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Baicalein could inhibit the growth and biofilm formation of Candida albicans, the most common clinical fungal pathogen. However, the antifungal mechanism of baicalein has not been elucidated. In this study, isobaric tags for relative and absolute quantification (iTRAQ) was used to verify the mechanism of antifungal fluconazole and baicalein. A total of 58 common proteins were detected in cells treated with fluconazole. These proteins encompassed fluconazole-targeted sterol synthesis pathway, including Erg11p, Erg6p, Erg3p, Erg25p, Erg5p, Erg10p, and Ncp1p. Next, iTRAQ was applied to the comparison of baicalein-treated C. albicans proteins, which detected 16 common proteins. The putative NADH dehydrogenase Cpd2p and the ATP-binding cassette transporter Snq2p were the most upregulated proteins with the treatment of baicalein. Our results showed that CPD2 disruption elevated C. albicans resistance to baicalein significantly both in vitro and in vivo. Further in-depth studies revealed that CPD2 disruption reduced the activation of C. albicans metacaspase and partially restored the mitochondrial membrane potential reduction caused by the treatment of baicalein, which indicated that CPD2 was involved in the apoptosis induced by baicalein. Consistently, under the treatment of baicalein, CPD2Δ/Δ mutant produced lower reactive oxygen species that was critical in causing oxidative damage and apoptosis in C. albicans. These results indicated that baicalein could increase intracellular oxidative damage by upregulating the expression of Cpd2p so as to inhibit the growth of C. albicans, which provides new insights for investigating the antifungal target of baicalein.In our study, isobaric tags for relative and absolute quantification (iTRAQ) was used to study the antifungal mechanisms of fluconazole and baicalein. Baicalein could enhance the oxidative stress of Candida albicans by upregulating CPD2 expression.

Aim: To investigate the role of SDH2 in Candida albicans filamentation and virulence. Materials & methods: Caenorhabditis elegans and mouse candidiasis models were used to assess the virulence of a sdh2Δ/Δ mutant. Various hypha-inducing media were used to evaluate the hyphal development of C. albicans. DCFH-DA was used to measure intracellular Reactive Oxygen Species (ROS) levels. Results: The sdh2Δ/Δ mutant was avirulent in the C. elegans model, hypovirulent in a murine candidiasis model, and defective to form filaments both in vitro and in vivo. Intracellular ROS level increased in the sdh2Δ/Δ mutant, and the filamentation defects of sdh2Δ/Δ were rescued by decreasing intracellular ROS. Conclusion: SDH2 plays an important role in C. albicans filamentation and virulence probably through affecting intracellular ROS. [Formula: see text]

Sterols are the basal components of the membranes of the fungal pathogen Candida albicans, and these membranes determine the susceptibility of C. albicans cells to a variety of stresses, such as ionic, osmotic and oxidative pressures, and treatment with antifungal drugs. The common antifungal azoles in clinical use are targeted to the biosynthesis of ergosterol. In the past years, the synthesis, storage and metabolism of ergosterol in Saccharomyces cerevisiae has been characterized in some detail; however, these processes has not been as well investigated in the human opportunistic pathogen C. albicans. In this review, we summarize the genes involved in ergosterol synthesis and regulation in C. albicans. As well, genes in S. cerevisiae implicated in ergosterol storage and conversions with other lipids are noted, as these provide us clues and directions for the study of the homologous genes in C. albicans. In this report we have particularly focused on the essential roles of ergosterol in the dynamic process of cell biology and its fundamental status in the biological membrane system that includes lipid rafts, lipid droplets, vacuoles and mitochondria. We believe that a thorough understanding of this classic and essential pathway will give us new ideas about drug resistance and morphological switching in C. albicans.

To ensure correct DNA replication, eukaryotes have signaling pathways that respond to replication-associated DNA damage and trigger repair. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, a complex of proteins, including the cullin protein Rtt101p and two adapter proteins Mms22p and Mms1p, is important for proper response to replication stress. We have investigated this system in Candida albicans. In this pathogen, Mms22p is important for recovery from DNA replication damage induced by agents including methylmethane sulfonate, camptothecin, and ionizing radiation. Although no clear ortholog of Mms1p has been identified in C. albicans, loss of either Mms22p or Rtt101p generates similar damage sensitivity, consistent with a common function. In S. cerevisiae, the Mrc1p−Csm3p−Tof1p complex stabilizes stalled replication forks and activates a replication checkpoint and interacts with Mms22p. A similar complex in S. pombe, consisting of the Tof1p and Csm3p orthologs Swi1p and Swi3p, along with the fission yeast Mrc1p, genetically also interacts with Mms22p. Intriguingly in C. albicans only Mrc1p and Csm3p appear involved in damage repair, and Mms22p is required for responding to DNA damage agents in MRC1 or CSM3 conditional mutants. In C. albicans, although the loss of RAD57 greatly impairs response in the pathogen to many DNA-damaging agents, lethality due to camptothecin damage requires concomitant loss of Rad57p and Mms22p, suggesting that Mms22p is only essential for homologous recombination induced by camptothecin. These results establish that although C. albicans uses conserved cellular modules to respond to DNA damage and replication blocks, the specific details of these modules differ significantly from the S. cerevisiae model.

There is currently a small number of classes of antifungal drugs, and these drugs are known to target a very limited set of cellular functions. We derived a set of approximately 900 nonessential, transactivator-defective disruption strains from the tetracycline-regulated GRACE collection of strains of the fungal pathogen

Antifungal azole drugs inhibit the synthesis of ergosterol and cause the accumulation of sterols containing a 14α-methyl group, which is related to the properties of cell membrane. Due to the frequent recurrence of fungal infections and clinical long-term prophylaxis, azole resistance is increasing rapidly. In our research, Nsg2p, encoded by the ORF19.273 in Candida albicans, is found to be involved in the inhibition of 14α-methylated sterols and resistance to azoles. Under the action of fluconazole, nsg2Δ/Δ mutants are seriously damaged in the integrity and functions of cell membranes with a decrease of ergosterol ratio and an increase of both obtusifoliol and 14α-methylfecosterol ratio. The balance between ergosterol and 14α-methyl sterols mediated by NSG2 plays an important role in C. albicans responding to azoles in vitro as well as in vivo. These phenotypes are completely different from those of Nsg2p in Saccharomyces cerevisiae, which is proved to increase the stability of HMG-CoA and resistance to lovastatin. Based on the evidence above, it is indicated that the decrease of 14α-methylated sterols is an azole-resistant mechanism in C. albicans, which may provide new strategies for overcoming the problems of azole resistance.

Candida albicans is one of the most common fungal pathogen in humans due to its high frequency as an opportunistic and pathogenic fungus causing superficial as well as invasive infections in immunocompromised patients. An understanding of gene function in C. albicans is necessary to study the molecular basis of its pathogenesis, virulence and drug resistance. Several manipulation techniques have been used for investigation of gene function in C. albicans, including gene disruption, controlled gene expression, protein tagging, gene reintegration, and overexpression. In this review, the main cassettes containing selectable markers used for gene manipulation in C. albicans are summarized; the advantages and limitations of these cassettes are discussed concerning the influences on the target gene expression and the virulence of the mutant strains.