RESUMEN
Mycobacterium tuberculosis is a highly specialized human pathogen. The success of M. tuberculosis is due to its ability to replicate within host macrophages, resist host immune responses, and ultimately enter a persistent state during a latent tuberculosis infection. Understanding how M. tuberculosis adapts to and replicates in the intracellular environment of the host is crucial for the development of novel, targeted therapeutics. We report the characterization of an M. tuberculosis mutant lacking Rv3249c, a TetR transcriptional regulator. We show that Rv3249c directly represses the adjacent alkB-rubA-rubB operon encoding an alkane hydroxylase/rubredoxin system. For consistency with related systems, we have named the rv3249c gene alkX. The alkX mutant survived better than wild-type M. tuberculosis inside macrophages. This could be phenocopied by overexpression of the alkB-rubA-rubB locus. We hypothesized that the improved intracellular survival phenotype is a result of increased fitness of the mutant; however, we found that the alkX mutant had a defect when grown on some host-associated carbon sources in vitro. We also found that the alkX mutant had a defect in biofilm formation, also linked to the overexpression of the alkB-rubAB genes. Combined, these results define the primary role of AlkX as a transcriptional repressor of the alkB-rubAB operon and suggest the operon contributes to intracellular survival of the pathogen. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the leading cause of death worldwide due to a single infectious agent. It is important to understand how M. tuberculosis adapts to and replicates in the intracellular environment of the host. In this study, we characterized the TetR transcriptional regulator Rv3249c and show that it regulates a highly conserved alkane hydroxylase/rubredoxin system. Our data demonstrate that the AlkBRubAB system contributes to the success of the bacterium in host macrophages.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Proteínas Bacterianas/genética , Biopelículas , Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Humanos , Macrófagos/microbiología , Mycobacterium tuberculosis/metabolismo , Rubredoxinas/genética , Tuberculosis/microbiologíaRESUMEN
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
The success of Mycobacterium tuberculosis as a human pathogen depends on the bacterium's ability to persist in a quiescent form in oxygen and nutrient-poor host environments. In vitro studies have demonstrated that these restricting environments induce a shift from bacterial replication to non-replicating persistence (NRP). Entry into NRP involves changes in bacterial metabolism and remodeling of the cell envelope. Findings consistent with the phenotypes observed in vitro have been observed in patient and animal model samples. This review focuses on the cell envelope differences seen between replicating and NRP M. tuberculosis and summarizes the ways in which serine/threonine protein kinases (STPKs) may mediate this process.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Animales , División Celular , Membrana Celular/metabolismo , Pared Celular/metabolismo , Humanos , Tuberculosis/genética , Tuberculosis/metabolismoRESUMEN
The mycobacterial cell envelope is crucial to host-pathogen interactions as a barrier against antibiotics and the host immune response. In addition, cell envelope lipids are mycobacterial virulence factors. Cell envelope lipid biosynthesis is the target of a number of frontline tuberculosis treatments and has been the focus of much research. However, the transport mechanisms by which these lipids reach the mycomembrane remain poorly understood. Many envelope lipids are exported from the cytoplasm to the periplasmic space via the mycobacterial membrane protein large (MmpL) family of proteins. In other bacteria, lipoproteins can contribute to outer membrane biogenesis through direct binding of substrates and/or protein-protein associations with extracytoplasmic biosynthetic enzymes. In this report, we investigate whether the lipoprotein LpqN plays a similar role in mycobacteria. Using a genetic two-hybrid approach, we demonstrate that LpqN interacts with periplasmic loop domains of the MmpL3 and MmpL11 transporters that export mycolic acid-containing cell envelope lipids. We observe that LpqN also interacts with secreted cell envelope biosynthetic enzymes such as Ag85A via pulldown assays. The X-ray crystal structures of LpqN and LpqN bound to dodecyl-trehalose suggest that LpqN directly binds trehalose monomycolate, the MmpL3 and Ag85A substrate. Finally, we observe altered lipid profiles of the ΔlpqN mutant during biofilm maturation, pointing toward a possible physiological role for the protein. The results of this study suggest that LpqN may act as a membrane fusion protein, connecting MmpL transporters with periplasmic proteins, and provide general insight into the role of lipoproteins in Mycobacterium tuberculosis cell envelope biogenesis.