Bistable Expression Of EAP2, A Master Regulator Of White-opaque Switching In Candida Albicans

Candida albicans is an opportunistic pathogen, often causing both mucosaland systemic infections in immunocompromised people. The yeast switchesheritably and at high frequency between at least seven general phenotypesidentified by colony morphology on agar. The most intensive studiedmorphologic transitions are the yeast-hyphae transition and white-opaque switch.The ability of morphologic switching is closely related with its pathogenicity.Filamentous growth has also been observed in the most popular model S.cerevisiae. To determine whether a similar regulatary mechanism exists in C.albicans, a C. albicans genomic DNA library was introduced into a S. cerevisiaehaploid flo8 mutant. In this screening, we identified several genes including EAP2and CZF2 by suppressing the invasive growth defect of flo8 mutant. Eap2contains a Tos9 domain characterized by a conserved motif (⁶⁴KRWTDG⁶⁹),resembling a PKA1 phosphorylation site. Eap2 has a putative nuclear localizationsignal sequence in its N-terminal, and GFP-Eap2 fusion expression confirmed itsnuclear localization. Czf2 is a Gal4 family protein, which contains a Zn(II)2Cys6binuclear cluster DNA binding motif.Two signal transduction pathways, the Kss1 MAPK pathway and thecAMP-dependent PKA1 pathway, that regulate filamentous growth are conservedin C. albicans and S. cerevisiae. To study the functional role of Eap2 and Czf2,the EAP2 and CZF2 were ectopicly expressed in ste7, ste12, tec1 and flo8mutants of S. cerevisiae. We found that ectopic expression of EAP2 and CZF2suppressed the defects of these mutants in diploid filamentous development andhaploid invasive growth. These results suggested that Eap2 and Czf2 stimulated S.cerevisiae filamentous and invasive growth and bypassed requirement of the twopathways.EAP2 has two transcripts of ³.9 kb and ².5 kb. In diploid wild typeSC5314, the expession of EAP2 can hardly be detected. We examinedtranscription of EAP2 in the laboratorial strain WO-1. The two transcripts ofEAP2 were exclusively expressed in opaque cells, but not in white cells. Therefore,EAP2 expression displays an all-or-none pattern. These results suggest that Eap2may function in regulating white/opaque switch of C. albicans.To elucidate the function of EAP2 in C. albicans, we deleted both copies ofEAP2 by homologous recombination. Disruption of EAP2 blocks white-to-opaqueswitching in homozygous MTLa strain. EAP2 was reintroduced into the mutantwith a plasmid pACT1-EAP2. The ability of white-to-opaque switching could berecovered. These results suggested that Eap2 is required for white-to-opaquetransition in C.albicans. We found the MTLa eap2 strain also showed reducedmating efficiency, compared with that of wild type MTLa strain. In addition, themating efficiencies are similar in different media. Our results indicate the lowfrequency of white-to-opaque switching of eap2 mutant may result to the reducedmating efficiency.Overexpression of EAP2 in halploid strains locked the cells in opaque phaseat 25 °C. The cells also maintained in opaque-phase at 30 or 37°C. In diploidstrains, which have no white/opaque transition, overexpression of EAP2 alsopromoted white cells switching to opaque cells. Therefore, the role of Eap2bypassed the inhibitory effect of MTLa/α complex.White-opaque switching is a bistable transition that happens stochastically,and the phase is stable for many cell divisions after switching. In this study, wehave identified Eap2 as a master control of white-opaque switching. Deletion ofEAP2 blocks opaque cell formation. Ectopic expression of EAP2 from the ACT1promoter converts all white cells to stable opaque cells in MTL homozygotes, butis unable to establish the opaque phase in eap2 mutants. Importantly, EAP2expression displays an all-or-none pattern and ectopic expression of Eap2 inducesthe transcription of EAP2 from its own promoter. We propose that a feedbackregulation of EAP2 expression supports the bistability of EAP2 expression andprovides a mechanism for white-opaque switching.We also found overexpression of EAP2 inhibited haploid cells developinghyphae under all hyphae-induced conditions investigated. Northern blot analysisconfirmed that the expression of hyphal specific genes was down-regulated. Indiploid white cells, Eap2 partially inhibited hyphal development, while the abilitywas completely impaired in diploid opaque cells. Therefore, Eap2 could be animportant negative regulator of C. albicans filamentous formation.Deletion of Tos9 domain inactivated the roles of Eap2 in promotingwhite-to-opaque transition and inhibiting filamentous formation in homogyzousstrains. The conserved motif (64KRWTDG69) resembles a PKA1 phosphorylationsite. Replacement of Thr67 to Ala blocked the functions of Eap2. These resultsindicated Eap2 may be a target of PKA1 kinases and involved in cAMP/PKAcascade.Czf2 is a Zn(II)2Cys6 zinc-finger protein. In efg1, nrg1 and tec1 mutants,expression of CZF2 was obviously up-regulated. To study the function of Czf2 inC. albicans, we disrupted both copies of CZF2 alleles. The czf2 null mutantshowed no remarkable defect of filamentous formation both in liquid media andsolid mdia. Compared to the wild strains, the czf2 null mutant formed lessbranches on solid serum-induced condition. In homozygous MTLa strain, deletionof CZF2 increased white-to-opaque transition efficiency obviously. And we foundczf2 null mutant showed increased mating efficiency.