Unraveling FKS1 and FKS2 Regulation in Candida glabrata

For clinicians battling the persistent threat of fungal infections, Candida glabrata stands out as a formidable adversary, particularly due to its increasing resistance to frontline antifungal drugs like echinocandins. These drugs target 1,3-β-glucan synthase (GS), a crucial enzyme for fungal cell wall biosynthesis. In C. glabrata, GS is encoded by two functionally redundant paralogous genes, FKS1 and FKS2. Despite their importance as drug targets, the intricate regulation of their expression in various conditions – from lab cultures to the host environment – has remained poorly understood. A recent study by Irene Gonzalez-Jimenez, et al. in Erika Shor and David Perlin's Lab at the Center for Discovery & Innovation dives into this regulatory complexity, revealing insights that could reshape our understanding of C. glabrata pathogenesis and antifungal resistance.

            The researchers employed a multi-faceted approach, combining various techniques to unravel the layers of FKS1 and FKS2 regulation. Unlike previous studies that often relied solely on quantitative reverse-transcriptase PCR (qRT-PCR with single primer pairs), this investigation utilized fluorescent transcriptional reporters (degGFP, a short-lived variant of GFP, with a half-life of only 7 minutes), time-lapse microscopy, qRT-PCR with multiple primer pairs spanning the gene, protein analysis, and RNA-seq data mining. This comprehensive strategy allowed them to distinguish between transcriptional and post-transcriptional regulatory mechanisms, a distinction often missed in earlier work.

            Some of the study's key revelations:

• The study confirmed that both FKS1 and FKS2 promoter activities are regulated by the cell cycle, peaking during the Synthesis phase (S-phase). This makes biological sense, as GS is essential for building the cell wall of the growing bud. This finding is particularly notable for C. glabrata, as previous cell cycle synchronization methods used for Saccharomyces cerevisiae are not applicable.

• Differential Expression in Culture vs. Host:

  • Under standard laboratory culture conditions (e.g., exponential growth in YPD or SC medium), FKS1 is expressed at significantly higher levels than FKS2, with its promoter showing stronger activity. This corrects a previous contradictory report, which was found to have used misassigned primers.

  • Strikingly, this dynamic shifts dramatically in a host environment or under stress conditions. During the stationary phase (slow or non-growing phase) in culture and especially under host conditions (e.g., in macrophages or mouse kidneys), FKS2 is expressed at equivalent or even higher levels than FKS1. This suggests a role for FKS2 in adaptation to host-imposed stresses, such as nutrient limitation.

• Unveiling Post-Transcriptional Regulation of FKS2:

  • One of the most significant findings is the evidence for calcineurin-mediated post-transcriptional regulation of FKS2. While deletion of FKS1 (fks1∆ mutant) strongly induces FKS2 mRNA levels (over 10-fold in YPD culture using primers near the 5' end of the ORF), there was no corresponding increase in FKS2 promoter activity. This strongly suggests regulation occurs after transcription initiation, possibly through mRNA stabilization.

  • Further supporting post-transcriptional control, the observed increase in FKS2 mRNA in the fks1∆ mutant was not uniform across the gene's ORF, being more pronounced at the 5' end in YPD-cultured cells.

  • The calcineurin inhibitor FK506 significantly reduced FKS2 expression in fks1∆ cells but had little to no effect in wild-type cells with functional FKS1, reinforcing the calcineurin pathway's role in this post-transcriptional upregulation.

  • Analysis of Fks2 protein levels corroborated the post-transcriptional effect, showing an approximate 30% increase in fks1∆ mutants, which was abolished by FK506. Interestingly, in fks1∆ cells, Fks2 protein was also observed to be more diffusely localized throughout the daughter cell, rather than solely at the bud tip, where new cell wall synthesis primarily occurs.

    
    

            These findings have implications for understanding and combating echinocandin resistance in C. glabrata. The fact that FKS2 expression predominates in vivo under host-stress conditions helps explain why the majority of clinical echinocandin resistance mutations are found in FKS2 rather than FKS1. If Fks2 is the primary GS subunit expressed during infection, then mutations in it would logically have a greater impact on drug resistance and be preferentially selected during antifungal treatment.

            This study underscores the complexity of gene regulation in fungal pathogens and highlights the importance of studying gene expression in physiologically relevant host-like environments. Future research should aim to delineate the precise mechanisms of FKS2 post-transcriptional regulation, such as mRNA stabilization or processing, and further explore the interplay between Fks1 and Fks2 subunits in forming functional GS complexes in vivo. Understanding these regulatory networks is vital for developing more effective strategies against this challenging fungal pathogen.

Gonzalez-Jimenez I, Keniya MV, Aptekmann AA, Quinteros C, Wilkerson A, Arastehfar A, Daneshnia F, Perlin DS, Shor E. Expression of 1,3-β-glucan synthase subunits in Candida glabrata is regulated by the cell cycle and growth conditions and at both transcriptional and post-transcriptional levels. Antimicrob Agents Chemother. 2025 Aug 6;69(8):e0050025. doi: 10.1128/aac.00500-25. Epub 2025 Jun 17. PMID: 40525410; PMCID: PMC12326982.

https://journals.asm.org/doi/10.1128/aac.00500-25