Integrative Transcriptomic and Systems Biology Analyses Identify TCB1 as a Calcium-Responsive Gene in Cryptococcus neoformans

<i>Cryptococcus neoformans</i> is a pathogenic yeast and the leading cause of cryptococcosis in humans. The calcium-calcineurin signaling pathway plays a central role in stress adaptation and virulence. To identify the uncharacterized regulators of fungal adaptation, we utilized an integrative systems biology approach, combining differential gene expression and network analysis using transcriptomic data from three key components of the calcium-calcineurin pathway (Cna1, Crz1, and Pmc1). Our workflow identified the <i>CNAG_00522</i> gene product, which we designated tricalbin 1 (<i>TCB1</i>) due to its conserved calcium and lipid-binding C2 domains. <i>TCB1</i> expression was found to be regulated by both Cna1 and Pmc1. Network analyses positioned Tcb1 as a bottleneck linking general stress response and cellular processes. Further molecular characterization confirmed that <i>TCB1</i> expression is temperature and calcium-responsive. Functional studies of the <i>tcb1</i>Δ mutant revealed an enlarged capsule, increased GXM shedding, and enhanced viability under host-mimicking conditions. However, phenotypic screening demonstrated that the <i>tcb1</i>Δ mutant does not display sensitivity to cell wall or osmotic stressors, and <i>TCB1</i> deletion did not attenuate virulence in the <i>Tenebrio</i> larval model. These findings suggest that <i>TCB1</i> functions as a specialized regulator of fungal growth at 37 °C, capsule size, and GXM shedding. This study validates our integrative approach for guiding the identification of these complex regulators.