N-acetylglucosamine (GlcNAc-inducible gene GIG2 is a novel component of GlcNAc metabolism in Candida albicans

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

Role of carbon and nitrogen assimilation in Candida albicans survival and virulence

Candida albicans is a fungal pathogen that lives in both commensal and opportunistic lifestyles within the human host. Fungal metabolism has long been thought to play a role in virulence, while the nutrient sources used by human pathogenic fungus in vivo remain obscure. A significant factor of disease progression is the interaction between C. albicans and the innate immune system. C. albicans has a complicated response to phagocytosis, which is like carbon deprivation. This shows that in vivo, nutritional deficiency is a substantial stressor. The mechanisms of Carbon and nitrogen catabolite repressions (CCR, NCR) are crucial for virulence as they utilize favored carbon and nitrogen sources respectively in a host niche-dependent manner. The findings of various studies demonstrate linkages between carbon metabolic regulations, pathogenicity, and Snf1 (sucrose-nonfermenting 1), a conserved regulator of nutrient stress response. It also links Mig1 (Multicopy Inhibitor of GAL) and Mig2 to the C. albicans glucose restraint pathway. Carbon utilization abnormalities in mutants lacking the ICL1 (Isocitrate lyase 1), a glyoxylate enzyme are likewise more severe than expected. These findings show that C. albicans' regulation of alternative carbon metabolism differs dramatically from that of other fungi. C. albicans regularly encounters nitrogen deficiency and carbon-poor environments throughout its development in the host, and the ability to effectively use a variety of non-fermentable carbon and nitrogen sources is a virulence determinant. The knowledge obtained from these studies could be useful for developing effective therapeutic strategies for the control of fungal diseases

N-acetylglucosamine Signaling: Transcriptional Dynamics of a Novel Sugar Sensing Cascade in a Model Pathogenic Yeast, <i>Candida albicans</i>

The amino sugar, N-acetylglucosamine (GlcNAc), has emerged as an attractive messenger of signaling in the pathogenic yeast Candida albicans, given its multifaceted role in cellular processes, including GlcNAc scavenging, import and metabolism, morphogenesis (yeast to hyphae and white to opaque switch), virulence, GlcNAc induced cell death (GICD), etc. During signaling, the exogenous GlcNAc appears to adopt a simple mechanism of gene regulation by directly activating Ngs1, a novel GlcNAc sensor and transducer, at the chromatin level, to activate transcriptional response through the promoter acetylation. Ngs1 acts as a master regulator in GlcNAc signaling by regulating GlcNAc catabolic gene expression and filamentation. Ndt80-family transcriptional factor Rep1 appears to be involved in the recruitment of Ngs1 to GlcNAc catabolic gene promoters. For promoting filamentation, GlcNAc adopts a little modified strategy by utilizing a recently evolved transcriptional loop. Here, Biofilm regulator Brg1 takes up the key role, getting up-regulated by Ngs1, and simultaneously induces Hyphal Specific Genes (HSGs) expression by down-regulating NRG1 expression. GlcNAc kinase Hxk1 appears to play a prominent role in signaling. Recent developments in GlcNAc signaling have made C. albicans a model system to understand its role in other eukaryotes as well. The knowledge thus gained would assist in designing therapeutic interventions for the control of candidiasis and other fungal diseases

Transcriptome analysis of <i>HXK1</i> regulated genes.

<p>A) Partial Heat map of over-represented genes (p-values <0.05, False Discovery Rate <1% in GO term analysis) differentially expressed in a <i>HXK1</i> dependent manner in response to glucose. Two-color microarray data expressed as <i>hxk1</i>/<i>HXK1</i> ratio (1 and 2 are biological replicates with dye swap, average of 1 and 2, <i>Ave</i>) is plotted as heat map. The color scale at the bottom indicates the log2 ratio. B) Heat map of Hyphal specific genes (HSGs) differentially expressed in a <i>HXK1</i> dependent manner in response to glucose. C) Modulation of expression levels of GlcNAc catabolic genes by <i>HXK1.</i> Quantitative RT-PCR of GlcNAc catabolic gene transcripts <i>NGT1</i>, <i>NAG1</i> and <i>DAC1</i> in <i>C. albicans</i> wild type (CAF2–1) and <i>hxk1</i> mutant, cells in response to glycerol-6%, Glucose-2%, Glucose-5 mM or GlcNAc-5 mM. <i>ACT1</i> has been selected as the endogenous control. The error bars represent co-efficient of variation.</p

N-Acetylglucosamine Kinase, <em>HXK1</em> Is Involved in Morphogenetic Transition and Metabolic Gene Expression in <em>Candida albicans</em>

<div><p><em>Candida albicans,</em> a common fungal pathogen which diverged from the baker’s yeast <em>Saccharomyces cerevisiae</em> 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 <em>Saccharomyces cerevisiae</em> lacks it. We report <em>HXK1</em> 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 <em>EFG1</em>, <em>CPH1</em>, <em>RAS1</em>, <em>TPK2</em> or <em>TUP1</em>. 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 <em>HXK1</em> with <em>SIR2</em> 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.</p> </div

<i>hxk1</i> mutant is constitutively filamentous and hyperfilamentous in filamentation inducing conditions.

<p>A) <i>hxk1</i> mutant is hyperfilamentous in liquid filamentation inducing media like Spider and Serum as compared to wild type strain (CAF2–1) and showed germ tube like protuberances (28–35%) in YPD liquid medium grown at 30°C (details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053638#pone.0053638.s009" target="_blank">Text S1</a>). B) <i>hxk1</i> mutant is hyper filamentous and showed filamentation early in YPD and YPD+serum solid plates at 30°C after 3 and 2 days respectively whereas wild type and revertant colonies grown under similar conditions were completely smooth.</p

<i>hxk1</i> mutants are hyperfilamentous on filamentation inducing solid media.

<p>A) Hyphae formation on Spider and SLAD plates. All hxk1 mutants including <i>efg1</i>/<i>hxk1</i> showed hyperfilamentation. Strains were incubated at 37°C for 7 days in case of spider and 10 days in case of SLAD plates. Wild-type CAF2–1 (<i>HXK1</i>/<i>HXK1</i>); <i>hxk1</i> mutant, H8–1–103 (<i>hxk1/hxk1</i>); <i>cph1</i> mutant, A11–1 (<i>cph1/cph1</i>); double mutant of <i>cph1</i>/<i>hxk1,</i> AN8–1–16 (<i>cph1/cph1 hxk1/hxk1</i>); <i>efg1</i> mutant, HLC52 (<i>efg1/efg1</i>); double mutant of <i>efg1</i>/<i>hxk1</i> HLC67–16–1–9 (<i>efg1/efg1 hxk1/hxk1</i>); <i>tpk2</i> mutant, TPO7.4 (<i>tpk2/tpk2</i>); double mutant of <i>tpk2</i>/<i>hxk1</i>, AS1–3–1–8 (<i>tpk2/tpk2 hxk1/hxk1</i>) and their respective complemented strains in which one functional copy of <i>HXK1</i> was reintroduced in the native locus. B) <i>efg1/efg1 hxk1/hxk1</i> double mutant showed hyperfilamentation under embedded conditions<b>.</b> Cells of wild-type, CAF2–1, <i>hxk1/hxk1, efg1/efg1, efg1/efg1hxk1/hxk1,</i> mutants were grown in YPD for 5 hrs at 30°, washed in sterile water and mixed with molten CM Agar (with 1% Tween-80) plated and grown for 3 days at 25°C. <i>efg1 hxk1</i> double mutant showed hyperfilamentation when compared to <i>hxk1</i> or <i>efg1</i> single mutants (<i>EFG1</i> is reported to be a negative regulator of filamentation under micro-aerophillic/embedded conditions). C) <i>tup1</i>/<i>hxk1</i> double mutant showed growth pattern slightly different from <i>tup1</i> mutant. Wild type (i), <i>hxk1</i> mutant (ii), <i>tup1</i>(iii) and <i>tup1</i>/<i>hxk1</i>(iv) mutants colonies were grown on YPD solid and liquid medium for 3 and 2 days respectively at 30°C. <i>tup1/hxk1</i>double mutant shared most of the features with a <i>tup1</i> single mutant (iii, iv) on YPD plates, but in some colonies of the double mutant a wavy, afilamentous fringe could be observed at the centre(v). In YPD broth the <i>tup1</i> single mutant grew in clumps having the tendency to settle at the bottom, the <i>tup1/hxk1</i> double mutant showed uniform turbidity throughout the culture with clumps in the bottom (vi).</p