Analysis of a fungus-specific transcription factor family, the Candida albicans zinc cluster proteins, by artificial activation.

The zinc cluster proteins are a family of transcription factors that are unique to the fungal kingdom. In the pathogenic yeast Candida albicans, zinc cluster transcription factors control the expression of virulence-associated traits and play key roles in the development of antifungal drug resistance. Gain-of-function mutations in several zinc cluster transcription factors, which result in constitutive overexpression of their target genes, are a frequent cause of azole resistance in clinical C. albicans isolates. We found that zinc cluster proteins can also be artificially activated by C-terminal fusion with the heterologous Gal4 activation domain. We used this strategy to create a comprehensive library of C. albicans strains expressing all 82 zinc cluster transcription factors of this fungus in a potentially hyperactive form. Screening of this library identified regulators of invasive filamentous growth and other phenotypes that are important during an infection. In addition, the approach uncovered several novel mediators of fluconazole resistance, including the multidrug resistance regulator Mrr2, which controls the expression of the major C. albicans multidrug efflux pump CDR1. Artificial activation therefore is a highly useful method to study the role of zinc cluster transcription factors in C. albicans and other fungi of medical, agricultural and biotechnological importance.

Inducible and constitutive activation of two polymorphic promoter alleles of the Candida albicans multidrug efflux pump MDR1.

Overexpression of the multidrug efflux pump MDR1 confers resistance to the antifungal drug fluconazole on Candida albicans. It has been reported that two types of MDR1 promoters exist in C. albicans and that homozygosity for the allele with higher activity may promote fluconazole resistance. We found that the two MDR1 promoter alleles in strain SC5314 were equally well activated by inducing chemicals or hyperactive forms of the transcription factors Mrr1 and Cap1, which control MDR1 expression. In addition, no loss of heterozygosity at the MDR1 locus was observed in MDR1-overexpressing clinical C. albicans strains that developed fluconazole resistance during therapy.

The transcription factor Ndt80 does not contribute to Mrr1-, Tac1-, and Upc2-mediated fluconazole resistance in Candida albicans.

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80Δ mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80Δ mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.

Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito.

Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.