Browsing by Author "Ghosh, Swagata"

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Cloning, expression, purification and crystallization of a novel GlcNAc metabolic protein, gig2 (duf1479) from pathogenic fungus Candida albicans
(Proteomics Society, India (PSI), 2017) Rani, Priya; Gautam, Gunjan; Rao, Kongara Hanumantha; Ghosh, Swagata; Gourinath, Samudrala; Dhar, Suman Kumar; Datta, Asis
N-acetylglucosamine (GlcNAc), an alternative sugar, is emerging as an important molecule having a multifarious role in Candida albicans including a major role in signaling. GlcNAc Inducible Gene 2, GIG2 is one of the highly upregulated genes in GlcNAc grown cells in C. albicans. Our earlier studies show the involvement of Gig2 in the formation of N-acetylneuraminic (NANA) acid from GlcNAc-6-phosphate through an understudied route. The crystal structure of Gig2 would help us in determining the exact reaction that this enzyme catalyzes. Here the cloning, expression, purification and crystallization of this protein are reported along with preliminary X-ray crystallographic analysis at 2.4Å resolution. The crystal belonged to P2 1 space group, with unit cell parameters a=59.59, b= 54.43, c= 73.29Å; α = 90°, β = 102.7° and γ = 90°. The structure was solved using PDB ID 2CSG as a template which has only 27% identity. Molecular replacement yielded a solution with LLG score of 87. The structure is currently under further refinement.
Env7p associates with the golgin protein Imh1 at the trans-Golgi network in Candida albicans
(American Society for Microbiology, 2016) Rao, Kongara Hanumantha; Ghosh, Swagata; Datta, Asis
Vesicular dynamics is one of the very important aspects of cellular physiology, an imbalance of which leads to the disorders or diseases in higher eukaryotes. We report the functional characterization of a palmitoylated protein kinase from Candida albicans whose homologue in Saccharomyces cerevisiae has been reported to be involved in negative regulation of membrane fusion and was named Env7. However, the downstream target of this protein remains to be identified. Env7 in C. albicans (CaEnv7) could be isolated from the membrane fraction and localized to vesicular structures associated with the Golgi apparatus. Our work reports Env7 in C. albicans as a new player involved in maintaining the functional dynamics at the trans-Golgi network (TGN) by interacting with two other TGN-resident proteins, namely, Imh1p and Arl1p. Direct interaction could be detected between Env7p and the golgin protein Imh1p. Env7 is itself phosphorylated (Env7p) and phosphorylates Imh1 in vivo. An interaction between Env7 and Imh1 is required for the targeted localization of Imh1. CaEnv7 has a putative palmitoylation site toward both N and C termini. An N-terminal palmitoylation-defective strain retains its ability to phosphorylate Imh1 in vitro. An ENV7 homozygous mutant showed compromised filamentation in solid media and attenuated virulence, whereas an overexpressed strain affected cell wall integrity. Thus, Env7 plays a subtle but important role at the level of multitier regulation that exists at the TGN.
N-acetylglucosamine (GlcNAc) inducible gene, GIG2, is a novel component of GlcNAc metabolism in Candida albicans
(American Society for Microbiology, 2014) Ghosh, Swagata; Rao, Kongara Hanumantha; Bhavesh, Neel Sarovar; Das, Gobardhan; Dwivedi, Ved Prakash; Datta, Asis
Candida albicans is an opportunistic fungal pathogen that resides in the human body as a commensal and can turn pathogenic when the host is immunocompromised. Adaptation of C. albicans to host niche-specific conditions is important for the establishment of pathogenicity, where the ability of C. albicans to utilize multiple carbon sources provides additional flexibility. One alternative sugar is N-acetylglucosamine (GlcNAc), which is now established as an important carbon source for many pathogens and can also act as a signaling molecule. Although GlcNAc catabolism has been well studied in many pathogens, the importance of several enzymes involved in the formation of metabolic intermediates still remains elusive. In this context, microarray analysis was carried out to investigate the transcriptional responses induced by GlcNAc under different conditions. A novel gene that was highly upregulated immediately following the GlcNAc catabolic genes was identified and was named GIG2 (GlcNAc-induced gene 2). This gene is regulated in a manner distinct from that of the GlcNAc-induced genes described previously in that GlcNAc metabolism is essential for its induction. Furthermore, this gene is involved in the metabolism of N-acetylneuraminate (sialic acid), a molecule equally important for initial host-pathogen recognition. Mutant cells showed a considerable decrease in fungal burden in mouse kidneys and were hypersensitive to oxidative stress conditions. Since GIG2 is also present in many other fungal and enterobacterial genomes, targeted inhibition of its activity would offer insight into the treatment of candidiasis and other fungal or enterobacterial infections.
N-acetylglucosamine kinase, HXK1 contributes to White-Opaque morphological transition in Candida albicans
(Elsevier B.V., 2014) Rao, Kongara Hanumantha; Ruhela, Deepa; Ghosh, Swagata; Abdin, M.Z.; Datta, Asis
Morphological transition (yeast-hyphal and white-opaque) is an important biological process in the life cycle of pathogenic yeast, Candida albicans and is a major determinant of virulence. Earlier reports show that the amino sugar, N-acetylglucosamine (GlcNAc) induces white to opaque switching in this pathogen. We report here a new contributor to this switching phenomenon, namely N-acetylglucosamine kinase or HXK1, the first enzyme of the GlcNAc catabolic cascade. Microarray profile analysis of wild type vs. hxk1 mutant cells grown under switching inducing condition showed upregulation of opaque specific and cell wall specific genes along genes involved in the oxidative metabolism. Further, our qRT-PCR and immunoblot analysis revealed that the expression levels of Wor1, a master regulator of the white-opaque switching phenomenon remained unaltered during this HXK1 mediated transition. Thus the derepression of opaque specific gene expression observed in hxk1 mutant could be uncoupled to the expression of WOR1. Moreover, this regulation via HXK1 is independent of Ras1, a major regulator of morphogenetic transition and probably independent of MTL locus too. These results extend our understanding of multifarious roles of metabolic enzymes like Hxk1 and suggest an adaptive mechanism during host-pathogen interactions.
N-acetylglucosamine kinase, HXK1 is involved in morphogenetic transition and metabolic gene expression in Candida albicans
(PLOS, 2013) Rao, Kongara Hanumantha; Ghosh, Swagata; Natarajan, Krishnamurthy; Datta, Asis
Candida albicans, a common fungal pathogen which diverged from the baker’s yeast Saccharomyces cerevisiae has the unique ability to utilise N-acetylglucosamine, an amino sugar and exhibits phenotypic differences. It has acquired intricate regulatory mechanisms at different levels in accordance with its life style. N-acetylglucosamine kinase, a component of the N-acetylglucosamine catabolic cascade is an understudied gene since Saccharomyces cerevisiae lacks it. We report HXK1 to act as both positive and negative regulator of transcription of genes involved in maintaining cellular homeostasis. It is involved in repression of hyphal specific genes in addition to metabolic genes. Its regulation of filamentation and GlcNAc metabolism is independent of the known classical regulators like EFG1, CPH1, RAS1, TPK2 or TUP1. Moreover, Hxk1-GFP is localised to cytoplasm, nucleus and mitochondria in a condition specific manner. By employing two-step affinity purification, we report the interaction of HXK1 with SIR2 under filamentation inducing conditions. Our work highlights a novel regulatory mechanism involved in filamentation repression and attempts to decipher the GlcNAc catabolic regulatory cascade in eukaryotes.
Two gene clusters co-ordinate for a functional N-acetylglucosamine catabolic pathway in Vibrio cholerae
(Wiley-Blackwell, 2011) Ghosh, Swagata; Rao, K. Hanumantha; Sengupta, Manjistha; Bhattacharya, Sujit K.; Datta, Asis
Pathogenic microorganisms like Vibrio cholerae are capable of adapting to diverse living conditions, especially when they transit from their environmental reservoirs to human host. V. cholerae attaches to N-acetylglucosamine (GlcNAc) residues in glycoproteins and lipids present in the intestinal epithelium and chitinous surface of zoo-phytoplanktons in the aquatic environment for its survival and colonization. GlcNAc utilization thus appears to be important for the pathogen to reach sufficient titres in the intestine for producing clinical symptoms of cholera. We report here the involvement of a second cluster of genes working in combination with the classical genes of GlcNAc catabolism, suggesting the occurrence of a novel variant of the process of biochemical conversion of GlcNAc to Fructose-6-phosphate as has been described in other organisms. Colonization was severely attenuated in mutants that were incapable of utilizing GlcNAc. It was also shown that N-acetylglucosamine specific repressor (NagC) performs a dual role - while the classical GlcNAc catabolic genes are under its negative control, the genes belonging to the second cluster are positively regulated by it. Further application of tandem affinity purification to NagC revealed its interaction with a novel partner. Our results provide a genetic program that probably enables V. cholerae to successfully utilize amino - sugars and also highlights a new mode of transcriptional regulation, not described in this organism.