RESUMO
Mycobacterium leprae has a reduced genome size due to the reductive evolution over a long period of time. Lipid metabolism plays an important role in the life cycle and pathogenesis of this bacterium. In comparison to 26 lip genes (Lip A-Z) of M. tuberculosis, M. leprae retained only three orthologs indicating their importance in its life cycle. ML0314c (LipU) is one of them. It is conserved throughout the mycobacterium species. Bioinformatics analysis showed the presence of an α/ß hydrolase fold and 'GXSXG' characteristic of the esterases/lipases. The gene was expressed in E. coli and purified to homogeneity. It showed preference towards short chain esters with pNP-acetate as the preferred substrate. The enzyme showed optimal activity at 45°C and pH8.0. ML0314c protein was stable between temperatures ranging from 20 to 60°C and pH5.0-8.0, i.e., relatively acidic and neutral conditions. The active site residues predicted bioinformatically were confirmed to be Ser168, Glu267, and His297 by site directed mutagenesis. E-serine, DEPC and Tetrahydrolipstatin (THL) completely inhibited the activity of ML0314c. The protein was localized in cell wall and extracellular medium. Several antigenic epitopes were predicted in ML0314c. Protein elicited strong humoral immune response in leprosy patients, whereas, a reduced immune response was observed in the relapsed cases. No humoral response was observed in treatment completed patients. Overexpression of ml0314c in the surrogate host M. smegmatis showed marked difference in the colony morphology and growth rate. In conclusion, ML0314c is a secretary carboxyl esterase that could modulate the immune response in leprosy patients.
Assuntos
Lipólise/genética , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Hanseníase/metabolismo , Hanseníase/microbiologia , Lipase/genética , Metabolismo dos Lipídeos/genética , Lipídeos , Mutagênese Sítio-Dirigida/métodos , Mycobacterium tuberculosis/genética , Especificidade por Substrato/genética , Fatores de VirulênciaRESUMO
Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.
Assuntos
Artrite Juvenil/genética , Doença de Crohn/genética , Infecções/genética , Hanseníase/genética , Macrófagos/imunologia , Proteínas/genética , Choque Séptico/genética , Trifosfato de Adenosina/metabolismo , Animais , Bacteriólise , Células Cultivadas , Metabolismo Energético , Ácido Graxo Sintase Tipo I/metabolismo , Predisposição Genética para Doença , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/metabolismo , Oxirredução , Polimorfismo de Nucleotídeo Único , RiscoRESUMO
Intracellular pathogens survive by evading the host immune system and accessing host metabolic pathways to obtain nutrients for their growth. Mycobacterium leprae, the causative agent of leprosy, is thought to be the mycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesions of individuals with the lepromatous (also known as disseminated) form of human leprosy (L-lep), the origin and significance of these lipids remains unclear. Here we show that in human L-lep lesions, there was preferential expression of host lipid metabolism genes, including a group of phospholipases, and that these genes were virtually absent from the mycobacterial genome. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions, and 1 specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC), accumulated in macrophages infected with live mycobacteria. Mycobacterial infection and host-derived oxidized phospholipids both inhibited innate immune responses, and this inhibition was reversed by the addition of normal HDL, a scavenger of oxidized phospholipids, but not by HDL from patients with L-lep. The accumulation of host-derived oxidized phospholipids in L-lep lesions is strikingly similar to observations in atherosclerosis, which suggests that the link between host lipid metabolism and innate immunity contributes to the pathogenesis of both microbial infection and metabolic disease.