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1.
Microb Biotechnol ; 11(1): 238-247, 2018 01.
Article in English | MEDLINE | ID: mdl-29076636

ABSTRACT

A range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip-OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr , a strong Pip-repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild-type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip-OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole-cell screening assay to identify inhibitors of this enzyme.


Subject(s)
Gene Expression Regulation, Bacterial , Genes, Essential , Genetics, Microbial/methods , Molecular Biology/methods , Mutagenesis , Mycobacterium tuberculosis/genetics , Promoter Regions, Genetic , Alcohol Oxidoreductases/antagonists & inhibitors , Bacterial Proteins/antagonists & inhibitors , Biosensing Techniques , Enzyme Inhibitors/analysis
2.
Tuberculosis (Edinb) ; 100: 95-101, 2016 09.
Article in English | MEDLINE | ID: mdl-27553416

ABSTRACT

The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation.


Subject(s)
Antibiotics, Antitubercular/pharmacology , Bacteriocins/pharmacology , Mycobacterium tuberculosis/drug effects , Peptides/pharmacology , Animals , Antibiotics, Antitubercular/administration & dosage , Bacterial Proteins/biosynthesis , Bacteriocins/administration & dosage , Cells, Cultured , Colony Count, Microbial , Dose-Response Relationship, Drug , Drug Discovery , Drug Evaluation, Preclinical/methods , Drug Resistance, Bacterial/genetics , Humans , Macrophages/microbiology , Microbial Sensitivity Tests/methods , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/isolation & purification , Peptide Chain Elongation, Translational/drug effects , Peptides/administration & dosage , Ribosomal Proteins/genetics
3.
Bioorg Med Chem Lett ; 25(16): 3234-45, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-26087937

ABSTRACT

Whole cell based screens to identify hits against Mycobacterium tuberculosis (Mtb), carried out under replicating and non-replicating (NRP) conditions, resulted in the identification of multiple, novel but structurally related spiropiperidines with potent antitubercular properties. These compounds could be further classified into three classes namely 3-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[indene-1,4'-piperidine] (abbr. spiroindenes), 4-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[chromene-2,4'-piperidine] (abbr. spirochromenes) and 1'-benzylspiro[indole-1,4'-piperidin]-2(1H)-one (abbr. spiroindolones). Spiroindenes showed ⩾ 4 log10 kill (at 2-12 µM) on replicating Mtb, but were moderately active under non replicating conditions. Whole genome sequencing efforts of spiroindene resistant mutants resulted in the identification of I292L mutation in MmpL3 (Mycobacterial membrane protein Large), required for the assembly of mycolic acid into the cell wall core of Mtb. MIC modulation studies demonstrated that the mutants were cross-resistant to spirochromenes but not to spiroindolones. This Letter describes lead identification efforts to improve potency while reducing the lipophilicity and hERG liabilities of spiroindenes. Additionally, as deduced from the SAR studies, we provide insights regarding the new chemical opportunities that the spiroindolones can offer to the TB drug discovery initiatives.


Subject(s)
Antitubercular Agents/pharmacology , Piperidines/pharmacology , Spiro Compounds/pharmacology , Animals , Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacokinetics , Bacteria/drug effects , Drug Resistance, Bacterial/genetics , Genome, Bacterial , High-Throughput Screening Assays , Hypoxia , Lipids/chemistry , Matrix Metalloproteinase 13/biosynthesis , Matrix Metalloproteinase 13/genetics , Mice , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Piperidines/chemical synthesis , Piperidines/pharmacokinetics , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacokinetics , Structure-Activity Relationship
4.
J Bacteriol ; 196(19): 3441-51, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25049093

ABSTRACT

The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.


Subject(s)
Bacterial Proteins/metabolism , Mannosyltransferases/metabolism , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/growth & development , Tuberculosis/microbiology , Animals , Bacterial Proteins/genetics , Female , Humans , Macrophages/metabolism , Macrophages/microbiology , Mannosyltransferases/genetics , Mice , Mice, Inbred C57BL , Mycobacterium tuberculosis/genetics , Phosphatidylinositols/biosynthesis
5.
Mol Microbiol ; 92(1): 194-211, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24517327

ABSTRACT

In Mycobacterium tuberculosis the decaprenyl-phospho-d-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1-depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA-inhibitors supported the use of conditional knock-down strains for target-based whole-cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl-pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.


Subject(s)
Arabinose/analogs & derivatives , Genes, Bacterial , Mycobacterium tuberculosis/growth & development , Signal Transduction , Anti-Bacterial Agents/pharmacology , Arabinose/antagonists & inhibitors , Arabinose/biosynthesis , Bacterial Proteins , Cell Line, Tumor , Cell Wall/ultrastructure , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Genes, Bacterial/drug effects , Genes, Essential/drug effects , Humans , Lipoproteins , Macrophages/microbiology , Membrane Proteins , Microscopy , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/ultrastructure , Signal Transduction/drug effects
6.
Pathog Dis ; 69(3): 232-9, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24106104

ABSTRACT

PE_PGRSs are a large family of proteins identified in Mycobacterium tuberculosis complex and in few other pathogenic mycobacteria. The PE domain of PE_PGRS33 mediates localization of the protein on the mycobacterial cell surface, where the PGRS domain is available to interact with host components. In this study, PE_PGRS33 and its functional deletion mutants were expressed in M. smegmatis, and in vitro and in vivo assays were used to dissect the protein domains involved in the immunomodulatory properties of the protein. We demonstrate that PE_PGRS33-mediated secretion of TNF-α by macrophages occurs by extracellular interaction with TLR2. Our results also show that while the PGRS domain of the protein is required for triggering TNF-α secretion, mutation in the PE domain affects the pro-inflammatory properties of the protein. These results indicate that PE_PGRS33 is a protein with immunomodulatory activity and that protein stability and localization on the mycobacterial surface can affect these properties.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Mycobacterium tuberculosis/immunology , Protein Interaction Domains and Motifs , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Death/genetics , Extracellular Space , Female , Gene Expression , Immunomodulation , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Knockout , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Protein Binding , Protein Interaction Domains and Motifs/genetics , Splenomegaly/genetics , Splenomegaly/metabolism , Splenomegaly/pathology , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/metabolism , Tumor Necrosis Factor-alpha/biosynthesis
7.
Mol Microbiol ; 90(2): 356-66, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23962235

ABSTRACT

Alpha-ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α-ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non-pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient-dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Genes, Bacterial , Ketoglutaric Acids/metabolism , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Cell Line, Tumor , Citric Acid Cycle , Gene Expression Regulation, Bacterial , Glutamic Acid/metabolism , Humans , Macrophages/microbiology , Mutagenesis, Site-Directed , Mycobacterium smegmatis/metabolism , Phenotype , Phosphorylation , Recombinant Proteins/metabolism
8.
PLoS One ; 8(3): e57517, 2013.
Article in English | MEDLINE | ID: mdl-23469198

ABSTRACT

PPE represent a peculiar family of mycobacterial proteins characterized by a 180 aminoacids conserved N-terminal domain. Several PPE genes are co-transcribed with a gene encoding for a protein belonging to another family of mycobacterial specific proteins named PE. Only one PE-PPE couple has been extensively characterized so far (PE25-PPE41) and it was shown that these two proteins form a heterodimer and that this interaction is essential for PPE41 stability and translocation through the mycobacterial cell wall. In this study we characterize the PE11-PPE17 couple. In contrast with what was found for PE25-PPE41, we show that PPE17 is not secreted but surface exposed. Moreover, we demonstrate that the presence of PE11 is not necessary for PPE17 stability or for its localization on the mycobacterial surface. Finally, we show that the PPE domain of PPE17 targets the mycobacterial cell wall and that this domain can be used as a fusion partner to expose heterologous proteins on the mycobacterial surface.


Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Wall/genetics , Mycobacterium bovis/genetics , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Binding Sites , Cell Wall/metabolism , Gene Expression , Mycobacterium bovis/metabolism , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/metabolism , Protein Binding , Protein Stability , Protein Structure, Tertiary , Protein Transport
9.
PLoS One ; 6(11): e27713, 2011.
Article in English | MEDLINE | ID: mdl-22110736

ABSTRACT

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PE(Rv1818c)), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PE(Rv1818c) domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cell Wall/metabolism , Mycobacterium/cytology , Mycobacterium/metabolism , Amino Acid Substitution , Bacterial Proteins/genetics , Intracellular Space/metabolism , Mutation , Protein Stability , Protein Structure, Tertiary , Protein Transport , Species Specificity
10.
Neurotoxicology ; 31(5): 509-17, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20685220

ABSTRACT

Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico-chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.


Subject(s)
Copper/deficiency , Cuprizone/toxicity , Monoamine Oxidase Inhibitors/toxicity , Nerve Degeneration/etiology , Neurotoxicity Syndromes , Animals , Brain/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Ceruloplasmin/drug effects , Chemical Phenomena , Copper/metabolism , Cuprizone/chemistry , Disease Models, Animal , Dose-Response Relationship, Drug , Humans , Liver/metabolism , Mass Spectrometry/methods , Mice , Monoamine Oxidase Inhibitors/chemistry , Nerve Degeneration/metabolism , Neuroblastoma , Neurotoxicity Syndromes/complications , Neurotoxicity Syndromes/etiology , Neurotoxicity Syndromes/metabolism , Oxidoreductases/metabolism , Spectrophotometry, Atomic/methods
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