ltd.)

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure. IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer "myco" membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens., (Copyright © 2021 Pohane et al.)

Corynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-cell wall antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen, M. smegmatis . Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies., Competing Interests: Td, KW, MM, VM, DW No competing interests declared, (© 2020, de Wet et al.)

Outside of Mycobacterium tuberculosis and Mycobacterium leprae , nontuberculous mycobacteria (NTM) are environmental mycobacteria (>190 species) and are classified as slow- or rapid-growing mycobacteria. Infections caused by NTM show an increased incidence in immunocompromised patients and patients with underlying structural lung disease. The true global prevalence of NTM infections remains unknown because many countries do not require mandatory reporting of the infection. This is coupled with a challenging diagnosis and identification of the species. Current therapies for treatment of NTM infections require multidrug regimens for a minimum of 18 months and are associated with serious adverse reactions, infection relapse, and high reinfection rates, necessitating discovery of novel antimycobacterial agents. Robust drug discovery processes have discovered inhibitors targeting mycobacterial membrane protein large 3 (MmpL3), a protein responsible for translocating mycolic acids from the inner membrane to periplasm in the biosynthesis of the mycobacterial cell membrane. This review focuses on promising new chemical scaffolds that inhibit MmpL3 function and represent interesting and promising putative drug candidates for the treatment of NTM infections. Additionally, agents (FS-1, SMARt-420, C10) that promote reversion of drug resistance are also reviewed.

Background: The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant (aceE-mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis (M. smegmatis). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis., Results: We observed that the colony morphology of aceE-mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis. Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE-mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE-mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE-mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceE gene restored the composition of MA., Conclusions: Over all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.

Mycobacterium tuberculosis is the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug-development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we used genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates the export of MAs and promotes the remodeling of the mycobacterial cell envelope. In particular, we identified the essential MmpL3 as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way for novel antimycobacterial strategies., (Copyright © 2020 Le et al.)

The genomes of Corynebacteriales contain several genes encoding mycoloyltransferases (Myt) that are specific cell envelope enzymes essential for the biogenesis of the outer membrane. MytA is a major mycoloyltransferase of Corynebacterium glutamicum, displaying an N-terminal domain with esterase activity and a C-terminal extension containing a conserved repeated Leu-Gly-Phe-Pro (LGFP) sequence motif of unknown function. This motif is highly conserved in Corynebacteriales and found associated with cell wall hydrolases and with proteins of unknown function. In this study, we determined the crystal structure of MytA and found that its C-terminal domain is composed of five LGFP motifs and forms a long stalk perpendicular to the N-terminal catalytic α/β-hydrolase domain. The LGFP motifs are composed of a 4-stranded β-fold and occupy alternating orientations along the axis of the stalk. Multiple acetate binding pockets were identified in the stalk, which could correspond to putative ligand-binding sites. By using various MytA mutants and complementary in vitro and in vivo approaches, we provide evidence that the C-terminal LGFP domain interacts with the cell wall peptidoglycan-arabinogalactan polymer. We also show that the C-terminal LGFP domain is not required for the activity of MytA but rather contributes to the overall integrity of the cell envelope., (© 2020 John Wiley & Sons Ltd.)

Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F 420 )-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated Mycobacterium tuberculosis var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene ( ndh ) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type ndh Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM., (Copyright © 2020 American Society for Microbiology.)

Tuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis , primarily affecting the lungs. The M. tuberculosis strain of the Haarlem family named M was responsible for a large multidrug-resistant TB (MDR-TB) outbreak in Buenos Aires. This outbreak started in the early 1990s and in the mid 2000s still accounted for 29% of all MDR-TB cases in Argentina. By contrast, a clonal variant of strain M, named 410, has caused a single tuberculosis case since the onset of the outbreak. The molecular bases of the high epidemiological fitness of the M strain remain unclear. To assess its unique molecular properties, herein, we performed a comparative protein and lipid analysis of a representative clone of the M strain (Mp) and the nonprosperous M variant 410. We also evaluated their growth in low pH. The variant 410 had higher levels of latency proteins under standard conditions and delayed growth at low pH, suggesting that it is more sensitive to stress stimuli than Mp. Moreover, Mp showed higher levels of mycolic acids covalently attached to the cell wall and lower accumulation of free mycolic acids in the outer layer than the 410 strain. The low expression of latency proteins together with the reduced content of surface mycolic acids may facilitate Mp to evade the host immune responses., Competing Interests: The authors have no conflict of interest with this study., (Copyright © 2020 Jinlong Bei et al.)

Non-tuberculous mycobacterium (NTM) infections, such as those caused by Mycobacterium abscessus , are increasing globally. Due to their intrinsic drug resistance, M. abscessus pulmonary infections are often difficult to cure using standard chemotherapy. We previously demonstrated that a piperidinol derivative, named PIPD1, is an efficient molecule both against M. abscessus and Mycobacterium tuberculosis , the agent of tuberculosis, by targeting the mycolic acid transporter MmpL3. These results prompted us to design and synthesize a series of piperidinol derivatives and to determine the biological activity against M. abscessus . Structure-activity relationship (SAR) studies pointed toward specific sites on the scaffold that can tolerate slight modifications. Overall, these results identified FMD-88 as a new promising active analogue against M. abscessus . Also, we determined the pharmacokinetics properties of PIPD1 and showed that intraperitoneal administration of this compound resulted in promising serum concentration and an elimination half-life of 3.2 hours., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

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., Competing Interests: The authors declare no conflict of interest.

The fatty acid synthase type II (FAS-II) multienzyme system builds the main chain of mycolic acids (MAs), important lipid pathogenicity factors of Mycobacterium tuberculosis (Mtb). Due to their original structure, the identification of the (3 R)-hydroxyacyl-ACP dehydratases, HadAB and HadBC, of Mtb FAS-II complex required in-depth work. Here, we report the discovery of a third dehydratase protein, HadD Mtb (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs. HadD Mtb deletion triggered a marked change in Mtb keto-MA content and size distribution, deeply impacting the production of full-size molecules. Furthermore, abnormal MAs, likely generated from 3-hydroxylated intermediates, accumulated. These data strongly suggest that HadD Mtb catalyzes the 3-hydroxyacyl dehydratation step of late FAS-II elongation cycles during keto-MA biosynthesis. Phenotyping of Mtb hadD deletion mutant revealed the influence of HadD Mtb on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. Importantly, HadD Mtb has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadD Mtb observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies.

The final step in mycolic acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short-chain fatty acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short-chain dehydrogenases. In particular, the predicted structure revealed a unique α-helical C-terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow-growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature mycolic acid production and accumulation of intermediates derived from 3-oxo-mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies., (© 2019 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA , kasB , and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR , therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.

Mycobacterium tuberculosis , the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc 2 6230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis , suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3 , encoding an inner membrane-associated mycolic acid flippase in M. tuberculosis Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis , docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria., (© 2019 Dupont et al.)

The mycobacterial cell envelope is crucial to host-pathogen interactions as a barrier against antibiotics and the host immune response. In addition, cell envelope lipids are mycobacterial virulence factors. Cell envelope lipid biosynthesis is the target of a number of frontline tuberculosis treatments and has been the focus of much research. However, the transport mechanisms by which these lipids reach the mycomembrane remain poorly understood. Many envelope lipids are exported from the cytoplasm to the periplasmic space via the mycobacterial membrane protein large (MmpL) family of proteins. In other bacteria, lipoproteins can contribute to outer membrane biogenesis through direct binding of substrates and/or protein-protein associations with extracytoplasmic biosynthetic enzymes. In this report, we investigate whether the lipoprotein LpqN plays a similar role in mycobacteria. Using a genetic two-hybrid approach, we demonstrate that LpqN interacts with periplasmic loop domains of the MmpL3 and MmpL11 transporters that export mycolic acid-containing cell envelope lipids. We observe that LpqN also interacts with secreted cell envelope biosynthetic enzymes such as Ag85A via pulldown assays. The X-ray crystal structures of LpqN and LpqN bound to dodecyl-trehalose suggest that LpqN directly binds trehalose monomycolate, the MmpL3 and Ag85A substrate. Finally, we observe altered lipid profiles of the Δ lpqN mutant during biofilm maturation, pointing toward a possible physiological role for the protein. The results of this study suggest that LpqN may act as a membrane fusion protein, connecting MmpL transporters with periplasmic proteins, and provide general insight into the role of lipoproteins in Mycobacterium tuberculosis cell envelope biogenesis., (© 2019 Melly et al.)

1 H -benzo[ d ]imidazole derivatives exhibit antitubercular activity in vitro at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular Mycobacterium tuberculosis To identify their target, we selected drug-resistant M. tuberculosis mutants and then used whole-genome sequencing to unravel mutations in the essential mmpL3 gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the mmpL3 alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1 H -benzo[ d ]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from M. tuberculosis cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1 H -benzo[ d ]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis., (Copyright © 2019 American Society for Microbiology.)

The Mycobacterium tuberculosis mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel M. tuberculosis growth inhibitors for differential growth inhibition of wild-type M. tuberculosis compared to the growth of a pool of 24 unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular M. tuberculosis M. tuberculosis cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species M. abscessus Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors., (Copyright © 2019 American Society for Microbiology.)

Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603-1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division., Competing Interests: The authors have declared that no competing interests exist.

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.

Background: Extrapulmonary tuberculosis (EPTB), accounting for 10%-20% of all cases of tuberculosis (TB), is known to be determined by host immunity. However, the contribution of bacterial factors to the development of EPTB has not been studied extensively. Mycolic acids are predominant lipids constituting the cell wall of Mycobacterium tuberculosis, and keto-mycolic acid is involved in the synthesis of foamy macrophages that facilitate persistence of mycobacteria. Hence, the present study was performed to gain an insight into variable expression of mycolic acids in clinical isolates of M. tuberculosis under stress., Methods: Pansusceptible clinical isolates of M. tuberculosis from patients with lymph node TB (LNTB) (n = 10) and pulmonary TB (PTB) (n = 10) were subjected to sodium dodecyl sulfate (SDS) stress, and the expression of mycolic acid and its biosynthetic genes was compared. Any bias arising due to the genotype of the clinical isolates was ruled out by performing single-nucleotide polymorphism cluster grouping (SCG), wherein no significant difference was observed between the SCG of LNTB or PTB isolates., Results: The expression of α-mycolic acid during the exposure to SDS was high in 7/10 (70%) LNTB and 6/10 (60%) PTB isolates. Methoxy mycolic acid showed an increased expression in 7/10 (70%) LNTB isolates and 4/10 (40%) PTB isolates. Increased expression of keto-mycolic acid on exposure with SDS was observed in 8/10 (80%) M. tuberculosis LNTB and 3/10 (30%) PTB isolates. Similarly, the mycolic acid synthesis gene, fas, was upregulated more in LNTB isolates than PTB isolates in vitro and ex vivo. SCG 3a was the most common SCG observed in 40% (8/20) of the isolates, followed by SCG 3b in 30% (6/20) of the isolates. There was no significant difference between the SCG of LNTB or PTB isolates., Conclusion: The higher expression of keto-mycolic acid in LNTB as against PTB isolates may indicate better survival in LNTB isolates in the presence of stress., Competing Interests: None

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736 / Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named "TMM transport factor A", TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis IMPORTANCE The cell envelope of Mycobacterium tuberculosis , the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria., (Copyright © 2019 Fay et al.)

The cell envelope of Mycobacterium tuberculosis is notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure of Mycobacterium smegmatis MmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall., Competing Interests: The authors declare no conflict of interest.