Mycobacterial phenolic glycolipid synthesis is regulated by cAMP-dependent lysine acylation of FadD22

The mycobacterial cell envelope is unique in its chemical composition, and has an important role to play in pathogenesis. Phthiocerol dimycocerosates (PDIMs) and glycosylated phenolphthiocerol dimycocerosates, also known as phenolic glycolipids (PGLs), contribute significantly to the virulence of Mycobacterium tuberculosis. FadD22 is essential for PGL biosynthesis. We have recently shown in vitro that FadD22 is a substrate for lysine acylation by a unique cAMP-dependent, protein lysine acyltransferase found only in mycobacteria. The lysine residue that is acylated is at the active site of FadD22. Therefore, acylation is likely to inhibit FadD22 activity and reduce PGL biosynthesis. Here, we show accumulation of PGLs in a strain of M. bovis BCG deleted for the gene encoding the cAMP-dependent acyltransferase, katbcg, with no change seen in PDIM synthesis. Complementation using KATbcg mutants that are deficient in cAMP-binding or acyltransferase activity shows that PGL accumulation is regulated by cAMP-dependent protein acylation in vivo. Expression of FadD22 and KATbcg mutants in Mycobacterium smegmatis confirmed that FadD22 is a substrate for lysine acylation by KATbcg. We have therefore described a mechanism by which cAMP can regulate mycobacterial virulence as a result of the ability of this second messenger to modulate critical cell wall components that affect the host immune response

Synthesis of 1,8-dioxooctahydroxanthene C-nucleosides

Since reactions between carbohydrates and cyclic 1,3-dicarbonyl compounds do not produce 1,8-dioxooctahydroxanthenes in general, reaction strategies have been devised to generate new 1,8-dioxooctahydroxanthene C-nucleosides by reacting sugars masked with acid-labile protecting groups and with free hydroxyl groups with 1,3-cyclohexanedione or dimedone. Some of these compounds are more cytotoxic to the cancer cells than against normal fibroblasts

DNA damaging, cell cytotoxicity and serum albumin binding efficacy of the rutin–Cu(ii) complex

Flavonoids are widely used as anti-oxidants, anti-cancer agents and possess metal ion chelation properties. In this report we have investigated the DNA binding (and damaging), cell cytotoxicity and serum albumin (SA) binding efficacy of the rutin–Cu(II) complex using differential spectroscopic methods. The rutin–Cu(II) complex was able to intercalate into calf thymus DNA (ct-DNA) at lower concentrations and its DNA damaging properties were also confirmed from the agarose gel based assay, fluorescence and UV-vis studies. The copper complex was found to be effective against the growth of HeLa cells in vivo. The binding constants (Kb) of the rutin–Cu(II) complex towards HSA and BSA were found to be (0.98 ± 0.03) and (1.05 ± 0.02) × 105 M−1, respectively, at 299 K and observed to increase with the increase in temperature. Site selectivity studies revealed that the rutin–Cu(II) complex binds near site 1 (subdomain IIA) of SAs. Thermodynamic parameters indicated that the mode of interaction of rutin and its copper complex with SAs are different from each other. Both ΔH° and ΔS° were observed to be positive for the interaction of the rutin–Cu(II) complex with SAs, indicating the presence of hydrophobic association in binding. The values of ΔH° were estimated to be negative (−42.07 ± 2.92 and −23.29 ± 2.33 kJ mol−1 for HSA and BSA respectively) in the binding of rutin with SAs. It implies that after chelation with Cu(II) ion, rutin alters its binding mode which could have varying applications to its other physicochemical activities

Cell cytotoxicity and serum albumin binding capacity of the morin–Cu(ii) complex and its effect on deoxyribonucleic acid

The dietary components, flavonoids, are important for their anti-oxidant properties and the ability to act as metal ion chelators. The characterization of the morin–Cu(II) complex is executed using elemental analysis, FTIR and mass spectroscopy. DNA cleaving and cell cytotoxicity properties followed by serum albumin binding have been investigated in this report. The morin–Cu(II) complex was found to cleave plasmid pBR322 DNA via an oxidative pathway as revealed by agarose gel based assay performed in the presence of some scavengers and reactive oxygen species. The breaking of the deoxyribose ring of calf thymus DNA (ct-DNA) was also confirmed by the formation of thiobarbituric acid reacting species (TBARS) between thiobarbituric acid and malonaldehyde. The morin–Cu(II) complex is able to inhibit the growth of human HeLa cells. Fluorescence studies revealed that the morin–Cu(II) complex can quench the intrinsic fluorescence of serum albumins (SAs) via a static quenching method. The binding constants were found to be in the order of 105 M−1 and observed to increase with temperature. Both ΔH° and ΔS° are positive for the binding of the morin–Cu(II) complex with serum albumins which indicated the presence of hydrophobic forces. Site-selectivity studies reveal that the morin–Cu(II) complex binds to both site 1 (subdomain IIA) and site 2 (subdomain IIIA) of human serum albumin (HSA) and bovine serum albumin (BSA). Circular dichroism (CD) studies showed the structural perturbation of SAs during binding with the morin–Cu(II) complex. The results from binding studies confirmed that after complexation with the Cu(II) ion, morin alters its mode of interaction with SAs which could have differential implications on its other biological and pharmaceutical properties

A novel encystation specific protein kinase regulates chitin synthesis in Entamoeba invadens

Phosphorylation is an important post-translational modification of proteins and is involved in the regulation of a variety of cellular events. The proteome of Entamoeba invadens, the reptilian counterpart of Entamoeba histolytica consists of an overwhelming number of putative protein kinases, and some may have a role to play in Entamoeba encystation. In this study, we have identified a novel protein kinase named as EiCSpk (Entamoeba invadens cyst specific protein kinase) which expressed almost exclusively during encystation. It is an active Protein kinase C with a characteristic substrate phosphorylation and auto-phosphorylation property. Gene silencing study has unveiled its role as a regulator of chitin synthesis through transcriptional activation of the chitin synthesis pathway genes along with glycogen phosphorylases that are involved in the influx of glucose from glycogen breakdown for chitin synthesis

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB