Erg6 Acts as a Downstream Effector of the Transcription Factor Flo8 To Regulate Biofilm Formation in Candida albicans

ABSTRACT The yeast-to-hyphal morphotype transition and subsequent biofilm formation are important virulence factors of Candida albicans and are closely associated with ergosterol biosynthesis. Flo8 is an important transcription factor that determines filamentous growth and biofilm formation in C. albicans. However, the relationship between Flo8 and regulation of the ergosterol biosynthesis pathway remains elusive. Here, we analyzed the sterol composition of a flo8-deficient C. albicans strain by gas chromatography-mass spectrometry and observed the accumulation of the sterol intermediate zymosterol, the substrate of Erg6 (C-24 sterol methyltransferase). Accordingly, the transcription level of ERG6 was reduced in the flo8-deficient strain. Yeast one-hybrid experiments revealed that Flo8 physically interacted with the ERG6 promoter. Ectopic overexpression of ERG6 in the flo8-deficient strain partially restored biofilm formation and in vivo virulence in a Galleria mellonella infection model. These findings suggest that Erg6 is a downstream effector of the transcription factor Flo8 that mediates the cross talk between sterol synthesis and virulence factors in C. albicans. IMPORTANCE Biofilm formation by C. albicans hinders its eradication by immune cells and antifungal drugs. Flo8 is an important morphogenetic transcription factor that regulates the biofilm formation and in vivo virulence of C. albicans. However, little is known about how Flo8 regulates biofilm formation and fungal pathogenicity. Here, we determined that Flo8 directly binds to the promoter of ERG6 to positively regulate its transcriptional expression. Consistently, loss of flo8 results in the accumulation of the substrate of Erg6. Moreover, ectopic overexpression of ERG6 at least partially restores the biofilm formation and virulence of the flo8-deficient strain both in vitro and in vivo. This work provides a new perspective on the metabolic link between transcription factors and morphotypes in C. albicans.

FIG 1 Erg6 is a downstream protein of the transcription factor Flo8. (A) C. albicans CAF2-1, flo8D/D, flo8D/D1pBA1, flo8D/D1pBA1-FLO8, or flo8D/D1pBA1-ERG6 cells were cultured in RPMI 1640 medium for 12 h at 37°C. Sterol components were analyzed by gas chromatography-mass spectrometry. b-Sitosterol was added as a standard. Peak 1, zymosterol; peak 2, ergosterol; peak 3, b-sitosterol. (B) After calibration using the internal reference, the relative change in zymosterol content in the flo8D/D strain compared with CAF2-1 and flo8D/D1pBA1 strains or the flo8D/D1pBA1-ERG6 strain compared with the flo8D/D1pBA1-FLO8 strain was calculated. The bars represent means 6 standard deviations. The data obtained from 4 biological replicates are shown. ns, no significance. (C) C. albicans flo8D/D1pBA1, flo8D/D1pBA1-FLO8, or flo8D/D1pBA1-ERG6 cells were incubated in RPMI 1640 medium for 0, 6, 12, 24, or 48 h at 37°C. The transcription levels of ERG6 in flo8D/D1pBA1-FLO8, flo8D/D1pBA1, or flo8D/D1pBA1-ERG6 cells as determined by PCR are indicated as the fold change relative to the transcription level of ERG6 in flo8D/D1pBA1-FLO8 cells at 0 h. The bars represent the means 6 standard deviations from three independent experiments. *, P , 0.05; **, P , 0.01; ***, P , 0.001. ns, no significance. (D) Y1H experiment showing the binding of Flo8 to the promoter of ERG6. Yeast cells containing pGAD-Rec-53 and p53His2 were the positive-control group. Yeast cells containing pGAD-Rec-53 and pHis2 or pGADT7 and pHis2-ERG6promoter-Apart were the two negative-control groups. Sophisticated transcriptional regulatory networks allow C. albicans to respond to various host-specific environmental signals, and further exploration of the flexibility of these networks is required to better understand their regulatory mechanisms (10,11). The transcription factor Flo8 acts downstream of the cAMP-protein kinase A (PKA) pathway and interacts with Efg1 to regulate hyphal growth (12). Flo8 is also required for biofilm development within 48 h (13). flo8-deficient strains fail to form filaments and biofilms and are avirulent in systemically infected mouse models (12). Studies of Flo8 have mainly focused on the phenotypic changes in flo8-deficient strains or the function of Flo8 (14)(15)(16). In contrast, little attention has been given to the genes that Flo8 regulates, particularly those involved in ergosterol biosynthesis.
A previous genome-scale analysis revealed that the ergosterol biosynthesis pathway is crucial for the development of hyphae by C. albicans (5). To investigate whether Flo8 regulates hyphal formation via sterol biosynthesis, we first analyzed the sterol composition of the flo8D/D strain and the wild-type strain CAF2-1. The absence of flo8 resulted in the accumulation of the sterol intermediate zymosterol ( Fig. 1A and B), which is converted to fecosterol by C-24 sterol methyltransferase (Erg6). We further compared the sterol compositions of the flo8D/D1pBA1 strain and the reconstituted flo8D/D1pBA1-FLO8 strain; the accumulation of zymosterol could be observed in the flo8-deficient strain after incubation at 37°C for 12 or 24 h ( Fig. 1A and B). These results indicated that there is a high probability that the expression of ERG6 is reduced in the flo8-deficient strain. We then performed quantitative PCR (qPCR) to analyze the transcriptional abundance of ERG6. Upon induction of hyphal formation in RPMI 1640 medium at 37°C, the expression of ERG6 in the flo8D/D1pBA1 strain was reduced compared with that in the flo8D/D1pBA1-FLO8 strain during 48 h of growth (Fig. 1C). A previous study revealed that transcriptional repression or deletion of ERG6 leads to defects in C. albicans hyphal formation (5,17). Thus, we speculated that Flo8 regulates hyphal or biofilm formation by binding to the putative promoter region of ERG6. To test this hypothesis, we conducted a yeast one-hybrid (Y1H) assay in Saccharomyces cerevisiae Y187 cells. The recombinant pHIS2-ERG6promoter-Apart (2426 bp to 2942 bp) or pHIS2-ERG6promoter-Bpart (21 bp to 2427 bp) was linearized and used to transform S. cerevisiae Y187 for the self-activation test. The results suggested that S. cerevisiae Y187 transformed with pHIS2-ERG6promoter-Bpart showed strong selfactivating activity while S. cerevisiae Y187 transformed with pHIS2-ERG6promoter-Apart did not show self-activating activity (see Fig. S1 in the supplemental material). As shown in Fig. 1D, yeast cells containing the bait vector carrying pHIS2-ERG6promoter-Apart grew on SD/2Leu2Trp2His medium supplemented with 10 mM 3-amino-1,2,4-triazole (3-AT) when cotransformed with the pGADT7-Flo8 construct. In contrast, yeast cells containing the bait vector carrying the promoter region of ERG6 and the empty pGADT7 vector failed to grow on the same selective medium. These results suggested that Flo8 binds to the promoter of ERG6 in vivo and activates expression of a downstream reporter gene in yeast.
To explore whether Flo8 regulates C. albicans hyphal development and subsequent biofilm formation by modulating ERG6 expression, we ectopically overexpressed ERG6 in a flo8D/D strain. The results of qPCR showed that the transcription of ERG6 was upregulated in the flo8D/D1pBA1-ERG6 strain compared with both the flo8D/D1pBA1 strain and the flo8D/D1pBA1-FLO8 strain during 48 h of growth under hypha-inducing conditions (Fig. 1C). In addition, overexpression of ERG6 in the flo8-deficient strain decreased the level of zymosterol ( Fig. 1A and B). We then measured the hyphal development and biofilm formation of flo8D/D1pBA1, flo8D/D1pBA1-FLO8, and flo8D/D1pBA1-ERG6 strains. Ectopic overexpression of ERG6 did not obviously restore the hyphal elongation of the flo8D/ D1pBA1 strain during the first 12 h of growth. However, the defect in biofilm formation of the flo8D/D1pBA1 strain was greatly reversed upon ectopic overexpression of ERG6 ( Fig. 2A and B). The flo8D/D1pBA1-ERG6 strain also formed a considerable quantity of hyphae at 24 h compared with the flo8D/D1pBA1 strain (Fig. 2C). Consistent with these effects, overexpression of ERG6 significantly increased the expression of transcription factors that positively regulate filamentation and biofilm formation, including EFG1, TEC1, NDT80, and BCR1, after 24 h but not 12 h of induction (Fig. 2D). We further tested the biofilm-forming ability of an ERG6 knockout strain. The results shown in Fig. S2 suggested the ERG6 knockout strain was defective in biofilm formation and had changes in the transcript levels of biofilm-regulated genes such as ROB1, GAL4, TEC1, and BRG1 similar to those in the FLO8 knockout strain. These results suggested that Erg6 acts as an important downstream effector of Flo8 to regulate biofilm formation. ERG6 deletion also leads to zymosterol accumulation (18). We deduce that the accumulation of specific sterol intermediates such as zymosterol causes changes in the integrity of the membranes and affects the biofilm formation, but this hypothesis needs further investigation.
Hyphal growth and biofilm formation are important virulence factors in C. albicans. To further evaluate the role of ERG6 in the in vivo virulence of C. albicans, Galleria mellonella larvae were used as an infection model. Infection of larvae with the flo8D/D1pBA1-ERG6 or flo8D/D1pBA1-FLO8 strain resulted in a mortality rate exceeding 80% after 6 days (Fig. 2E). In contrast, 50% of the larvae infected with the flo8D/D1pBA1 strain survived more than 6 days (Fig. 2E). Moreover, the fungal burden caused by the flo8D/D1pBA1-ERG6 strain was similar to that caused by the flo8D/D1pBA1-FLO8 strain and higher than that caused by the flo8D/D1pBA1 strain (Fig. 2F). Consistent with these observations, histological analysis using periodic acid-Schiff (PAS) staining revealed a higher number of melanized nodules in larvae infected with the flo8D/D1pBA1-ERG6 strain than in larvae infected with the flo8D/D1pBA1 strain (Fig. 2G). These results suggested that Erg6 is an important effector of Flo8 in the regulation of C. albicans virulence.
In summary, we determined that Flo8 directly binds to the putative promoter of ERG6 to regulate sterol synthesis. Disruption of flo8 leads to the accumulation of the intermediate zymosterol due to the low expression of ERG6, as well as reduced biofilm formation and in vivo virulence. Erg6 appears to be an important downstream effector of Flo8 in the regulation of C. albicans biofilm formation and in vivo virulence. This study provides a perspective for understanding the physiological effects of transcriptional regulators.

SUPPLEMENTAL MATERIAL
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