Yaf9 conditionally contributes to cell size control in Candida albicans

Abstract

Candida albicans is an opportunistic fungal pathogen renowned for its ability to transition between distinct phenotypic states, such as the yeast-hyphae transition and the white-opaque switching. This morphological plasticity allows the organism to adapt to various host environments and evade immune responses. The white state is characterized by yeast-like cells with high proliferative capacity, whereas the opaque state features elongated cells with enhanced mating ability. The regulation of white-opaque switching is primarily controlled by a complex network of transcription factors. White-Opaque Regulator 1 (Wor1) serves as a master regulator crucial for the establishment and maintenance of the opaque state by activating the expression of genes required for opaque cell formation [1–3]. Conversely, the Mating-Type Like (MTL) locus in C. albicans acts as a critical barrier to white-opaque switching. The genes present at this locus strictly repress the white-to-opaque transition by the formation of a1/α2 complex; therefore, only MTLa/a or MTLα/α strains frequently switch to the opaque state [4,5]. Although the MTLa/α lab strain CAI4 is typically locked in the white state, some MTLa/α clinical isolates can switch to opaque [6]. Several genes were found to modulate this repression. For example, loss of HBR1, which is an activator of MTLALPHA1 and MTLALPHA2 gene expression, enables switching in MTLa/α cells [7]. Deletion of transcriptional repressors of the opaque state such as TUP1 also facilitates white-to-opaque switching [8]. The SWR1 complex incorporates H2A.Z into chromatin, and loss of Swr1 enhances switching and stabilizes the opaque state in MTL homozygous cells [9]. Our previous work revealed that the NuA4 histone acetyltransferase complex and the SWR1 complex merge into a supercomplex via Yaf9 in white-state yeast cells in C. albicans [10]. Here, we first tested whether Yaf9 is involved in white-to-opaque switching in MTLa/α heterozygous cells. The knockout of the YAF9 gene was validated by genotyping and qRT-PCR, confirming its loss at both the genomic and transcriptional levels (Supplementary Figure S1). The yaf9 null mutant cells were spread onto YPD plates and incubated in 20% CO2 at 25°C. After eight days of growth, sectors containing opaque cells were observed (Figure 1A). The frequency of opaque cell formation in the yaf9 mutant exceeded that in wild-type (WT) cells overexpressing WOR1 (Figure 1B). qRT-PCR analysis confirmed significant upregulation of opaque cell-specific markers, including WOR1 and OP4, in yaf9 mutant opaque cells, whereas the white cell marker WH11 was downregulated (Figure 1C). To examine whether YAF9 deletion affects the expression of MTL genes, we performed qRT-PCR for MTLA1 and MTLALPHA2 in white WT cells and in both white and opaque yaf9 mutant cells. The expression of both genes remained unchanged in yaf9 mutant cells (Figure 1D), suggesting that Yaf9-mediated repression of white-to-opaque switching occurs independently of MTL gene regulation. As Yaf9 is a component of the NuA4 and SWR1 complexes, we next investigated the roles of the NuA4 core enzyme Esa1 and the SWR1 core enzyme Swr1 in white-to-opaque switching in MTLa/α heterozygous cells. As shown in Figure 1E (upper panel), esa1 cells failed to switch to the opaque form under 20% CO2 stimulation, indicating that Esa1 activity is essential for opaque cell formation under the tested conditions. In contrast, swr1 cells readily underwent white-to-opaque switching (Figure 1E, lower panel), similar to the yaf9 mutant. These results indicate that Yaf9 functions as a repressor of white-to-opaque switching and that its deletion bypasses the repression imposed by the MTLa/α configuration. We then examined the role of YAF9 in white-to-opaque switching in MTLa/a cells, where MTL repression is removed. In air, yaf9 cells remained white; however, when exposed to 20% CO2, they frequently (> 50%) switched to the opaque form, which occurred at a significantly higher frequency than WT cells (Figure 2A,B). Notably, yaf9 cells exhibited a novel elongated opaque morphology, which we term e-Op cells. Quantification revealed that e-Op cells had similar width but were two to three times longer than WT opaque cells (Figure 2C). At the transcriptional level, e-Op cells displayed comparable upregulation of WOR1 and OP4 and downregulation of WH11 (Figure 2D). Notably, WH11 expression in yaf9 white cells was slightly higher than that in WT white cells. Like white cells, opaque cells are also capable of forming filaments under specific conditions [11]. To determine whether e-Op cells represent a filamentous form of opaque cells, we examined their gene expression and morphological stability. Multiple lines of evidence indicate that e-Op cells are distinct from these filamentous forms. First, when cultured on SOR medium, which promotes filamentous g

1. Introduction

Candida albicans is an opportunistic fungal pathogen renowned for its ability to transition between distinct phenotypic states, such as the yeast-hyphae transition and the white-opaque switching. This morphological plasticity allows the organism to adapt to various host environments and evade immune responses. The white state is characterized by yeast-like cells with high proliferative capacity, whereas the opaque state features elongated cells with enhanced mating ability.

The regulation of white-opaque switching is primarily controlled by a complex network of transcription factors. White-Opaque Regulator 1 (Wor1) serves as a master regulator crucial for the establishment and maintenance of the opaque state by activating the expression of genes required for opaque cell formation [1–3]. Conversely, the Mating-Type Like (MTL) locus in C. albicans acts as a critical barrier to white-opaque switching. The genes present at this locus strictly repress the white-to-opaque transition by the formation of a1/α2 complex; therefore, only MTLa/a or MTLα/α strains frequently switch to the opaque state [4,5].