RESUMEN
C-type lectin receptors (CLRs) are a family of pattern recognition receptors, which detect a broad spectrum of ligands via small carbohydrate-recognition domains (CRDs). CLEC12A is an inhibitory CLR that recognizes crystalline structures such as monosodium urate crystals. CLEC12A also recognizes mycolic acid, a major component of mycobacterial cell walls, and suppresses host immune responses. Although CLEC12A could be a therapeutic target for mycobacterial infection, structural information on CLEC12A was not available. We report here the crystal structures of human CLEC12A (hCLEC12A) in ligand-free form and in complex with 50C1, its inhibitory antibody. 50C1 recognizes human-specific residues on the top face of hCLEC12A CRD. A comprehensive alanine scan demonstrated that the ligand-binding sites of mycolic acid and monosodium urate crystals may overlap with each other, suggesting that CLEC12A utilizes a common interface to recognize different types of ligands. Our results provide atomic insights into the blocking and ligand-recognition mechanisms of CLEC12A and leads to the design of CLR-specific inhibitors.
Asunto(s)
Lectinas Tipo C , Receptores Mitogénicos , Lectinas Tipo C/inmunología , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Humanos , Receptores Mitogénicos/química , Receptores Mitogénicos/inmunología , Receptores Mitogénicos/metabolismo , Cristalografía por Rayos X , Ligandos , Unión Proteica , Sitios de Unión , Modelos Moleculares , Ácido Úrico/química , Ácido Úrico/metabolismo , Ácido Úrico/inmunologíaRESUMEN
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 ß-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 C16 to C24, 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium/metabolismo , Ácidos Micólicos/metabolismo , Racemasas y Epimerasas/metabolismo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/fisiología , Pared Celular/metabolismo , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos/fisiología , Infecciones por Mycobacterium , Mycobacterium tuberculosis/metabolismo , Racemasas y Epimerasas/fisiología , Factores de Transcripción/metabolismoRESUMEN
The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium. Mycobacterium smegmatis is a nonpathogenic and fast growing species within this genus. In 1990, a mutant of M. smegmatis, designated mc2155, that could be transformed with episomal plasmids was isolated, elevating M. smegmatis to model status as the ideal surrogate for mycobacterial research. Classical bacterial models, such as Escherichia coli, were inadequate for mycobacteria research because they have low genetic conservation, different physiology, and lack the novel envelope structure that distinguishes the Mycobacterium genus. By contrast, M. smegmatis encodes thousands of conserved mycobacterial gene orthologs and has the same cell architecture and physiology. Dissection and characterization of conserved genes, structures, and processes in genetically tractable M. smegmatis mc2155 have since provided previously unattainable insights on these same features in its slow-growing relatives. Notably, tuberculosis (TB) drugs, including the first-line drugs isoniazid and ethambutol, are active against M. smegmatis, but not against E. coli, allowing the identification of their physiological targets. Furthermore, Bedaquiline, the first new TB drug in 40 years, was discovered through an M. smegmatis screen. M. smegmatis has become a model bacterium, not only for M. tuberculosis, but for all other Mycobacterium species and related genera. With a repertoire of bioinformatic and physical resources, including the recently established Mycobacterial Systems Resource, M. smegmatis will continue to accelerate mycobacterial research and advance the field of microbiology.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium smegmatis/genética , Escherichia coli/genética , Mycobacterium tuberculosis/genética , IsoniazidaRESUMEN
Mycobacterium fortuitum represents one of the most clinically relevant rapid-growing mycobacterial species. Treatments are complex due to antibiotic resistance and to severe side effects of effective drugs, prolonged time of treatment, and co-infection with other pathogens. Herein, we explored the activity of NITD-916, a direct inhibitor of the enoyl-ACP reductase InhA of the type II fatty acid synthase in Mycobacterium tuberculosis. We found that this compound displayed very low MIC values against a panel of M. fortuitum clinical strains and exerted potent antimicrobial activity against M. fortuitum in macrophages. Remarkably, the compound was also highly efficacious in a zebrafish model of infection. Short duration treatments were sufficient to significantly protect the infected larvae from M. fortuitum-induced killing, which correlated with reduced bacterial burdens and abscesses. Biochemical analyses demonstrated an inhibition of de novo synthesis of mycolic acids. Resolving the crystal structure of the InhAMFO in complex with NAD and NITD-916 confirmed that NITD-916 is a direct inhibitor of InhAMFO. Importantly, single nucleotide polymorphism leading to a G96S substitution in InhAMFO conferred high resistance levels to NITD-916, thus resolving its target in M. fortuitum. Overall, these findings indicate that NITD-916 is highly active against M. fortuitum both in vitro and in vivo and should be considered in future preclinical evaluations for the treatment of M. fortuitum pulmonary diseases.
Asunto(s)
Mycobacterium fortuitum , Mycobacterium tuberculosis , Animales , Pez Cebra , Ácidos Micólicos/farmacología , OxidorreductasasRESUMEN
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 MmpL11TB 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.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Pared Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/fisiología , Ácidos Micólicos/metabolismo , Periplasma/metabolismo , Fosforilación , Sideróforos/metabolismo , Tuberculosis/microbiología , Factores de Virulencia/metabolismoRESUMEN
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 Δcg1246 mutant strain, suggesting that in vivo, the phosphatase activity is not involved in mycolic acid biosynthesis.
Asunto(s)
Corynebacterium glutamicum , Ácidos Micólicos , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Corynebacterium glutamicum/metabolismo , Ácidos Micólicos/metabolismo , Nucleósidos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Trehalosa/metabolismoRESUMEN
Mycolic acids (MAs) are unique components of cell envelope of Mycobacterium or Corynebacterium and are key factors of their virulence to human. In order to develop new anti-Tuberculosis (TB) drugs, many efforts have paid on investigation of structures and functions of proteins involved in the biosynthesis pathway of MAs. FadD32 and polyketide synthase 13 (pks13) catalyze the last step of MAs synthesis. Here we present the crystal structures of FadD32 with substrates and holo-form of ACP-domain from Corynebacterium diphtheriae. The crystal structures and in vitro biochemical assays provide new insights into the assembly of FadD32 and pks13.
Asunto(s)
Proteínas Bacterianas/química , Corynebacterium diphtheriae/metabolismo , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Unión Proteica , Dominios ProteicosRESUMEN
The acylation of sugars, most commonly via acetylation, is a widely used mechanism in bacteria that uses a simple chemical modification to confer useful traits. For structures like lipopolysaccharide, capsule and peptidoglycan, that function outside of the cytoplasm, their acylation during export or post-synthesis requires transport of an activated acyl group across the membrane. In bacteria this function is most commonly linked to a family of integral membrane proteins - acyltransferase-3 (AT3). Numerous studies examining production of diverse extracytoplasmic sugar-containing structures have identified roles for these proteins in O-acylation. Many of the phenotypes conferred by the action of AT3 proteins influence host colonisation and environmental survival, as well as controlling the properties of biotechnologically important polysaccharides and the modification of antibiotics and antitumour drugs by Actinobacteria. Herein we present the first systematic review, to our knowledge, of the functions of bacterial AT3 proteins, revealing an important protein family involved in a plethora of systems of importance to bacterial function that is still relatively poorly understood at the mechanistic level. By defining and comparing this set of functions we draw out common themes in the structure and mechanism of this fascinating family of membrane-bound enzymes, which, due to their role in host colonisation in many pathogens, could offer novel targets for the development of antimicrobials.
Asunto(s)
Aciltransferasas , Peptidoglicano , Acetilación , Acilación , Aciltransferasas/genética , Aciltransferasas/metabolismo , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Peptidoglicano/metabolismoRESUMEN
Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. KEY POINTS: ⢠Improved vector for homologous recombination in R. opacus. ⢠Mycolic acid is important for survival of R. opacus like it is for Mycobacterium. ⢠Similar conditional suicide plasmids may be constructed for other bacteria.
Asunto(s)
Ácidos Micólicos , Rhodococcus , Mutagénesis Sitio-Dirigida , Ácidos Micólicos/metabolismo , Plásmidos/genética , Recombinasas/genética , Rhodococcus/genética , Rhodococcus/metabolismo , ARN Guía de Kinetoplastida , Triglicéridos/metabolismoRESUMEN
The development of amine-functionalized graphene quantum dots (GQDs) linked to mycolic acids (MAs) as a potential fluorescent biosensor to detect tuberculosis (TB) biomarkers is described. GQDs have attractive properties: high fluorescence, excellent biocompatibility, good water solubility, and low toxicity. MAs are lipids that are found in the cell wall of Mycobacterium tuberculosis that are antigenic, however, they are soluble only in chloroform and hexane. Chloroform-soluble MAs were covalently linked to synthesized water-soluble GQDs using an amide connection to create a potential fluorescent water-soluble TB biosensor: MA-GQDs. Fluorescence results showed that GQDs had a narrow emission spectrum with the highest emission at 440 nm, while MA-GQDs had a broader spectrum with the highest emission at 470 nm, after exciting at 360 nm. The appearance of the peptide bond (amide linkage) in the Fourier-transform infrared spectrum of MA-GQDs confirmed the successful linking of MAs to GQDs. Powder X-ray diffraction exhibited an increase in the number of peaks for MA-GQDs relative to GQDs, suggesting that linking MAs to GQDs changed the crystal structure thereof. The linked MA-GQDs showed good solubility in water, high fluorescence, and visual flow through a nitrocellulose membrane. These properties are promising for biomedical fluorescence sensing applications.
Asunto(s)
Técnicas Biosensibles , Grafito , Puntos Cuánticos , Tuberculosis , Humanos , Puntos Cuánticos/química , Grafito/química , Ácidos Micólicos , Cloroformo , Agua/química , AmidasRESUMEN
Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the serine/threonine phosphatase PstP, in the model organism Mycobacterium smegmatis We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phosphomimetic mutation, pstP T171E, slows growth, misregulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis.IMPORTANCE Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Pared Celular/fisiología , Mycobacterium smegmatis/efectos de los fármacos , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Modelos Moleculares , Mycobacterium smegmatis/fisiología , Peptidoglicano/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosforilación , Conformación ProteicaRESUMEN
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.
Asunto(s)
Mycobacterium , Ácidos Micólicos/metabolismo , Oxidorreductasas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Modelos Moleculares , Mycobacterium/genética , Mycobacterium/crecimiento & desarrollo , Mycobacterium/metabolismo , Mycobacterium bovis/genética , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium bovis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismoRESUMEN
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/ß-hydrolase domain. The LGFP motifs are composed of a 4-stranded ß-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.
Asunto(s)
Aciltransferasas/metabolismo , Membrana Externa Bacteriana/metabolismo , Pared Celular/metabolismo , Corynebacterium glutamicum/metabolismo , Dominios Proteicos/fisiología , Aciltransferasas/genética , Sitios de Unión/fisiología , Corynebacterium glutamicum/genética , Cristalografía por Rayos X , Galactanos/metabolismo , Ácidos Micólicos/metabolismo , Oligopéptidos/metabolismo , Peptidoglicano/metabolismo , Conformación ProteicaRESUMEN
Tuberculosis is one of the leading causes of death across the world. The treatment regimens for tuberculosis are well established, but still the control of the disease faces many challenges such as lengthy treatment protocols, drug resistance and toxicity. In the present work, mycolic acid methyl transferase (MmaA1), a protein involved in the maturation of mycolic acids in the biochemical pathway of the Mycobacterium, was studied for novel drug discovery. The homology model of the MmaA1 protein was built and validated by using computational techniques. The MmaA1 protein has 286 amino acid residues consisting of 10 α-helices and 7 ß-sheets. The active site of the MmaA1 protein was identified using CASTp, SiteMap and PatchDock. Virtual screening studies were performed with two small molecule ligand databases: Asinex synergy and Diverse_Elite_Gold_Platinum databases having a total of 43,446 molecules and generated 1,30,814 conformers against the predicted and validated active site of the MmaA1 protein. Binding analysis showed that the residues ASP 19, PHE 22, TRP 30, TYR 32, TRP 74 and ALA 77 of MmaA1 protein have consistent interactions with the ligands. The hit ligands were further filtered by in silico ADME properties to eliminate potentially toxic molecules. Of the top 10 molecules, 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl) benzamide was synthesised and screened for in vitro anti-TB activity against Mtb H37Rv using MABA assay. The compound and its intermediates exhibited good in vitro anti-TB activity which can be taken up for future lead optimisation studies. Structure based virtual screening study was performed using a validated homology model against small molecules from two virtual compound libraries. Synthesised the lead compound 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl)benzamide obtained from virtual screening. In vitro activity against Mtb H37Rv has given a promising result.
Asunto(s)
Antituberculosos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Metiltransferasas/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/análisis , Ligandos , Metiltransferasas/química , Metiltransferasas/metabolismo , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Estructura Secundaria de Proteína , Reproducibilidad de los Resultados , Relación Estructura-ActividadRESUMEN
Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.
Asunto(s)
Pared Celular/metabolismo , Factores Cordón/metabolismo , Corynebacterium glutamicum/crecimiento & desarrollo , Colorantes Fluorescentes/química , Procesamiento de Imagen Asistido por Computador/métodos , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/diagnóstico , Proteínas Bacterianas/metabolismo , División Celular , Fluorescencia , Humanos , Peptidoglicano/metabolismo , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
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.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Proteínas de la Membrana/metabolismo , Mutación , Mycobacterium smegmatis/fisiología , Ácidos Micólicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Prueba de Complementación Genética , Espectrometría de Masas , Proteínas de la Membrana/genética , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica , Mutagénesis Sitio-Dirigida , Mycobacterium smegmatis/metabolismoRESUMEN
Actinobacteria is a group of diverse bacteria. Most species in this class of bacteria are filamentous aerobes found in soil, including the genus Streptomyces perhaps best known for their fascinating capabilities of producing antibiotics. These bacteria typically have a Gram-positive cell envelope, comprised of a plasma membrane and a thick peptidoglycan layer. However, there is a notable exception of the Corynebacteriales order, which has evolved a unique type of outer membrane likely as a consequence of convergent evolution. In this chapter, we will focus on the unique cell envelope of this order. This cell envelope features the peptidoglycan layer that is covalently modified by an additional layer of arabinogalactan . Furthermore, the arabinogalactan layer provides the platform for the covalent attachment of mycolic acids , some of the longest natural fatty acids that can contain ~100 carbon atoms per molecule. Mycolic acids are thought to be the main component of the outer membrane, which is composed of many additional lipids including trehalose dimycolate, also known as the cord factor. Importantly, a subset of bacteria in the Corynebacteriales order are pathogens of human and domestic animals, including Mycobacterium tuberculosis. The surface coat of these pathogens are the first point of contact with the host immune system, and we now know a number of host receptors specific to molecular patterns exposed on the pathogen's surface, highlighting the importance of understanding how the cell envelope of Actinobacteria is structured and constructed. This chapter describes the main structural and biosynthetic features of major components found in the actinobacterial cell envelopes and highlights the key differences between them.
Asunto(s)
Actinobacteria/citología , Membrana Celular/química , Membrana Celular/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Animales , Humanos , Mycobacterium tuberculosis/patogenicidad , Ácidos Micólicos/metabolismo , Peptidoglicano/metabolismoRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Proteínas de Transporte de Membrana/metabolismo , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Micobacterias no Tuberculosas/metabolismo , Antibacterianos/uso terapéutico , Proteínas Bacterianas/metabolismo , Transporte Biológico/efectos de los fármacos , Descubrimiento de Drogas , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Yodóforos/farmacología , Yodóforos/uso terapéutico , Isoxazoles/farmacología , Isoxazoles/uso terapéutico , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Ácidos Micólicos/metabolismo , Micobacterias no Tuberculosas/efectos de los fármacos , Polisacáridos/farmacología , Polisacáridos/uso terapéutico , Compuestos de Espiro/farmacología , Compuestos de Espiro/uso terapéuticoRESUMEN
Cell-penetrating peptides might have great potential for enhancing the therapeutic effect of drug molecules against such dangerous pathogens as Mycobacterium tuberculosis (Mtb), which causes a major health problem worldwide. A set of cationic cell-penetration peptides with various hydrophobicity were selected and synthesized as drug carrier of isoniazid (INH), a first-line antibacterial agent against tuberculosis. Molecular interactions between the peptides and their INH-conjugates with cell-membrane-forming lipid layers composed of DPPC and mycolic acid (a characteristic component of Mtb cell wall) were evaluated, using the Langmuir balance technique. Secondary structure of the INH conjugates was analyzed and compared to that of the native peptides by circular dichroism spectroscopic experiments performed in aqueous and membrane mimetic environment. A correlation was found between the conjugation induced conformational and membrane affinity changes of the INH-peptide conjugates. The degree and mode of interaction were also characterized by AFM imaging of penetrated lipid layers. In vitro biological evaluation was performed with Penetratin and Transportan conjugates. Results showed similar internalization rate into EBC-1 human squamous cell carcinoma, but markedly different subcellular localization and activity on intracellular Mtb.
Asunto(s)
Antituberculosos/administración & dosificación , Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos/metabolismo , Isoniazida/administración & dosificación , Lípidos de la Membrana/metabolismo , Secuencia de Aminoácidos , Antituberculosos/química , Antituberculosos/farmacocinética , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Portadores de Fármacos/química , Humanos , Isoniazida/química , Isoniazida/farmacocinética , Membrana Dobles de Lípidos/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológicoRESUMEN
In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters ("cord factor") form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram-scale synthesis of four stereo-isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn-methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material.