RESUMEN
A growing body of evidence indicates that MmpL (mycobacterial membrane protein large) transporters are dedicated to cell wall biosynthesis and transport mycobacterial lipids. How MmpL transporters function and the identities of their substrates have not been fully elucidated. We report the characterization of Mycobacterium smegmatis MmpL11. We showed previously that M. smegmatis lacking MmpL11 has reduced membrane permeability that results in resistance to host antimicrobial peptides. We report herein the further characterization of the M. smegmatis mmpL11 mutant and identification of the MmpL11 substrates. We found that biofilm formation by the M. smegmatis mmpL11 mutant was distinct from that by wild-type M. smegmatis. Analysis of cell wall lipids revealed that the mmpL11 mutant failed to export the mycolic acid-containing lipids monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. In addition, analysis of total lipids indicated that the mycolic acid-containing precursor molecule mycolyl phospholipid accumulated in the mmpL11 mutant compared with wild-type mycobacteria. MmpL11 is encoded at a chromosomal locus that is conserved across pathogenic and nonpathogenic mycobacteria. Phenotypes of the M. smegmatis mmpL11 mutant are complemented by the expression of M. smegmatis or M. tuberculosis MmpL11, suggesting that MmpL11 plays a conserved role in mycobacterial cell wall biogenesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Pared Celular/metabolismo , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/fisiología , Ácidos Micólicos/metabolismo , Transporte Biológico , Factores Cordón/metabolismo , Ésteres/química , Galactanos/biosíntesis , Modelos Biológicos , Mutación/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/ultraestructura , Plancton/crecimiento & desarrollo , Espectrometría de Masa por Ionización de Electrospray , Ceras/químicaRESUMEN
The mycobactericidal properties of macrophages include the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal ubiquitin-derived peptides (Ub-peptides). To improve our understanding of interactions of ubiquitin-derived peptides with mycobacteria, we further characterized the structure and function of bactericidal Ub-peptide Ub2. We found that Ub2 adopts a ß-sheet conformation in the context of sodium dodecyl sulfate micelles and phospholipid (1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) vesicles that was dependent upon the primary sequence of the peptide. Point mutations in Ub2 that reduced the net charge of the peptide decreased Ub2 bactericidal activity. We investigated Ub-peptide function in the context of model membranes and intact bacteria. Differential scanning calorimetry analysis demonstrated that Ub2 inserts into and perturbs model phospholipid vesicles. In addition, we demonstrate that Ub2 disrupts the integrity of the mycobacterial membrane, equilibrates the transmembrane potential, and is localized within both the mycobacterial membrane and cytoplasm of treated bacteria. Finally, we identified additional bactericidal Ub-peptides and characterized their activity and structure. This study provides new insight into the mycobactericidal mechanisms of Ub-peptides.
Asunto(s)
Membranas Artificiales , Mycobacterium smegmatis/química , Péptidos/química , Péptidos/fisiología , Ubiquitina/química , Secuencia de Aminoácidos , Western Blotting , Rastreo Diferencial de Calorimetría , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Espectrometría de Masas , Datos de Secuencia Molecular , Fracciones Subcelulares/químicaRESUMEN
The mycobactericidal properties of macrophages include the generation of reactive oxygen intermediates and the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal ubiquitin-derived peptides (Ub-peptides). To better understand the interactions of ubiquitin-derived peptides with mycobacteria and identify putative mycobacterial intrinsic resistance mechanisms, we screened for transposon mutants with increased susceptibility to the bactericidal Ub-peptide Ub2. We isolated 27 Mycobacterium smegmatis mutants that were hypersusceptible to Ub2. Two mutants were isolated that possessed mutations in the msmeg_0166 gene, which encodes a transcriptional regulator. The msmeg_0166 mutants were also hypersusceptible to other host antimicrobial peptides and oxidative stress. In characterizing msmeg_0166, we found that it encodes a repressor of oxyS, and therefore we have renamed the gene roxY. We demonstrate that RoxY and OxyS contribute to M. smegmatis resistance to oxidative stress. An ahpD transposon mutant was also isolated in our screen for Ub-peptide hypersusceptibility. Overexpression of oxyS in M. smegmatis reduced transcription of the ahpCD genes, which encode a peroxide detoxification system. Our data indicate that RoxY, OxyS, and AhpD play a role in the mycobacterial oxidative stress response and are important for resistance to host antimicrobial peptides.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Regulación hacia Abajo , Mycobacterium smegmatis/metabolismo , Estrés Oxidativo , Péptidos/farmacología , Proteínas Represoras/metabolismo , Ubiquitina/metabolismo , Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Péptidos/metabolismo , Proteínas Represoras/genética , Ubiquitina/farmacologíaRESUMEN
Non-human primates, notably rhesus macaques (Macaca mulatta, RM), provide a robust experimental model to investigate the immune response to and effective control of Mycobacterium tuberculosis infections. Changes in the function of immune cells and immunosenescence may contribute to the increased susceptibility of the elderly to tuberculosis. The goal of this study was to examine the impact of age on M. tuberculosis host-pathogen interactions following infection of primary alveolar macrophages derived from young and aged rhesus macaques. Of specific interest to us was whether the mycobactericidal capacity of autophagic macrophages was reduced in older animals since decreased autophagosome formation and autophagolysosomal fusion has been observed in other cells types of aged animals. Our data demonstrate that alveolar macrophages from old RM are as competent as those from young animals for autophagic clearance of M. tuberculosis infection and controlling mycobacterial replication. While our data do not reveal significant differences between alveolar macrophage responses to M. tuberculosis by young and old animals, these studies are the first to functionally characterize autophagic clearance of M. tuberculosis by alveolar macrophages from RM.