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1.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-35165190

RESUMEN

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/metabolismo
2.
Biomacromolecules ; 25(1): 413-424, 2024 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-38124388

RESUMEN

Bacteriophages have many biotechnological and therapeutic applications, but as with other biologics, cryopreservation is essential for storage and distribution. Macromolecular cryoprotectants are emerging for a range of biologics, but the chemical space for polymer-mediated phage cryopreservation has not been explored. Here we screen the cryoprotective effect of a panel of polymers against five distinct phages, showing that nearly all the tested polymers provide a benefit. Exceptions were poly(methacrylic acid) and poly(acrylic acid), which can inhibit phage-infection with bacteria, making post-thaw recovery challenging to assess. A particular benefit of a polymeric cryopreservation formulation is that the polymers do not function as carbon sources for the phage hosts (bacteria) and hence do not interfere with post-thaw measurements. This work shows that phages are amenable to protection with hydrophilic polymers and opens up new opportunities for advanced formulations for future phage therapies and to take advantage of the additional functionality brought by the polymers.


Asunto(s)
Bacteriófagos , Productos Biológicos , Polímeros/farmacología , Polímeros/química , Criopreservación , Bacterias , Crioprotectores/farmacología , Crioprotectores/química
3.
Biochem Biophys Res Commun ; 624: 120-126, 2022 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-35940124

RESUMEN

Cysteine plays a versatile role in cellular physiology and has previously been shown to be instrumental to Mycobacterium tuberculosis (M.tb) pathophysiology. In this study, we have generated mutants deficient in CysK2 and CysH, the key Cysteine, biosynthetic enzymes. In contrast to the ΔcysH mutant, the ΔcysK2 mutant is not an auxotroph and as such not essential for cysteine biosynthesis. Interestingly, the ΔcysK2 mutant shows increased sensitivity to cumene hydroperoxide, vitamin C, diamide, rifampicin and Vancomycin and shows alterations in phospholipid profile of Mtb cell wall. Our findings suggest that alteration in phospholipids content of M.tb cell wall by CysK2 may form a mode of defence against selected antibiotics and oxidative stress.


Asunto(s)
Mycobacterium tuberculosis , Pared Celular , Cisteína/genética , Mycobacterium tuberculosis/genética , Fosfolípidos , Vancomicina/farmacología
4.
Mol Syst Biol ; 17(5): e10280, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33943004

RESUMEN

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.


Asunto(s)
Carbono/metabolismo , Colesterol/metabolismo , Glicerol/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , Técnicas Bacteriológicas , Ciclo del Ácido Cítrico , Glioxilatos/metabolismo , Marcaje Isotópico , Metabolismo de los Lípidos , Redes y Vías Metabólicas , Mycobacterium tuberculosis/metabolismo , Fenotipo
5.
FASEB J ; 35(4): e21475, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33772870

RESUMEN

Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes are not entirely understood. Herein we present the report of a c-di-GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c-di-GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS-PdtaR (Rv1626) two-component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems' approach of transcriptomics, unbiased metabolomics, and lipid analyses.


Asunto(s)
Carbono/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Histidina Quinasa/metabolismo , Bacterias , Proteínas Bacterianas/metabolismo , Simulación del Acoplamiento Molecular/métodos , Mycobacterium/metabolismo , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/fisiología
6.
Mol Microbiol ; 113(2): 521-533, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31785114

RESUMEN

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/metabolismo
7.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669411

RESUMEN

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.


Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/metabolismo , Coinfección/metabolismo , Glucolípidos/metabolismo , VIH-1/fisiología , Liposomas/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculosis/metabolismo , Infecciones Oportunistas Relacionadas con el SIDA/virología , Moléculas de Adhesión Celular/metabolismo , Pared Celular/metabolismo , Células HEK293 , Humanos , Lectinas Tipo C/metabolismo , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium bovis/metabolismo , Mycobacterium smegmatis/metabolismo , Receptores de Superficie Celular/metabolismo , Tuberculosis/microbiología , Internalización del Virus
8.
Microbiology (Reading) ; 166(9): 817-825, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32678058

RESUMEN

Mycobacterial cells elongate via polar deposition of cell wall material, similar to the filamentous Streptomyces species, which contain a tip-organizing centre. Coiled-coiled proteins such as DivIVA play an important role in this process. The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes many coiled-coil proteins that are homologous to DivIVA with a potential role in mycobacterial cell elongation. Here we describe studies on Mycobacterium smegmatis MSMEG_2416, a homologue of M. tuberculosis Rv2927c. Two previous independent studies showed that MSMEG_2416 was involved in septation (subsequently referred to as sepIVA). Contrary to these previous reports, we found sepIVA to be dispensable for growth in laboratory media by generating a viable null mutant. The mutant strain did, however, show a number of differences, including a change in colony morphology and biofilm formation that could be reversed on complementation with sepIVA as well as Rv2927c, the sepIVA homologue from M. tuberculosis. However, analysis of cell wall lipids did not reveal any alterations in lipid profiles of the mutant strain. Microscopic examination of the mutant revealed longer cells with more septa, which occurred at irregular intervals, often generating mini-compartments, a profile similar to that observed in the previous studies following conditional depletion, highlighting a role for sepIVA in mycobacterial growth.


Asunto(s)
Proteínas Bacterianas/metabolismo , División Celular , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Pared Celular/química , Eliminación de Gen , Genes Bacterianos , Lípidos/análisis , Mutación , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Dominios Proteicos
9.
Mol Syst Biol ; 15(3): e8584, 2019 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-30833303

RESUMEN

The success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA Using Path-seq and regulatory network analyses, we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/desA2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection.


Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Redes Reguladoras de Genes , Interacciones Huésped-Patógeno , Macrófagos/inmunología , Mycobacterium tuberculosis/fisiología , Tuberculosis/microbiología , Adaptación Fisiológica , Animales , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Macrófagos/microbiología , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Ácidos Micólicos/metabolismo , Biología de Sistemas , Tuberculosis/inmunología
10.
J Biol Chem ; 293(15): 5695-5704, 2018 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-29475946

RESUMEN

Enzymes at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and 13C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, Mycobacterium tuberculosis (Mtb). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO2 through the ANA node is essential for intracellular survival of Mtb and that Mtb possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of Mtb, which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.


Asunto(s)
Proteínas Bacterianas , Macrófagos , Viabilidad Microbiana , Mycobacterium tuberculosis , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/patología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Células THP-1
11.
BMC Genomics ; 20(1): 431, 2019 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-31138110

RESUMEN

BACKGROUND: BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG's efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host's immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria. RESULTS: To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. CONCLUSION: These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host's innate immune system to allow it to persist, a property that is important for its protective efficacy.


Asunto(s)
Elementos Transponibles de ADN , Mycobacterium bovis/genética , Animales , Vacuna BCG , Bovinos , Colesterol/metabolismo , Biblioteca de Genes , Genes Bacterianos , Aptitud Genética , Mycobacterium bovis/metabolismo , Oxazoles , Azúcares/metabolismo , Sulfatos/metabolismo , Tuberculosis Bovina/microbiología
12.
Infect Immun ; 85(7)2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28483856

RESUMEN

Nontoxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans cause invasive disease in humans and animals. Host sensing of corynebacteria is largely uncharacterized, albeit the recognition of lipoglycans by Toll-like receptor 2 (TLR2) appears to be important for macrophage activation by corynebacteria. The members of the order Corynebacterineae (e.g., mycobacteria, nocardia, and rhodococci) share a glycolipid-rich cell wall dominated by mycolic acids (termed corynomycolic acids in corynebacteria). The mycolic acid-containing cord factor of mycobacteria, trehalose dimycolate, activates the C-type lectin receptor (CLR) Mincle. Here, we show that glycolipid extracts from the cell walls of several pathogenic and nonpathogenic Corynebacterium strains directly bound to recombinant Mincle in vitro Macrophages deficient in Mincle or its adapter protein Fc receptor gamma chain (FcRγ) produced severely reduced amounts of granulocyte colony-stimulating factor (G-CSF) and of nitric oxide (NO) upon challenge with corynebacterial glycolipids. Consistently, cell wall extracts of a particular C. diphtheriae strain (DSM43989) lacking mycolic acid esters neither bound Mincle nor activated macrophages. Furthermore, TLR2 but not TLR4 was critical for sensing of cell wall extracts and whole corynebacteria. The upregulation of Mincle expression upon encountering corynebacteria required TLR2. Thus, macrophage activation by the corynebacterial cell wall relies on TLR2-driven robust Mincle expression and the cooperative action of both receptors.


Asunto(s)
Pared Celular/inmunología , Corynebacterium/inmunología , Glucolípidos/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Pared Celular/química , Corynebacterium/química , Glucolípidos/aislamiento & purificación , Factor Estimulante de Colonias de Granulocitos/metabolismo , Macrófagos/inmunología , Ratones , Ratones Noqueados , Óxido Nítrico/metabolismo , Unión Proteica
13.
Mol Microbiol ; 102(1): 168-82, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27349932

RESUMEN

Persistent R-loops lead to replicative stress due to RNA polymerase stalling and DNA damage. RNase H enzymes facilitate the organisms to survive in the hostile condition by removing these R-loops. MS_RHII-RSD was previously identified to be the second (p)ppGpp synthetase in Mycobacterium smegmatis. The unique presence of an additional RNase HII domain raises an important question regarding the significance of this bifunctional protein. In this report, we demonstrate its ability to hydrolyze R-loops in Escherichia coli exposed to UV stress. MS_RHII-RSD gene expression was upregulated under UV stress, and this gene deleted strain showed increased R-loop accumulation as compared to the wild type. The domains in isolation are known to be inactive, and the full length protein is required for its function. Domain interdependence studies using active site mutants reveal the necessity of a hexamer form with high alpha helical content. In previous studies, bacterial RNase type HI has been mainly implicated in R-loop hydrolysis, but in this study, the RNase HII domain containing protein showed the activity. The prospective of this differential RNase HII activity is discussed. This is the first report to implicate a (p)ppGpp synthetase protein in R-loop-induced stress response.


Asunto(s)
Ligasas/metabolismo , Mycobacterium smegmatis/metabolismo , Proteínas Bacterianas/metabolismo , Dominio Catalítico , ARN Polimerasas Dirigidas por ADN/metabolismo , Hidrólisis , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Dominios Proteicos , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Estrés Fisiológico/fisiología , Especificidad por Sustrato
14.
Microbiology (Reading) ; 163(3): 373-382, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28141495

RESUMEN

The mycobacterial cell envelope is unique in its chemical composition, and has an important role to play in pathogenesis. Phthiocerol dimycocerosates (PDIMs) and glycosylated phenolphthiocerol dimycocerosates, also known as phenolic glycolipids (PGLs), contribute significantly to the virulence of Mycobacterium tuberculosis. FadD22 is essential for PGL biosynthesis. We have recently shown in vitro that FadD22 is a substrate for lysine acylation by a unique cAMP-dependent, protein lysine acyltransferase found only in mycobacteria. The lysine residue that is acylated is at the active site of FadD22. Therefore, acylation is likely to inhibit FadD22 activity and reduce PGL biosynthesis. Here, we show accumulation of PGLs in a strain of M. bovis BCG deleted for the gene encoding the cAMP-dependent acyltransferase, katbcg, with no change seen in PDIM synthesis. Complementation using KATbcg mutants that are deficient in cAMP-binding or acyltransferase activity shows that PGL accumulation is regulated by cAMP-dependent protein acylation in vivo. Expression of FadD22 and KATbcg mutants in Mycobacterium smegmatis confirmed that FadD22 is a substrate for lysine acylation by KATbcg. We have therefore described a mechanism by which cAMP can regulate mycobacterial virulence as a result of the ability of this second messenger to modulate critical cell wall components that affect the host immune response.


Asunto(s)
Proteínas Bacterianas/metabolismo , Glucolípidos/biosíntesis , Ligasas/metabolismo , Lisina Acetiltransferasas/metabolismo , Mycobacterium bovis/genética , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/patogenicidad , Acilación , Antígenos Bacterianos/biosíntesis , Membrana Celular/metabolismo , Pared Celular/metabolismo , AMP Cíclico/metabolismo , Lisina/metabolismo , Lisina Acetiltransferasas/genética , Mycobacterium bovis/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Factores de Virulencia/genética
15.
Mol Microbiol ; 98(1): 7-16, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26135034

RESUMEN

Mycolic acids are unique long chain fatty acids found in the lipid-rich cell walls of mycobacteria including the tubercle bacillus Mycobacterium tuberculosis. Essential for viability and virulence, enzymes involved in the biosynthesis of mycolic acids represent novel targets for drug development. This is particularly relevant to the impact on global health given the rise of multidrug resistant and extensively drug resistant strains of M. tuberculosis. In this review, we discuss recent advances in our understanding of how mycolic acid are synthesised, especially the potential role of specialised fatty acid synthase complexes. Also, we examine the role of a recently reported mycolic acid transporter MmpL3 with reference to several reports of the targeting of this transporter by diverse compounds with anti-M. tuberculosis activity. Additionally, we consider recent findings that place mycolic acid biosynthesis in the context of the cell biology of the bacterium, viz its localisation and co-ordination with the bacterial cytoskeleton, and its role beyond maintaining cell envelope integrity.


Asunto(s)
Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Descubrimiento de Drogas , Ácido Graso Sintasas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/patogenicidad , Ácidos Micólicos/química , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Virulencia
16.
J Biol Chem ; 289(9): 6177-87, 2014 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-24446451

RESUMEN

Benzothiazinones (BTZs) are a new class of sulfur containing heterocyclic compounds that target DprE1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (DPR) to decaprenyl-phosphoarabinose (DPA) in the Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. As a result, BTZ inhibition leads to inhibition of cell wall arabinan biosynthesis. Previous studies have demonstrated the essentiality of dprE1. In contrast, Cg-UbiA a ribosyltransferase, which catalyzes the first step of DPR biosynthesis prior to DprE1, when genetically disrupted, produced a viable mutant, suggesting that although BTZ biochemically targets DprE1, killing also occurs through chemical synthetic lethality, presumably through the lack of decaprenyl phosphate recycling. To test this hypothesis, a derivative of BTZ, BTZ043, was examined in detail against C. glutamicum and C. glutamicum::ubiA. The wild type strain was sensitive to BTZ043; however, C. glutamicum::ubiA was found to be resistant, despite possessing a functional DprE1. When the gene encoding C. glutamicum Z-decaprenyl-diphosphate synthase (NCgl2203) was overexpressed in wild type C. glutamicum, resistance to BTZ043 was further increased. This data demonstrates that in the presence of BTZ, the bacilli accumulate DPR and fail to recycle decaprenyl phosphate, which results in the depletion of decaprenyl phosphate and ultimately leads to cell death.


Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Corynebacterium glutamicum/metabolismo , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Fosfatos de Poliisoprenilo/metabolismo , Compuestos de Espiro/farmacología , Tiazinas/farmacología , Oxidorreductasas de Alcohol , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crecimiento & desarrollo , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Compuestos de Espiro/química , Tiazinas/química
17.
J Med Chem ; 67(4): 2529-2548, 2024 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-38331432

RESUMEN

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.


Asunto(s)
Azetidinas , Tuberculosis Extensivamente Resistente a Drogas , Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Azetidinas/farmacología , Azetidinas/uso terapéutico , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Extensivamente Resistente a Drogas/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana
18.
J Biol Chem ; 287(24): 20417-29, 2012 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-22505711

RESUMEN

The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence.


Asunto(s)
Proteínas Bacterianas/metabolismo , Lipopolisacáridos/metabolismo , Mycobacterium marinum/metabolismo , Animales , Proteínas Bacterianas/genética , Transporte Biológico Activo/fisiología , Elementos Transponibles de ADN/fisiología , Enfermedades de los Peces/genética , Enfermedades de los Peces/metabolismo , Lipopolisacáridos/genética , Mutación , Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/veterinaria , Mycobacterium marinum/genética , Pez Cebra/microbiología
19.
Microbiology (Reading) ; 159(Pt 4): 726-736, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23412844

RESUMEN

Mycobacterium tuberculosis possesses a complex cell wall that is unique and essential for interaction of the pathogen with its human host. Emerging evidence suggests that the biosynthesis of complex cell-wall lipids is mediated by serine/threonine protein kinases (STPKs). Herein, we show, using in vivo radiolabelling, MS and immunostaining analyses, that targeted deletion of one of the STPKs, pknH, attenuates the production of phthiocerol dimycocerosates (PDIMs), a major M. tuberculosis virulence lipid. Comparative protein expression analysis revealed that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted pknH strain. Furthermore, we analysed the composition of the major lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), and found a twofold higher LAM/LM ratio in the mutant strain. Thus, we provide experimental evidence that PknH contributes to the production and synthesis of M. tuberculosis cell-wall components.


Asunto(s)
Pared Celular/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Lípidos/biosíntesis , Mycobacterium tuberculosis/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular , Pared Celular/química , Humanos , Monocitos/microbiología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Proteínas Serina-Treonina Quinasas/genética , Virulencia
20.
PLoS Pathog ; 7(2): e1001299, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21383969

RESUMEN

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT)) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT), linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.


Asunto(s)
Galactanos/metabolismo , Lectinas/metabolismo , Lipopolisacáridos/metabolismo , Mycobacterium tuberculosis/enzimología , Pentosiltransferasa/química , Pentosiltransferasa/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Cristalografía por Rayos X , Mutagénesis Sitio-Dirigida , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/genética , Pentosiltransferasa/genética , Conformación Proteica
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