RESUMEN
Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.
Asunto(s)
Proteínas Bacterianas , Glicósido Hidrolasas , Lipopolisacáridos , Macrófagos , Mananos , Mycobacterium tuberculosis , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , Lipopolisacáridos/metabolismo , Mananos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Glicósido Hidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Animales , Ratones , Humanos , Fosfatidilinositoles/metabolismo , Cápsulas Bacterianas/metabolismoRESUMEN
In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-ß-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.
Asunto(s)
Mycobacterium tuberculosis , Fosfatos de Poliisoprenilo , Mycobacterium tuberculosis/genética , Fosforribosil Pirofosfato/metabolismo , Antituberculosos/metabolismo , Pared Celular/metabolismoRESUMEN
Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important amongst these are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are both trafficked out of the bacterium to the host via unknown mechanisms. An important class of exported LM/LAM is the capsular derivative of these molecules which is devoid of its lipid anchor. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures where arabinomannan acts as a signal for growth phase transition. Finally, we demonstrate that LamH is important for Mycobacterium tuberculosis survival in macrophages. These data provide a new framework for understanding the biological role of LAM in mycobacteria.
RESUMEN
Mycobacterium tuberculosis is one of the global leading causes of death due to a single infectious agent. Pretomanid and delamanid are new antitubercular agents that have progressed through the drug discovery pipeline. These compounds are bicyclic nitroimidazoles that act as pro-drugs, requiring activation by a mycobacterial enzyme; however, the precise mechanisms of action of the active metabolite(s) are unclear. Here, we identify a molecular target of activated pretomanid and delamanid: the DprE2 subunit of decaprenylphosphoribose-2'-epimerase, an enzyme required for the synthesis of cell wall arabinogalactan. We also provide evidence for an NAD-adduct as the active metabolite of pretomanid. Our results highlight DprE2 as a potential antimycobacterial target and provide a foundation for future exploration into the active metabolites and clinical development of pretomanid and delamanid.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Nitroimidazoles , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Terapia Molecular Dirigida , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Nitroimidazoles/farmacología , Nitroimidazoles/uso terapéutico , Pared Celular/metabolismo , Resistencia a Medicamentos , Profármacos/química , Profármacos/metabolismo , Espectrofotometría , NAD/metabolismo , CinéticaRESUMEN
Arabinogalactan (AG) is an essential cell wall component in mycobacterial species, including the deadly human pathogen Mycobacterium tuberculosis. It plays a pivotal role in forming the rigid mycolyl-AG-peptidoglycan core for in vitro growth. AftA is a membrane-bound arabinosyltransferase and a key enzyme involved in AG biosynthesis which bridges the assembly of the arabinan chain to the galactan chain. It is known that AftA catalyzes the transfer of the first arabinofuranosyl residue from the donor decaprenyl-monophosphoryl-arabinose to the mature galactan chain (i.e., priming); however, the priming mechanism remains elusive. Herein, we report the cryo-EM structure of Mtb AftA. The detergent-embedded AftA assembles as a dimer with an interface maintained by both the transmembrane domain (TMD) and the soluble C-terminal domain (CTD) in the periplasm. The structure shows a conserved glycosyltransferase-C fold and two cavities converging at the active site. A metal ion participates in the interaction of TMD and CTD of each AftA molecule. Structural analyses combined with functional mutagenesis suggests a priming mechanism catalyzed by AftA in Mtb AG biosynthesis. Our data further provide a unique perspective into anti-TB drug discovery.
Asunto(s)
Mycobacterium tuberculosis , Humanos , Galactanos , Pentosiltransferasa/genéticaRESUMEN
Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Mycobacterium smegmatis/enzimología , Pentosiltransferasa/química , Pentosiltransferasa/ultraestructura , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Etambutol/farmacología , Pentosiltransferasa/antagonistas & inhibidores , Pentosiltransferasa/metabolismoRESUMEN
The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo-electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.
Asunto(s)
Antituberculosos/química , Pared Celular/enzimología , Etambutol/química , Mycobacterium tuberculosis/enzimología , Pentosiltransferasa/química , Microscopía por Crioelectrón , Farmacorresistencia Bacteriana Múltiple , Conformación ProteicaRESUMEN
The arabinan-containing polysaccharides, arabinogalactan (AG) and lipoarabinomannan (LAM), are key cell wall components of the Corynebacterineae, which include Corynebacteria, Norcadia and Mycobacteria. Both AG and LAM contain elaborate arabinan domains composed of distinct structural motifs. Mycobacterial EmbA, EmbB and EmbC, collectively known as the Emb proteins, have been identified as arabinosyltransferases (ArafTs), which are targeted by the front-line anti-tubercular drug ethambutol. Previous studies have established that EmbA and EmbB play a role in the synthesis of the characteristic terminal hexa-arabinosuranosyl motif, whilst EmbC is involved exclusively in the biosynthesis of LAM. Herein, we have investigated the role of the singular Emb protein from Corynebacterium glutamicum through the detailed biochemical and chemical analysis of a double ΔaftAΔemb mutant, where the priming Cg-AftA protein, which generates the substrate for Cg-Emb has been deleted. Analysis of its cell wall revealed a complete absence of arabinose resulting in a truncated cell wall containing only a galactan backbone accompanied with complete loss of cell wall bound mycolates. In vitro cell-free assays using C. glutamicumΔaftA, C. glutamicumΔemb, C. glutamicumΔaftAΔemb and C. glutamicumΔaftBΔaftD and two synthetic acceptors, which mimick the arabinofuranose (Araf) "primed" galactan chain, demonstrated that Cg-Emb is able to transfer an Araf residue to the C5 of the Araf positioned on the synthetic acceptor(s). These results indicate that Cg-Emb acts as an α(1â¯ââ¯5) ArafT and elongates the arabinan core during the early stages of arabinan biosynthesis in C. glutamicum.
RESUMEN
Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and ß-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes.
Asunto(s)
Membrana Celular/metabolismo , Euglena gracilis/enzimología , Fluorescencia , Glicosiltransferasas/metabolismo , Manósidos/metabolismo , Microsomas/metabolismo , Cinética , Manósidos/química , Especificidad por SustratoRESUMEN
Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis ß-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related ß-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Indazoles/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Sulfonamidas/farmacología , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Farmacorresistencia Bacteriana/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple/genética , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/prevención & controlRESUMEN
Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos Esenciales/metabolismo , Genes Esenciales , Mycobacterium tuberculosis/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/genética , Ratones , Pruebas de Sensibilidad Microbiana , Mutación Missense , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mutación Puntual , Unión Proteica , Mapeo de Interacción de Proteínas , Tuberculosis/microbiología , Tuberculosis/patología , Técnicas del Sistema de Dos HíbridosRESUMEN
The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at (535)GAC>(535)AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.
Asunto(s)
Antituberculosos/farmacología , Aspartato-ARNt Ligasa/metabolismo , Mycobacterium bovis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Piperidinas/farmacología , Tiazoles/farmacología , Secuencia de Aminoácidos , Antituberculosos/uso terapéutico , Aspartato-ARNt Ligasa/química , Aspartato-ARNt Ligasa/genética , Clonación Molecular , Cristalografía por Rayos X , Dimerización , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mycobacterium bovis/enzimología , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Piperidinas/química , Piperidinas/uso terapéutico , Polimorfismo de Nucleótido Simple , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Tiazoles/química , Tiazoles/uso terapéutico , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/patologíaRESUMEN
Two complementary methods for the synthesis of fluorinated exo-glycals have been developed, for which previously no general reaction had been available. First, a Selectfluor-mediated fluorination was optimized after detailed analysis of all the reaction parameters. A dramatic effect of molecular sieves on the course of the reaction was observed. The reaction was generalized with a set of biologically relevant furanosides and pyranosides. A second direct approach involving carbanionic chemistry and the use of N-fluorobenzenesulfonimide (NFSI) was performed and this method gave better diastereoselectivities. Assignment of the Z/E configuration of all the fluorinated exo-glycals was achieved based on the results of HOESY experiments. Furthermore, fluorinated exo-glycal analogues of UDP-galactofuranose were prepared and assayed against GlfT2, which is a key enzyme involved in the cell-wall biosynthesis of major pathogens. The fluorinated exo-glycals proved to be potent inhibitors as compared with a series of C-glycosidic analogues of UDP-Galf, thus demonstrating the double beneficial effect of the exocyclic enol ether functionality and the fluorine atom.
Asunto(s)
Compuestos de Diazonio/química , Inhibidores Enzimáticos/química , Galactosa/análogos & derivados , Galactosiltransferasas/antagonistas & inhibidores , Sulfonamidas/química , Uridina Difosfato/análogos & derivados , Antituberculosos/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Compuestos de Diazonio/síntesis química , Compuestos de Diazonio/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Galactosa/síntesis química , Galactosa/química , Galactosa/farmacología , Galactosiltransferasas/metabolismo , Halogenación , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Tuberculosis/tratamiento farmacológico , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Difosfato/farmacologíaRESUMEN
Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1ß/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1ß processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1ß expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores Cordón/inmunología , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Mycobacterium/inmunología , Peptidoglicano/inmunología , Células Th17/inmunología , Células Th17/metabolismo , Adyuvantes Inmunológicos , Animales , Proteínas Adaptadoras de Señalización CARD , Diferenciación Celular/inmunología , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Noqueados , Mycobacterium/química , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-18/metabolismo , Transducción de Señal , Células Th17/citología , Receptores Toll-Like/metabolismoRESUMEN
ABSTRACT The chronic nature of tuberculosis (TB), its requirement of long duration of treatment, its ability to evade immune intervention, and its propensity to relapse after drug treatment is discontinued are reminiscent of other chronic, biofilm-associated bacterial diseases. Historically, Mycobacterium tuberculosis was grown as a pellicle, a biofilm-like structure, at the liquid-air interface in a variety of synthetic media. Notably, the most widely administered human vaccine, BCG, is grown as a pellicle for vaccine production. However, the molecular requirements for this growth remain ill defined. Here, we demonstrate that keto-mycolic acids (keto-MA) are essential for pellicle growth, and mutants lacking in or depleted of this MA species are unable to form a pellicle. We investigated the role of the pellicle biofilm in the reduction of antibiotic sensitivity known as drug tolerance using the pellicle-defective ΔmmaA4 mutant strain. We discovered that the ΔmmaA4 mutant, which is both pellicle defective and highly sensitive to rifampicin (RIF) under planktonic growth, when incorporated within the wild-type pellicle biofilm, was protected from the bactericidal activity of RIF. The observation that growth within the M. tuberculosis pellicle biofilm can confer drug tolerance to a drug-hypersensitive strain suggests that identifying molecular requirements for pellicle growth could lead to development of novel interventions against mycobacterial infections. Our findings also suggest that a class of drugs that can disrupt M. tuberculosis biofilm formation, when used in conjunction with conventional antibiotics, has the potential to overcome drug tolerance. IMPORTANCE Two of the most important questions in tuberculosis (TB) research are (i) how does Mycobacterium tuberculosis persist in the human host for decades in the face of an active immune response and (ii) why does it take six months and four drugs to treat uncomplicated TB. Both these aspects of M. tuberculosis biology are reminiscent of infections caused by organisms capable of forming biofilms. M. tuberculosis is capable of growing as a biofilm-like structure called the pellicle. In this study, we demonstrate that a specific cell wall component, keto-mycolic acid, is essential for pellicle growth. We also demonstrate that a strain of M. tuberculosis that is both drug sensitive and pellicle defective exhibits commensal behavior and becomes drug tolerant by becoming part of a heterogeneous pellicle, a characteristic of multispecies biofilms. These observations could have important implications for identifying novel pathways for M. tuberculosis drug tolerance and the design of new modalities to rapidly treat TB.
Asunto(s)
Antituberculosos/farmacología , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Tolerancia a Medicamentos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/fisiología , Ácidos Micólicos/metabolismo , Proteínas Bacterianas/genética , Eliminación de GenRESUMEN
The biosynthesis of mycobacterial mannose-containing lipoglycans, such as lipomannan (LM) and the immunomodulator lipoarabinomanan (LAM), is carried out by the GT-C superfamily of glycosyltransferases that require polyprenylphosphate-based mannose (PPM) as a sugar donor. The essentiality of lipoglycan synthesis for growth makes the glycosyltransferase that synthesizes PPM, a potential drug target in Mycobacterium tuberculosis, the causative agent of tuberculosis. In M. tuberculosis, PPM has been shown to be synthesized by Ppm1 in enzymatic assays. However, genetic evidence for its essentiality and in vivo role in LM/LAM and PPM biosynthesis is lacking. In this study, we demonstrate that MSMEG3859, a Mycobacterium smegmatis gene encoding the homologue of the catalytic domain of M. tuberculosis Ppm1, is essential for survival. Depletion of MSMEG3859 in a conditional mutant of M. smegmatis resulted in the loss of higher order phosphatidyl-myo-inositol mannosides (PIMs) and lipomannan. We were also able to demonstrate that two other M. tuberculosis genes encoding glycosyltransferases that either had been shown to possess PPM synthase activity (Rv3779), or were involved in synthesizing similar polyprenol-linked donors (ppgS), were unable to compensate for the loss of MSMEG3859 in the conditional mutant.
Asunto(s)
Proteínas Bacterianas/genética , Lipopolisacáridos/biosíntesis , Manosiltransferasas/genética , Mycobacterium smegmatis/genética , Proteínas Bacterianas/fisiología , Genes Esenciales , Prueba de Complementación Genética , Manosiltransferasas/fisiología , Viabilidad Microbiana , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/crecimiento & desarrollo , Fenotipo , Eliminación de SecuenciaRESUMEN
Tuberculosis (TB) remains a leading cause of mortality worldwide. With the emergence of multidrug resistant TB, extensively drug resistant TB and HIV-associated TB it is imperative that new drug targets be identified. The potential of Mycobacterium tuberculosis inosine monophosphate dehydrogenase (IMPDH) as a novel drug target was explored in the present study. IMPDH exclusively catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD(+)). Although the enzyme is a dehydrogenase, the enzyme does not catalyze the reverse reaction i.e. the conversion of XMP to IMP. Unlike other bacteria, M. tuberculosis harbors three IMPDH-like genes, designated as Mt-guaB1, Mt-guaB2 and Mt-guaB3 respectively. Of the three putative IMPDH's, we previously confirmed that Mt-GuaB2 was the only functional ortholog by characterizing the enzyme kinetically. Using an in silico approach based on designed scaffolds, a series of novel classes of inhibitors was identified. The inhibitors possess good activity against M. tuberculosis with MIC values in the range of 0.4 to 11.4 µg mL(-1). Among the identified ligands, two inhibitors have nanomolar K(i)s against the Mt-GuaB2 enzyme.
Asunto(s)
Antituberculosos/farmacología , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Animales , Sitios de Unión , Humanos , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/aislamiento & purificación , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/genética , Unión Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/farmacología , Especificidad por Sustrato , Tuberculosis/tratamiento farmacológicoRESUMEN
The hallmark of tuberculosis (TB) is the formation of granulomas, which are clusters of infected macrophages surrounded by additional macrophages, neutrophils and lymphocytes. Although it has long been thought that granulomas are beneficial for the host, there is evidence that mycobacteria also promote the formation of these structures. In this study, we aimed to identify new mycobacterial factors involved in the initial stages of granuloma formation. We exploited the zebrafish embryo Mycobacterium marinum infection model to study initiation of granuloma formation and developed an in vivo screen to select for random M. marinum mutants that were unable to induce granuloma formation efficiently. Upon screening 200 mutants, three mutants repeatedly initiated reduced granuloma formation. One of the mutants was found to be defective in the espL gene, which is located in the ESX-1 cluster. The ESX-1 cluster is disrupted in the Mycobacterium bovis BCG vaccine strain and encodes a specialized secretion system known to be important for granuloma formation and virulence. Although espL has not been implicated in protein secretion before, we observed a strong effect on the secretion of the ESX-1 substrates ESAT-6 and EspE. We conclude that our zebrafish embryo M. marinum screen is a useful tool to identify mycobacterial genes involved in the initial stages of granuloma formation and that we have identified a new component of the ESX-1 secretion system. We are confident that our approach will contribute to the knowledge of mycobacterial virulence and could be helpful for the development of new TB vaccines.
Asunto(s)
Proteínas Bacterianas/metabolismo , Genes Bacterianos/genética , Granuloma/microbiología , Mycobacterium marinum/genética , Pez Cebra/embriología , Pez Cebra/microbiología , Animales , Elementos Transponibles de ADN/genética , Embrión no Mamífero/microbiología , Prueba de Complementación Genética , Granuloma/patología , Humanos , Espacio Intracelular/microbiología , Mutación/genética , Mycobacterium marinum/crecimiento & desarrollo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2-0.5 µg ml(-1). When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo.
Asunto(s)
Antituberculosos/metabolismo , Carbanilidas/metabolismo , Inhibidores Enzimáticos/metabolismo , IMP Deshidrogenasa/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Antituberculosos/química , Carbanilidas/química , Clonación Molecular , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/química , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Concentración de Iones de Hidrógeno , IMP Deshidrogenasa/química , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/metabolismo , Inosina Monofosfato/metabolismo , Pruebas de Sensibilidad Microbiana , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
Aiming at developing inhibitors of mannosyltransferases, the enzymes that participate in the biosynthesis of the cell envelope of Mycobacterium tuberculosis, the synthesis of a range of designed triazole-linked 1,6-oligomannosides up to a hexadecamer has been accomplished by a modular approach centered on the Cu(I)-catalyzed azide-alkyne cycloaddition as key process. The efficiency and fidelity of the cycloaddition are substantiated by high yields (76-96%) and exclusive formation of the expected 1,4-disubstituted triazole ring in all oligomer assembling reactions. Key features of oligomers thus prepared are the anomeric carbon-carbon bond of all mannoside residues and the 6-deoxymannoside capping residue. Suitable bioassays with dimer, tetramer, hexamer, octamer, decamer, and hexadecamer showed variable inhibitor activity against mycobacterial α-(1,6)-mannosyltransferases, the highest activity (IC(50) = 0.14-0.22 mM) being registered with the hexamannoside and octamannoside.