Biology subjects

Varahan, S.

Publications and source records attributed to Varahan, S..

6 recordsLinked to original sources

bioRxiv2026

Systematic Screening of a Glycolysis-targeting Small-molecule Library Identifies Novel Inhibitors of Fungal Morphogenesis in Candida albicans

Fungal infections are an increasing global health concern, with Candida albicans emerging as a leading cause of mucosal and life-threatening systemic infections. C. albicans relies on yeast-to-hyphae transition, to establish systemic infections in the host. Our recent work demonstrated that glycolysis is a key regulator of hyphal differentiation in C. albicans. Leveraging this knowledge, we screened a small-molecule compound library containing glycolysis inhibitors for their ability to block fungal morphogenesis and identified multiple inhibitors of glycolysis that robustly block hyphal differentiation without compromising overall growth. While early glycolysis pathway inhibitors showed moderate effects, two compounds (NPD10084 and PKM2-IN-6) targeting pyruvate kinase activity emerged as the most potent inhibitors of fungal morphogenesis, completely blocking hyphal differentiation, in multiple filamentation-inducing conditions. Comparative RNA-Seq analysis revealed that pyruvate kinase inhibition using PKM2-IN-6 resulted in the repression of multiple genes associated with fungal morphogenesis, biofilm formation, and virulence. These transcriptional changes were accompanied by a significant reduction in biofilm formation and increased sensitivity to conventional antifungal drugs, including amphotericin B and fluconazole, in the presence of PKM2-IN-6. Furthermore, administration of PKM2-IN-6 significantly improved the survival of mice in a systemic model of murine candidiasis underscoring the in vivo efficacy of this compound. Molecular docking analysis showed stable binding of both compounds to the ATP-binding pocket of pyruvate kinase, suggesting ATP-competitive inhibition as the mode of action. Collectively, our study has identified novel inhibitors of fungal morphogenesis in C. albicans that target pyruvate kinase activity to increase antifungal susceptibility and attenuate the virulence of C. albicans. ImportanceCandida albicans is a major fungal pathogen that causes fatal infections, particularly in immunocompromised individuals. The increasing prevalence of antifungal resistance and the limited availability of effective antifungal therapies underscore the need for new treatment strategies. In this study, we identify pyruvate kinase, a key glycolytic enzyme, as an important regulator of fungal morphogenesis and virulence. Pharmacological inhibition of pyruvate kinase blocked hyphal differentiation without affecting fungal growth, impaired biofilm formation, and enhanced the activity of clinically relevant antifungal drugs. Transcriptomic analysis further revealed broad repression of genes involved in filamentation, biofilm formation, cell adhesion, antifungal resistance, and virulence, providing a molecular basis for these phenotypes. Importantly, pyruvate kinase inhibition also improved host survival in a murine model of systemic candidiasis. Together, our findings identify pyruvate kinase as a promising therapeutic target and demonstrate the potential of targeting fungal metabolism to effectively combat C. albicans infections.

microbiology↗

bioRxiv2025

A Conserved Metabolic Network Regulates Titan Cell Formation in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes pulmonary infections and life-threatening meningoencephalitis in immunocompromised individuals. In addition to the polysaccharide capsule and melanin, titan cell formation is a key virulence trait that promotes immune evasion and disease progression. Despite the established role of titan cells in C. neoformans pathogenesis, the molecular mechanisms governing their formation remain poorly understood. Here, we demonstrate that glycolysis is critical for titan cell formation in C. neoformans. Pharmacological inhibition or genetic disruption of glycolysis significantly impaired titanization, whereas exogenous cAMP add-back restored the defect. To elucidate the underlying mechanism, we performed comparative RNA-seq analyses of wild- type, hxk2{Delta}, and hxk2{Delta} supplemented with cAMP under titan cell inducing conditions. Transcriptomic analyses revealed significant downregulation of a large subset of calcineurin- responsive genes in the hxk2{Delta} mutant, many of which were restored upon cAMP supplementation. Consistent with these findings, pharmacological inhibition of calcineurin using FK506 or cyclosporin A markedly reduced titan cell formation, establishing an essential role for calcineurin signaling in this morphological transition. Furthermore, supplementation with CaCl2 rescued the titanization defect of the hxk2{Delta} mutant, whereas chelation of extracellular calcium with EGTA significantly inhibited titanization in wild-type cells. Using the calcium-sensitive dye, we found that intracellular calcium levels were substantially reduced in the hxk2{Delta} mutant and were restored by CaCl2 or cAMP supplementation. Collectively, our findings uncover a previously unrecognized glycolysis-calcineurin regulatory axis that governs titan cell formation and establishes a direct mechanistic link between central carbon metabolism, calcium homeostasis and fungal morphogenesis. SummaryTitan cell formation is a critical virulence trait that promotes immune evasion and disease progression of Cryptococcus neoformans. However, the mechanisms governing this morphological transition remain poorly understood. Here, we demonstrate that glycolysis is essential for titan cell formation and uncover a previously unrecognized link between glycolysis, calcium homeostasis, and calcineurin signaling. Comparative transcriptomics, pharmacological perturbation, calcium rescue experiments, and intracellular calcium measurements reveal that disruption of glycolysis attenuates calcineurin signaling by reducing intracellular calcium levels, thereby impairing titanization. These findings establish a novel glycolysis-calcineurin regulatory axis controlling fungal morphogenesis in C. neoformans.

microbiology↗

bioRxiv2025

Glycolysis-dependent Sulfur Metabolism Orchestrates Morphological Plasticity and Virulence in Fungi

Fungi exhibit remarkable morphological plasticity, which allows them to undergo reversible transitions between distinct cellular states in response to changes in their environment. This phenomenon, termed fungal morphogenesis, is critical for fungi to survive and colonize diverse ecological niches and establish infections in a variety of hosts. Despite significant advancements in the field with respect to understanding the gene regulatory networks that control these transitions, the metabolic determinants of fungal morphogenesis remain poorly characterized. In this study, we uncover a previously uncharacterized, conserved dependency between central carbon metabolism and de novo biosynthesis of sulfur-containing amino acids that is critical for fungal morphogenesis, in two key fungal species. Using a multidisciplinary approach, we demonstrate that glycolytic flux is crucial to drive fungal morphogenesis in a cAMP-independent manner and perturbation of this pathway leads to a significant downregulation in the expression of genes involved in de novo biosynthesis of sulfur-containing amino acids. Remarkably, exogenous supplementation of sulfur-containing amino acids robustly rescues the morphogenesis defect induced by the perturbation of glycolysis in both Saccharomyces cerevisiae and Candida albicans, underscoring the pivotal role of de novo biosynthesis of sulfur-containing amino acid as a downstream effector of morphogenesis. Furthermore, a C. albicans mutant lacking the glycolytic enzyme, phosphofructokinase-1 (Pfk1) exhibited significantly reduced survival within murine macrophages and attenuated virulence in a murine model of systemic candidiasis. Overall, our work elucidates a previously uncharacterized coupling between glycolysis and sulfur metabolism that is critical for driving fungal morphogenesis, contributing to our understanding of this conserved phenomenon.

microbiology↗

bioRxiv2022

Phosphate budgeting to mitochondria controls glucose-mediated mitochondrial repression

Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing{Delta} ubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in{Delta} ubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic-flux dependent intracellular Pi budgeting is a key constraint for mitochondrial repression.

biochemistry↗

bioRxiv2020

Metabolite plasticity drives carbon-nitrogen resource budgeting to enable division of labor in a clonal community

Previously, we discovered that in glucose-limited yeast colonies, metabolic constraints drive cells into groups exhibiting gluconeogenic and glycolytic metabolic states. Here, threshold amounts of trehalose - a limiting, produced resource, controls the emergence and self-organization of the cells exhibiting the glycolytic state, by acting as a carbon source to fuel these metabolic demands (Varahan et al., 2019). We now discover that the plasticity of use of a non-limiting resource, aspartate, controls both resource production and the emergence of heterogeneous cell states, based on differential cellular metabolic budgeting. In gluconeogenic cells, aspartate provides carbon for trehalose production, while in glycolytic cells using trehalose for carbon, aspartate supplies nitrogen to drive nucleotide synthesis. This metabolic plasticity of aspartate enables carbon-nitrogen budgeting, thereby driving the biochemical self-organization of distinct cell states. Through this organization, cells in each state exhibit true division of labor, providing bet-hedging and growth/survival advantages for the whole community.

cell biology↗

bioRxiv2019

Metabolic constraints drive self-organization of specialized cell groups

How phenotypically distinct states in isogenic cell populations appear and stably co-exist remains an unresolved question. We find that within a clonal yeast colony developing in low glucose, cells arrange into metabolically disparate cell groups. Using this system, we model and experimentally identify metabolic constraints sufficient to drive such assembly. Beginning in a gluconeogenic state, cells in a contrary state, exhibiting high pentose phosphate pathway activity, spontaneously appear and proliferate, in a spatially constrained manner. The gluconeogenic cells in the developing colony produce a resource, which we identify as trehalose. At threshold concentrations of trehalose, cells in the new metabolic state emerge and proliferate. A self-organized system establishes, where cells in this new state are sustained by trehalose consumption, which thereby restrains other cells in the trehalose producing, gluconeogenic state. Our work suggests simple physico-chemical principles that determine how isogenic cells spontaneously self-organize into structured assemblies in complimentary, specialized states.

cell biology↗