ABSTRACT
Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a â¼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis.
Subject(s)
Bacterial Proteins/genetics , Mycobacterium tuberculosis/metabolism , Protein Kinases/genetics , Transcription Factors/physiology , Tuberculosis/microbiology , Animals , Bacterial Proteins/metabolism , Cells, Cultured , DNA-Binding Proteins , Escherichia coli , Female , Gene Expression Regulation, Bacterial , Macrophages/microbiology , Mice, Inbred BALB C , Mycobacterium smegmatis , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Oxidation-Reduction , Protein Binding , Protein Kinases/metabolism , Transcription, Genetic , Virulence , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Sorting of luminal and membrane proteins into phagosomes is critical for the immune function of this organelle. However, little is known about the mechanisms that contribute to the spatiotemporal regulation of this process. Here, we investigated the role of the proneurotrophin receptor sortilin during phagosome maturation and mycobacterial killing. We show that this receptor is acquired by mycobacteria-containing phagosomes via interactions with the adaptor proteins AP-1 and GGAs. Interestingly, the phagosomal association of sortilin is critical for the delivery of acid sphingomyelinase (ASMase) and required for efficient phagosome maturation. Macrophages from Sort1(-/-) mice are less efficient in restricting the growth of Mycobacterium bovis BCG and M. tuberculosis. In vivo, Sort1(-/-) mice showed a substantial increase in cellular infiltration of neutrophils in their lungs and higher bacterial burden after infection with M. tuberculosis. Altogether, sortilin defines a pathway required for optimal intracellular mycobacteria control and lung inflammation in vivo.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Macrophages/immunology , Mycobacterium tuberculosis/immunology , Phagosomes/metabolism , Phagosomes/microbiology , Adaptor Proteins, Vesicular Transport/deficiency , Animals , Bacterial Load , Disease Models, Animal , Lung/microbiology , Mice , Mice, Knockout , Microbial Viability , Mycobacterium bovis/immunology , Tuberculosis, Pulmonary/microbiology , Tuberculosis, Pulmonary/pathologyABSTRACT
This study describes how bkaR, a highly conserved mycobacterial TetR-like transcriptional repressor, regulates a number of nearby genes that have associations with branched-chain keto-acid metabolism. bkaR (MSMEG_4718) was deleted from the nonpathogenic species Mycobacterium smegmatis, and changes in global gene expression were assessed using microarray analysis and reporter gene studies. bkaR was found to directly control the expression of 10 genes in M. smegmatis, and its ortholog in Mycobacterium tuberculosis (Rv2506) is predicted to control at least 12 genes. A conserved operator motif was identified, and binding of purified recombinant M. tuberculosis BkaR to the motif was demonstrated. Analysis of the stoichiometry of binding showed that BkaR binds to the motif as a dimer.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Keto Acids/metabolism , Mycobacterium smegmatis/genetics , Operon , Repressor Proteins/metabolism , Amino Acid Motifs , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Mycobacterium smegmatis/chemistry , Mycobacterium smegmatis/metabolism , Promoter Regions, Genetic , Repressor Proteins/chemistry , Repressor Proteins/geneticsABSTRACT
p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor alpha, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.
Subject(s)
Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Parabens/metabolism , Animals , Cells, Cultured , Colony Count, Microbial , Cytokines/biosynthesis , DNA Transposable Elements , Disease Models, Animal , Female , Lung/microbiology , Lung/pathology , Macrophages/immunology , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Mutagenesis, Insertional , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/metabolism , Tuberculosis/microbiologyABSTRACT
Understanding the molecular mechanism of host-pathogen interactions is the basis for drug design and vaccine development. The fine composition of mycolic acids (MA), the major constituents of Mycobacterium tuberculosis (Mtb) cell envelope, as well as other cell wall-associated lipids, contribute to determine the virulence of a given strain. However, endogenous receptors for mycolic acids on susceptible cells exposed to mycobacterial infections have not been fully identified. Here, we show that galectin-3, a multifunctional beta-galactoside binding lectin present mainly in the cytoplasm of inflammatory cells and also present on the cell surface, can recognize mycobacterial mycolic acids. MA can inhibit the lectin self-association but not its carbohydrate-binding abilities and can selectively interfere in the interaction of the lectin with its receptors on temperature-sensitive dendritic cell line, suggesting that galectin-3 could be involved in the recognition of trafficking mycolic acids and participate in their interaction with host cells.
Subject(s)
Galectin 3/metabolism , Lectins/metabolism , Lipids/physiology , Mycobacterium/metabolism , Mycolic Acids/metabolism , Animals , Antibodies, Monoclonal/metabolism , Carbohydrate Sequence , Cell Line , Cell Membrane/chemistry , Cricetinae , Dendritic Cells/cytology , Dendritic Cells/metabolism , Galectin 3/chemistry , Galectin 3/genetics , Interleukin-6/analysis , Kinetics , Laminin/metabolism , Ligands , Lipopolysaccharides/metabolism , Mice , Mycobacterium/chemistry , Mycobacterium tuberculosis/metabolism , Protein Structure, Tertiary , Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solubility , Surface Plasmon Resonance , Temperature , fas Receptor/metabolismABSTRACT
Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies and, at high doses, to treat a range of autoimmune and inflammatory disorders. With high-dose IVIg (hdIVIg), immunomodulatory mechanisms act on a range of cells, including T cells, B cells, and dendritic cells. Here, we demonstrate that the treatment of M. tuberculosis-infected mice with a single cycle of hdIVIg resulted in substantially reduced bacterial loads in the spleen and lungs when administered at either an early or late stage of infection. Titration of the IVIg showed a clear dose-response effect. There was no reduction in bacterial load when mice were given equimolar doses of another human protein, human serum albumin, or maltose, the stabilizing agent in the IVIg preparation. HdIVIg in vitro had no inhibitory effect on the growth of M. tuberculosis in murine bone marrow-derived macrophages. In addition, the effect of hdIVIg on bacterial loads was not observed in nude mice, suggesting the involvement of conventional T cells. Analysis of T cells infiltrating the lungs revealed only small increases in CD8(+) but not CD4(+) T-cell numbers in hdIVIg-treated mice. The mechanism of action of hdIVIg against tuberculosis in mice remains to be determined. Nevertheless, since hdIVIg is already widely used clinically, the magnitude and long duration of the therapeutic effect seen here suggest that IVIg, or components of it, may find ready application as an adjunct to therapy of human tuberculosis.