Marcello Ventura

Assessing the essentiality of the decaprenyl-phospho-D-arabinofuranose pathway in Mycobacterium tuberculosis using conditional mutants

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.

Functional Dissection of the PE Domain Responsible for Translocation of PE_PGRS33 across the Mycobacterial Cell Wall

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 (PERv1818c), 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 PERv1818c 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.

Cuprizone neurotoxicity, copper deficiency and neurodegeneration

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.

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

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.

The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

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.

The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface

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.

Promoter mutagenesis for fine-tuning expression of essential genes in Mycobacterium tuberculosis

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.

Erratum for Boldrin et al., The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

Reference EPFL-ARTICLE-204012doi:10.1128/Jb.02332-14View record in Web of Science Record created on 2014-12-30, modified on 2017-05-12

The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo (vol 196, pg 3441, 2014)

Reference EPFL-ARTICLE-204012doi:10.1128/Jb.02332-14View record in Web of Science Record created on 2014-12-30, modified on 2017-05-12