Transcriptional expression of PHR2 is positively controlled by the calcium signaling transcription factor Crz1 through its binding motif in the promoter

ABSTRACT GH72 family of β-(1,3)-glucanosyltransferases is required for cell wall biogenesis, morphogenesis, and virulence of fungi. Candida albicans PHR2 is an acidic pH-induced gene that encodes a GH72 enzyme involved in cross-linking of β−1,3- and β−1,6-glucans in the cell wall. In response to environmental pH changes, the expression of PHR2 is negatively regulated by the transcription factor CaRim101 through the pH-responsive Rim101 pathway. In this study, we have generated the inactivation mutant for PHR2 through the CRISPR/Cas9 approach. The PHR2 mutant is sensitive to both membrane- and cell wall-disturbing agents, SDS, Calcofluor white and casponfungi, but resistant to two azole antifungals, ketoconazole and fluconazole. Although the PHR2 mutant is not sensitive to calcium stress, it becomes sensitive to high levels of calcium stress in the presence of cyclosporin A, the specific inhibitor of calcineurin. In addition, we have demonstrated that the expression of PHR2, the downstream target of CaRim101, is positively controlled by the transcription factor CaCrz1 through its binding motif (5′ A-315TAGGCACAG 3′) in the PHR2 promoter. Furthermore, the deletion of PHR2 increases the cell precipitation rate in cell culture under acidic conditions. IMPORTANCE The fungal cell wall consists of glucans, mannoproteins, and chitin and is essential for cell viability, morphogenesis, and pathogenesis. The enzymes of the GH72 family are responsible for ß-(1,3)-glucan elongation and branching, which is crucial for the formation of the glucan-chitin polymer at the bud neck of yeast cells. In the human fungal pathogen Candida albicans, there are five GH72 enzyme-encoding genes: PHR1, PHR2, PHR3, PGA4, and PGA5. It is known that expression of PHR1 and PHR2 is controlled by the pH-responsive Rim101 pathway through the transcription factor Rim101. In this study, we have demonstrated that the transcription expression of PHR2 is also controlled by the transcription factor Crz1 through its binding motif in the promoter. Therefore, we have uncovered a dual-control mechanism by which PHR2 expression is negatively regulated via CaRim101 through the pH-responsive pathway and positively modulated by CaCrz1 through the calcium/calcineurin signaling pathway.