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Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex (1,2). Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi (3,4). The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata (5,6). The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent (7,8). Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active (9-14), suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease., On the basis of our previous findings that deletion of the KIX domain of S. cerevisiae GAL11 or C. glabrata GAL11A abrogates Pdrl-dependent transcriptional responses and xenobiotic tolerance we hypothesized [...]

Current increases in antifungal drug resistance in Candida spp. and clinical treatment failures are of concern, as invasive candidiasis is a significant cause of mortality in intensive care units (ICUs). This trend reflects the large and expanding use of newer broad-spectrum antifungal agents, such as triazoles and echinocandins. In this review, we firstly present an overview of the mechanisms of action of the drugs and of resistance in pathogenic yeasts, subsequently focusing on recent changes in the epidemiology of antifungal resistance in ICU. Then, we emphasize the clinical impacts of these current trends. The emergence of clinical treatment failures due to resistant isolates is described. We also consider the clinical usefulness of recent advances in the interpretation of antifungal susceptibility testing and in molecular detection of the mutations underlying acquired resistance. We pay particular attention to practical issues relating to ICU patient management, taking into account the growing threat of antifungal drug resistance., Author(s): Danièle Maubon [sup.1] [sup.2], Cécile Garnaud [sup.1] [sup.2], Thierry Calandra [sup.3], Dominique Sanglard [sup.4], Muriel Cornet [sup.1] [sup.2] Author Affiliations: (1) grid.410529.b, 0000000107924829, Parasitologie-Mycologie, Institut de Biologie et de [...]

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was ~35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-[alpha], were recovered from AS (MTL-a/MTL-[alpha]), while a single type of PAP1 allele (PAP1-[alpha]) was recovered from AR isolates (MTL-[alpha]/MTL-[alpha]). Among the heterozygous deletions of PAP1-a ([DELTA]pap1-a/PAP1-[alpha]) and PAP1-[alpha] (PAP1-a/[DELTA]pap1-[alpha]), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance. DOI 10.1099/mic.0.035154-0

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

Upregulation of the MDR1 ([m.bar]ulti[d.bar]rug [r.bar]esistance 1) gene is involved in the development of resistance to antifungal agents in clinical isolates of the pathogen Candida albicans. To better understand the molecular mechanisms underlying the phenomenon, the cis-acting regulatory elements present in the MDR1 promoter were characterized using a [beta]-galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or [H.sub.2][O.sub.2], whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements within the MDR1 promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (CDR2). One, a [b.bar]enomyl [r.bar]esponse [e.bar]lement (BRE), is situated at position -296 to -260 with respect to the ATG start codon. It is required for benomyl-dependent MDR1 upregulation and is also necessary for constitutive high expression of MDR1. A second element, termed [[H.bar].sub.2][O.sub.2] [r.bar]esponse [e.bar]lement (HRE), is situated at position -561 to -520. The HRE is required for [H.sub.2][O.sub.2]-dependent MDR1 upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p (Candida AP-1 protein) lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to [H.sub.2][O.sub.2]. Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a trans-acting and positive regulatory role in the [H.sub.2][O.sub.2]-dependent transcription of MDR1. A minimal BRE (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that MDR1 may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the cis-acting elements of the MDR1 promoter should now serve to elucidate the nature of the components that modulate its activity. DOI 10.1099/mic.0.29277-0

TAC1, a Candida albicans transcription factor situated near the mating-type locus on chromosome 5, is necessary for the upregulation of the ABC-transporter genes CDR1 and CDR2, which mediate azole resistance. We showed previously the existence of both wild-type and hyperactive TAC1 alleles. Wild-type alleles mediate upregulation of CDR1 and CDR2 upon exposure to inducers such as fluphenazine, while hyperactive alleles result in constitutive high expression of CDR1 and CDR2. Here we recovered TAC1 alleles from two pairs of matched azole-susceptible (DSY294; FH1: heterozygous at mating-type locus) and azole-resistant isolates (DST296; FH3: homozygous at mating-type locus). Two different TAC1 wild-type alleles were recovered from DSN294 (TAC1-3 and TAC1-4) while a single hyperactive allele (TAC1-5) was isolated from DSY296. A single amino acid (aa) difference between TAC1-4 and TAC1-5 (Asn977 to Asp or N977D) was observed in a region corresponding to the predicted activation domain of Taclp. Two TAC1 alleles were recovered from FH1 (TAC1-6 and TAC1-7) and a single hyperactive allele (TAC1-7) was recovered from FH3. The N977D change was seen in TAC1-7 in addition to several other aa differences. The importance of N977D in conferring hyperactivity to TAC1 was confirmed by site-directed mutagenesis. Both hyperactive alleles TAC1-5 and TAC1-7 were codominant with wild-type alleles and conferred hyperactive phenotypes only when homozygous. The mechanisms by which hyperactive alleles become homozygous was addressed by comparative genome hybridization and single nucleotide polymorphism arrays and indicated that loss of TAC1 heterozygosity can occur by recombination between portions of chromosome 5 or by chromosome 5 duplication.

Phospholipases C are known to be important regulators of cellular processes but may also act as virulence factors of pathogenic microbes. At least three genes in the genome of the human-pathogenic fungus Candida albicans encode phospholipases with conserved phospholipase C (Plc) motifs. None of the deduced protein sequences contain N-terminal signal peptides, suggesting that these phospholipases are not secreted. In contrast to its orthologue in Sacharomyces cerevisiae, CaPLC1 seems to be an essential gene. However, a conditional mutant with reduced transcript levels of CaPLC1 had phenotypes similar to Plc1p-deficient mutants in S. cerevisiae, including reduced growth on media causing increased osmotic stress, on media with a non-glucose carbon source, or at elevated or lower temperatures, suggesting that CaPlc1p, like the Plc1p counterpart in S. cerevisiae, may be involved in multiple cellular processes. Furthermore, phenotypic screening of the heterozygous [DELTA]Caplc1/ CaPLC1 mutant showed additional defects in hyphal formation. The loss of CaPLC1 cannot be compensated by two additional PLC genes of C. albicans (CaPLC2 and CaPLC3) encoding two almost identical phospholipases C with no counterpart in S. cerevisiae but containing structural elements found in bacterial phospholipases C. Although the promoter sequences of CaPLC2 and CaPLC3 differed dramatically, the transcriptional pattern of both genes was similar. In contrast to CaPLC1, CaPLC2 and CaPLC3 are not essential. Although Caplc2/3 mutants had reduced abilities to produce hyphae on solid media, these mutants were as virulent as the wild-type in a model of systemic infection. These data suggest that C. albicans contains two different classes of phospholipases C which are involved in cellular processes but which have no specific functions in pathogenicity.

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast-hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast-hypha morphogenesis in C. albicans.