RESUMEN
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. tuberculosisM. 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.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/genética , Benzamidas/farmacología , Benzotiazoles/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Mycobacterium tuberculosis/efectos de los fármacos , Piridinas/farmacología , Antituberculosos/síntesis química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Benzamidas/síntesis química , Benzotiazoles/síntesis química , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Factores Cordón/antagonistas & inhibidores , Factores Cordón/biosíntesis , Factores Cordón/metabolismo , Farmacorresistencia Bacteriana/genética , Galactanos/metabolismo , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mutación , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/genética , Mycobacterium abscessus/crecimiento & desarrollo , Mycobacterium abscessus/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Piridinas/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuenciación Completa del GenomaRESUMEN
1H-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 1H-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 1H-benzo[d]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/genética , Bencimidazoles/farmacología , Factores Cordón/antagonistas & inhibidores , Farmacorresistencia Bacteriana/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Mycobacterium tuberculosis/efectos de los fármacos , Secuencias de Aminoácidos , Antituberculosos/síntesis química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bencimidazoles/síntesis química , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Clonación Molecular , Factores Cordón/biosíntesis , Factores Cordón/metabolismo , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Galactanos/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuenciación Completa del GenomaRESUMEN
Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.
Asunto(s)
Hidrolasas de Éster Carboxílico/química , Factores Cordón/biosíntesis , Esterasas/química , Mycobacterium/enzimología , Adenosina Trifosfato/química , Antibacterianos/farmacología , Antituberculosos/farmacología , Hidrolasas de Éster Carboxílico/metabolismo , Catálisis , Factores Cordón/química , Relación Dosis-Respuesta a Droga , Esterasas/metabolismo , Membrana Dobles de Lípidos/química , Lípidos/química , Trehalosa/químicaRESUMEN
The antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosis due to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosis virulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosis division in broth culture. I3-AG85 also inhibited M. tuberculosis survival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosis strains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosis envelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosis envelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosis strain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Factores Cordón/antagonistas & inhibidores , Factores Cordón/biosíntesis , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Animales , Antígenos Bacterianos , Células de la Médula Ósea/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Farmacorresistencia Bacteriana , Farmacorresistencia Bacteriana Múltiple , Femenino , Lípidos/biosíntesis , Macrófagos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Oxazinas , Proteínas Recombinantes/biosíntesis , Tioglucósidos/farmacología , Uracilo/metabolismo , XantenosRESUMEN
Cord formation of Mycobacterium tuberculosis complex is very uncommon in smear specimen prepared directly from sputum, although such a finding is well known in solid or liquid media and has recently been evaluated as a rapid method for presumptive identification in special liquid media (BACTEC or MGIT). We examined 308 (Mycobacterium tuberculosis 271 and Nontuberculous mycobacteria 37) positive smear specimens prepared directly from sputum in our hospital. These specimens all showed a "modified Gaffky scale" as +2 or more and this cord formation was found in four cases (five specimens). Each of these specimens was from a patient with severe lung tuberculosis showing cavity formation and each patient was complicated severe diabetes mellitus. The morphology of cord formation on smear specimens prepared directly from sputum was similar to that in liquid or solid media, and consequently the relevant bacilli were identified as Mycobacterium tuberculosis complex by PCR examination. In this study, we assessed "cord formation" in smear specimen prepared directly from sputum as a more rapid presumptive identification of Mycobacterium tuberculosis complex based on microscopic morphology, as well as cord formation in liquid or solid media.
Asunto(s)
Técnicas Bacteriológicas/métodos , Factores Cordón/biosíntesis , Mycobacterium tuberculosis/aislamiento & purificación , Esputo/microbiología , Anciano , Complicaciones de la Diabetes , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/metabolismo , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/microbiologíaRESUMEN
Ligation of mycolic acids to structural components of the mycobacterial cell wall generates a hydrophobic, impermeable barrier that provides resistance to toxic compounds such as antibiotics. Secreted proteins FbpA, FbpB, and FbpC attach mycolic acids to arabinogalactan, generating mycolic acid methyl esters (MAME) or trehalose, generating alpha,alpha'-trehalose dimycolate (TDM; also called cord factor). Our studies of Mycobacterium smegmatis showed that disruption of fbpA did not affect MAME levels but resulted in a 45% reduction of TDM. The fbpA mutant displayed increased sensitivity to both front-line tuberculosis-targeted drugs as well as other broad-spectrum antibiotics widely used for antibacterial chemotherapy. The irregular, hydrophobic surface of wild-type M. smegmatis colonies became hydrophilic and smooth in the mutant. While expression of M. smegmatis fbpA restored defects of the mutant, heterologous expression of the Mycobacterium tuberculosis fbpA gene was less effective. A single mutation in the M. smegmatis FbpA esterase domain inactivated its ability to provide antibiotic resistance. These data show that production of TDM by FbpA is essential for the intrinsic antibiotic resistance and normal colonial morphology of some mycobacteria and support the concept that FbpA-specific inhibitors, alone or in combination with other antibiotics, could provide an effective treatment to tuberculosis and other mycobacterial diseases.
Asunto(s)
Aciltransferasas/genética , Aciltransferasas/metabolismo , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Factores Cordón/metabolismo , Resistencia a Múltiples Medicamentos/genética , Mycobacterium smegmatis/genética , Aciltransferasas/química , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Pared Celular/fisiología , Factores Cordón/biosíntesis , Elementos Transponibles de ADN , Esterasas/química , Esterasas/genética , Esterasas/metabolismo , Biblioteca de Genes , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Mutación , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/metabolismo , Fenotipo , Estructura Terciaria de ProteínaRESUMEN
By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C. glutamicum strain ATCC 17965. The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 ( cmt from corynebacterium mycolyl transferases). cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids. Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains. Proteome analyses of the cell envelope of C. glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4. All six mycolyltransferase genes were used for mutational analysis. cmt4 could not be mutated and is considered to be essential. cop1 was found to play an additional role in cell shape formation. A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium. This mutant was also no longer able to synthesize trehalose di coryno mycolate (TDCM). Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis. The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Factores Cordón/biosíntesis , Corynebacterium/enzimología , ADN (Citosina-5-)-Metiltransferasas , Metilasas de Modificación del ADN/metabolismo , Ácidos Micólicos/química , Ubiquitina-Proteína Ligasas , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Corynebacterium/citología , Corynebacterium/genética , Metilasas de Modificación del ADN/aislamiento & purificación , Eliminación de Gen , Genoma Bacteriano , FilogeniaRESUMEN
A total of 1208 positive BACTEC vials were examined for the presence or absence of serpentine cording. A very high (92.9%) rate of laboratory prevalence was obtained for Mycobacterium tuberculosis complex. The sensitivity, specificity, positive and negative predictive values of this test were 92.7%, 95.3%, 99.6% and 50.0%, respectively. It was concluded that testing cord formation in laboratories that have a high prevalence of Mycobacterium tuberculosis complex is an exceptionally reliable method for preliminary reporting of cording-positive cases; however, for cording-negative cases, preliminary reports based solely on cord formation are not reliable. It was also observed that the length of the incubation period has a significant effect on cord formation. Incubation periods of 4 days or less are not sufficient to determine noncording in smears prepared from positive BACTEC vials.
Asunto(s)
Técnicas Bacteriológicas/métodos , Factores Cordón/biosíntesis , Medios de Cultivo/metabolismo , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/metabolismo , Humanos , Laboratorios , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Tuberculosis/diagnósticoRESUMEN
We evaluated cord formation in BACTEC media as a criterion for presumptive identification of Mycobacterium tuberculosis complex. Auramine-rhodamine smears from 673 radiometrically positive BACTEC vials were examined. The presence of cording had a sensitivity, a specificity, and positive and negative predictive values of 22.9, 98.5, 82.9, and 80.3%, respectively. Before cord formation is used to report presumptive identification of M. tuberculosis complex, workers should determine the reliability of this finding in their own laboratories.
Asunto(s)
Factores Cordón/biosíntesis , Mycobacterium tuberculosis/aislamiento & purificación , Medios de Cultivo , Humanos , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismoRESUMEN
We evaluated cord formation in BACTEC 7H12 medium as a criterion for rapid identification of Mycobacterium tuberculosis complex. Kinyoun-stained smears, prepared from 270 radiometrically positive BACTEC 7H12 bottles, were examined independently by three observers. Smears from 93.2, 88.6, and 83.0% of the M. tuberculosis complex cultures were read as cord positive, and smears from 97.3, 97.8, and 99.5% of the mycobacteria other than M. tuberculosis cultures were read as cord negative by the three observers, respectively. There was 93.3% agreement between the observers. The presence of cords in BACTEC 7H12 medium can be a reliable criterion for rapid, presumptive identification of M. tuberculosis complex.
Asunto(s)
Factores Cordón/biosíntesis , Medios de Cultivo , Glucolípidos/biosíntesis , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/metabolismo , Tuberculosis/diagnóstico , Humanos , Radiometría , Sensibilidad y EspecificidadRESUMEN
When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids.
Asunto(s)
Actinomycetaceae/metabolismo , Glucosa/fisiología , Ácidos Micólicos/biosíntesis , Actinomycetaceae/ultraestructura , Sitios de Unión , Carbohidratos/fisiología , Pared Celular/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Factores Cordón/biosíntesis , Glucolípidos/biosíntesis , Microscopía Electrónica , Factores de TiempoRESUMEN
The fluffy layer fraction prepared from Bacterionema matruchotii was found to possess high activity for the biosynthesis of mycolic acids which were bound to an unknown compound by an alkali-labile linkage [T. Shimakata, M. Iwaki, and T. Kusaka (1984) Arch. Biochem. Biophys. 229, 329-339]. To determine the structure of the mycolate-containing compound, it was purified and analyzed by field desorption (FD) and secondary ion mass spectrometry (SI-MS). When non-labelled palmitic acid was used as a precursor in the in vitro biosynthetic system, the underivatized product had a cationized molecular ion, [M + Na]+, at m/z 843 in FD-MS and a protonated ion, [M + H]+, at m/z 821 in SI-MS, corresponding to the quasimolecular ion of trehalose monomycolate (C32:0). In SI-MS, characteristic fragment ions due to cleavage of glycosidic linkages were clearly detected in addition to the molecular ion. If [1-13C]palmitic acid was the precursor, 2 mass unit increases in both the quasimolecular and fragment ions were observed, indicating that two molecules of palmitate were incorporated into the product. alpha-Trehalose was found in the aqueous phase after saponification of the product. By the electron impact mass spectrometry of the trimethylsilylated product, the mycolate was found to be esterified with an hydroxyl group at position 6 of the trehalose molecule. These results clearly demonstrated that the predominant product synthesized by the fluffy layer fraction with palmitate as substrate was 6-monomycolate (C32:0) of alpha-D-trehalose. Because newly synthesized mycolic acid was mainly in the form of trehalose monomycolate instead of free mycolate or trehalose dimycolate, the role of trehalose in the biosynthesis of mycolic acid is discussed.
Asunto(s)
Actinomycetaceae/metabolismo , Factores Cordón/biosíntesis , Glucolípidos/biosíntesis , Sistema Libre de Células , Espectrometría de Masas , Ácidos Micólicos/metabolismoRESUMEN
Appropriate solvolysis of 2,3,2',3'-tetra-O-benzyl-4,6,4', 6'-tetra-O-mesyl-alpha,alpha-trehalose gave 2,3,2',3' -tetra-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (2). Selective tosylation or mesylation of 2 respectively gave the 6, 6'-ditosylate (3) and 6,6'-dimesylate (4), the structures of which were confirmed by the 1H-n.m.r. spectra of the corresponding 4,4'-di-O-acetyl derivatives. Treatment of 3 with potassium mycolate in toluene, and subsequent hydrogenolysis, gave the 6'-mycolate 6-tosylate derivative. Treatment of 3 with potassium mycolate or potassium corynomycolate in hexamethylphosphoric triamide, followed by catalytic hydrogenolysis, yielded the respective cord-factor analogs 6,6'-di-O-mycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) and 6,6'-di-O-corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside). The same 6,6'-diesters were obtained from the 6,6'-dimesylate 4. Putative 4,6-anhydro-6'-monomycolates are also described.