Browsing by Author "Ruhela, Deepa"

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Characterization of a putative spindle assembly checkpoint kinase Mps1, suggests its involvement in cell division, morphogenesis and oxidative stress tolerance in Candida albicans
(PLOS, 2014) Kamthan, Mohan; Nalla, Vijaya Kumar; Ruhela, Deepa; Kamthan, Ayushi; Maiti, Protiti; Datta, Asis
In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1 mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1 and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus.
In vivo role of Candida albicans β-hexosaminidase (HEX1) in carbon scavenging
(John Wiley & Sons, 2015) Ruhela, Deepa; Kamthan, Mohan; Saha, Paramita; Majumdar, Subeer S.; Datta, Kasturi; Abdin, Malik Zainul; Datta, Asis
The capability to utilize of N-acetylglucosamine (GlcNAc) as a carbon source is an important virulence attribute of Candida albicans. But there is a lack of information about the in vivo source of GlcNAc for the pathogen within the host environment. Here, we have characterized the GlcNAc-inducible β-hexosaminidase gene (HEX1) of C. albicans showing a role in carbon scavenging. In contrast to earlier studies, we have reported HEX1 to be a nonessential gene as shown by homozygous trisomy test. Virulence study in the systemic mouse murine model showed that Δhex1 strain is significantly less virulent in comparison to the wild-type strain. Moreover, Δhex1 strain also showed a higher susceptibility to peritoneal macrophages. In an attempt to determine possible substrates of Hex1, hyaluronic acid (HA) was treated with purified Hex1 enzyme. A significant release of GlcNAc was observed by gas chromatography-mass spectrometry analysis analysis suggesting HA degradation. Interestingly, immunohistochemistry analysis showed significant accumulation of HA in the mice kidney infected with the wild-type strain of C. albicans. Northern blot analysis showed that C. albicans HEX1 is expressed during mice renal colonization. Thus, C. albicans can obtain GlcNAc during organ colonization by secreting Hex1 via degradation of host HA.
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.
Upregulation of galactose metabolic pathway by N-Acetylglucosamine induced endogenous synthesis of galactose in Candida albicans
(Elsevier, 2013) Kamthan, Mohan; Kamthan, Ayushi; Ruhela, Deepa; Maiti, Protiti; Bhavesh, Neel Sarovar; Datta, Asis
N-Acetylglucosamine (GlcNAc) is an important signaling molecule that plays multiple roles in Candida albicans. Induction of galactose metabolic pathway by GlcNAc is an intriguing aspect of C. albicans biology. In order to investigate the role of galactose metabolic genes (GAL genes) in presence of GlcNAc, we created knockouts of galactokinase (GAL1) and UDP galactose epimerase (GAL10) genes. These mutants failed to grow on galactose and also showed lower growth rate in presence of GlcNAc. Interestingly, expression of GAL genes in presence of GlcNAc was higher in gal1Δ strain relative to that of wild type strain. Moreover, no GlcNAc induced upregulation of GAL genes was observed in the gal10Δ strain suggesting that UDP galactose epimerase is essential for GlcNAc induced activation of GAL genes. GlcNAc induced expression of GAL genes was also investigated in GlcNAc metabolic pathway triple mutant N216 (hxk1Δ nag1Δ dac1Δ). Interestingly, in this mutant the GAL genes are neither induced nor repressed and remain derepressed as found on a neutral carbon source such as glycerol, suggesting that catabolism of GlcNAc play an important role in the expression of GAL genes. GC/MS analysis of derivatized metabolites revealed a significant accumulation of galactose in the gal1Δ strain while no galactose was detected in gal10Δ and N216 strain. Solution-state NMR spectroscopy using N-acetyl-¹³C₁-glucosamine confirmed the flow of ¹³C label from GlcNAc to galactose. Thus, internal galactose synthesized via UDP galactose pathway from GlcNAc metabolites acts as the inducer of GAL genes in presence of GlcNAc.