Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the ␣1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4 -6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyltransferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.
Lipomannan (LM) and lipoarabinomannan (LAM) are phosphatidylinositol-anchored glycans present in the mycobacterial cell wall. In Mycobacterium smegmatis, the mannan core of LM/LAM constitutes a linear chain of 20 -25 ␣1,6-mannoses elaborated by 8 -9 ␣1,2-monomannose side branches. At least two ␣1,6-mannosyltransferases mediate the linear mannose chain elongation, and one branching ␣1,2-mannosyltransferase (encoded by MSMEG_4247) transfers monomannose branches. An MSMEG_4247 deletion mutant accumulates branchless LAM and interestingly fails to accumulate LM, suggesting an unexpected role of mannose branching for LM synthesis or maintenance. To understand the roles of MSMEG_4247-mediated branching more clearly, we analyzed the MSMEG_4247 deletion mutant in detail. Our study showed that the deletion mutant restored the synthesis of wild-type LM and LAM upon the expression of MSMEG_4247 at wild-type levels. In striking contrast, overexpression of MSMEG_4247 resulted in the accumulation of dwarfed LM/LAM, although monomannose branching was restored. The dwarfed LAM carried a mannan chain less than half the length of wild-type LAM and was elaborated by an arabinan that was about 4 times smaller. Induced overexpression of an elongating ␣1,6-mannosyltransferase competed with the overexpressed branching enzyme, alleviating the dwarfing effect of the branching enzyme. In wild-type cells, LM and LAM decreased in quantity in the stationary phase, and the expression levels of branching and elongating mannosyltransferases were reduced in concert, presumably to avoid producing abnormal LM/LAM. These data suggest that the coordinated expressions of branching and elongating mannosyltransferases are critical for mannan backbone elongation.Mycobacterium tuberculosis is an etiologic agent of tuberculosis, an infectious disease that remains a global problem.Glycoconjugates from the mycobacterial cell wall are involved in pathogenesis and immune modulation. In particular, phosphatidylinositol mannosides (PIMs), 4 lipomannan (LM), and lipoarabinomannan (LAM) form a class of glycoconjugates found in all species of mycobacteria, including pathogenic M. tuberculosis and saprophytic and experimentally tractable Mycobacterium smegmatis, and are known to have potent immunomodulatory activities (1-3). PIMs are anchored to the plasma membrane by a phosphatidylinositol (PI) and carry two or six mannoses, which are directly linked to the 2-OH and 6-OH of the inositol residue (4, 5) (Fig. 1A). Monomannose attached to the 2-OH of inositol is further modified by a fatty acid, making triacylated PIMs the major lipid species. The 3-OH of inositol can be further modified by a fatty acid to become a tetra-acylated species (6). LM carries a much longer chain of mannoses. For example, in M. smegmatis, 20 -25 ␣1,6-mannose residues were thought to form a linear chain, which is elaborated by 8 -9 ␣1,2-mannose monomer branches (7). A more recent report estimated M. smegmatis LM to carry 21-34 mannose residues (8), highlighting even greater heterogeneity of ...
During tuberculosis, Mycobacterium uses host macrophage cholesterol as a carbon and energy source. To mimic these conditions, Mycobacterium smegmatis can be cultured in minimal medium (MM) to induce cholesterol consumption in vitro. During cultivation, M. smegmatis consumes MM cholesterol and changes the accumulation of cell wall compounds, such as PIMs, LM, and LAM, which plays an important role in its pathogenicity. These changes lead to cell surface hydrophobicity modifications and H2O2 susceptibility. Furthermore, when M. smegmatis infects J774A.1 macrophages, it induces granuloma-like structure formation. The present study aims to assess macrophage molecular disturbances caused by M. smegmatis after cholesterol consumption, using proteomics analyses. Proteins that showed changes in expression levels were analyzed in silico using OmicsBox and String analysis to investigate the canonical pathways and functional networks involved in infection. Our results demonstrate that, after cholesterol consumption, M. smegmatis can induce deregulation of protein expression in macrophages. Many of these proteins are related to cytoskeleton remodeling, immune response, the ubiquitination pathway, mRNA processing, and immunometabolism. The identification of these proteins sheds light on the biochemical pathways involved in the mechanisms of action of mycobacteria infection, and may suggest novel protein targets for the development of new and improved treatments.
Summary Inflammation is an attempt by the body to remove noxious stimuli and initiate thus a cascade of responses in order to promote healing. There are a variety of inflammatory mechanisms involved in infections, chronic diseases and other tissue damage. Understanding these mechanisms and the search for new anti-inflammatory drugs with greater specificity and fewer side effects, underlying the development and improvement of new protocols and standardization of experimental inflammatory models to understand better these issues. The aim of this study was to evaluate the anti-inflammatory and analgesic activity of 3-benzoyl-propionic acid (3BPA) and its potential toxicological effect. To test the 3BPA as new anti-inflammatory and analgesic drug, the use carrageenan air pouch model 1% by in vitro model of cell culture to test genocytotoxicity. In the in vitro model the 3BPA presented low level of genotoxic and low cytotoxicity risk, shown by comet assay and no damage to the plasma membrane by hemolytic test erythrocytes. In the study of anti-inflammatory activity in vivo by the air pouch method were conducted nitrite dose trials, PGE 2 levels and cell migration. To verify analgesic effects of 3BPA drug in vivo tests of abdominal contortions induced by acetic acid and formalin were performed. Regard to the anti-inflammatory activity, 3BPA showed intense activity shown in marked reduction of cell migration and levels of NO, with large populations of neutrophils and reduction of PGE 2 values at a dose of 0.5mg/kg. In studies of antinociceptive activity, 3BPA reduced the number of writhes and the time lick the neurogenic and inflammatory phases of the formalin test. The results of this study also advanced substantially with respect to anti-inflammatory and analgesic properties of 3BPA by providing evidence of their likely mechanism of action, through the evaluation of antinociceptive activity, as well as the anti-inflammatory activity in vitro and in vivo, where the 3BPA showed no genotoxic effect.
Gliomas are the most common primary malignant brain tumors in adults, and have a poor prognosis, despite the different types of treatment available. There is growing demand for new therapies to treat this life-threatening tumor. Quinone derivatives from plants have received increased interest as potential anti-glioma drugs, due to their diverse pharmacologic activities, such as inhibiting cell growth, inflammation, tumor invasion, and promoting tumor regression. Previous studies have demonstrated the anti-glioma activity of Eleutherine plicata, which is related to three main naphthoquinone compounds—eleutherine, isoeleutherine, and eleutherol—but their mechanism of action remains elusive. Thus, the aim of this study was to investigate the mechanism of action of eleutherine on rat C6 glioma. In vitro cytotoxicity was evaluated by MTT assay; morphological changes were evaluated by phase-contrast microscopy. Apoptosis was determined by annexin V–FITC–propidium iodide staining, and antiproliferative effects were assessed by wound migration and colony formation assays. Protein kinase B (AKT/pAKT) expression was measured by western blot, and telomerase reverse transcriptase mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Eleutherine reduced C6 cell proliferation in a dose-dependent manner, suppressed migration and invasion, induced apoptosis, and reduced AKT phosphorylation and telomerase expression. In summary, our results suggest that eleutherine has potential clinical use in treating glioma.
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