RESUMEN
Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the Mycobacterium tuberculosis (M. tuberculosis) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase stf3, includes a putative cytochrome P450 gene (cyp128) and a gene of unknown function, rv2269c. We demonstrate that cyp128 and stf3 are sufficient for the biosynthesis of SMK from menaquinone and rv2269c exhibits promoter activity in M. tuberculosis. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in M. tuberculosis. Finally, we showed in a mouse model of infection that the loss of cyp128 exhibits a hypervirulent phenotype similar to that in previous studies of the stf3 mutant. These findings provide a platform for defining the molecular basis of SMK's role in M. tuberculosis pathogenesis.
Asunto(s)
Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/microbiología , Vitamina K 2/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Ratones , Mycobacterium tuberculosis/genética , Operón , VirulenciaRESUMEN
Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Lípidos/biosíntesis , Lípidos/química , Mycobacterium tuberculosis/metabolismo , Trehalosa/análogos & derivados , Aciltransferasas/genética , Proteínas Bacterianas/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Lactonas/farmacología , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Orlistat , Estructura Terciaria de ProteínaRESUMEN
Mycobacteria, including the pathogen Mycobacterium tuberculosis, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome.
Asunto(s)
Mycobacterium/metabolismo , Trehalosa/química , Trehalosa/metabolismo , Alquinos/química , Azidas/química , Colorantes Fluorescentes/química , Glucolípidos/metabolismoRESUMEN
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.
Asunto(s)
Interacciones Huésped-Patógeno , Lípidos/biosíntesis , Macrófagos/microbiología , Mycobacterium tuberculosis/fisiología , Animales , Péptidos Catiónicos Antimicrobianos , Catelicidinas/farmacología , Línea Celular , Humanos , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Glucolípidos/biosíntesis , Mycobacterium tuberculosis/metabolismo , Factores de Virulencia/biosíntesis , Acilación/fisiología , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/fisiología , Inhibidores Enzimáticos/farmacología , Glucolípidos/genética , Lactonas/farmacología , Mycobacterium tuberculosis/genética , Orlistat , Factores de Virulencia/genéticaRESUMEN
Mycothiol (MSH), the primary low-molecular weight thiol produced in mycobacteria, acts to protect the cell from oxidative stress and to maintain redox homeostasis, notably in the pathogenic Mycobacterium tuberculosis in the course of human infection. The mycothiol disulfide reductase (Mtr) enzyme reduces the oxidized form of mycothiol, mycothione (MSSM), back to MSH, however its role in bacterial viability is not clear. In this study, we sought to determine the MSH levels of wild-type (WT) and Mtr mutant mycobacteria during oxidative stress. We describe a rapid method for the relative quantification of MSH using high-sensitivity mass spectrometry (MS) with selected ion monitoring (SIM). This method uses only minimal sample cleanup, and does not require advanced chromatographic equipment or fluorescent compounds. MSH levels decreased in the Mtr mutant only upon treatment with peroxide, and the results were consistent between our method and previously-described thiol quantification methods. Our results indicate that our MS-based method is a useful, high-throughput alternative tool for the quantification of MSH from mycobacteria.
RESUMEN
CysQ is a 3'-phosphoadenosine-5'-phosphatase that dephosphorylates intermediates from the sulfate assimilation pathway of Mycobacterium tuberculosis (Mtb). Here, we demonstrate that cysQ disruption attenuates Mtb growth in vitro and decreases the biosynthesis of sulfated glycolipids but not major thiols, suggesting that the encoded enzyme specifically regulates mycobacterial sulfation.
Asunto(s)
Glucolípidos/biosíntesis , Mycobacterium tuberculosis/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Sulfatos/química , Cromatografía Liquida , Glucolípidos/química , Mycobacterium tuberculosis/crecimiento & desarrolloRESUMEN
Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.
Asunto(s)
Aciltransferasas/metabolismo , Mycobacterium tuberculosis/enzimología , Trehalosa/metabolismo , Aciltransferasas/genética , Glucolípidos/metabolismo , Lipoilación , Mycobacterium tuberculosis/genética , Palmitoil Coenzima A/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Trehalosa/análogos & derivadosRESUMEN
Mycobacterium tuberculosis, the causative agent of tuberculosis, produces unique sulfated metabolites associated with virulence. One such metabolite from M. tuberculosis lipid extracts, S881, has been shown to negatively regulate the virulence of M. tuberculosis in mouse infection studies, and its cell-surface localization suggests a role in modulating host-pathogen interactions. However, a detailed structural analysis of S881 has remained elusive. Here we use high-resolution, high-mass-accuracy, and tandem mass spectrometry to characterize the structure of S881. Exact mass measurements showed that S881 is highly unsaturated, tandem mass spectrometry indicated a polyisoprene-derived structure, and characterization of synthetic structural analogs confirmed that S881 is a previously undescribed sulfated derivative of dihydromenaquinone-9, the primary quinol electron carrier in M. tuberculosis. To our knowledge, this is the first example of a sulfated menaquinone produced in any prokaryote. Together with previous studies, these findings suggest that this redox cofactor may play a role in mycobacterial pathogenesis.