Inhibitor-Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition.

Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20 Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant "hot spot 1" mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.

Identification and functional characterization of Penicillium marneffei major facilitator superfamily (MFS) transporters.

PENICILLIUM MARNEFFEI: is a thermally dimorphic fungus that causes penicilliosis, and become the third-most-common opportunistic fungal infection in immunocompromised patients in Southeast Asia. Azoles and amphotericin B have been introduced for the treatment, however, it is important to investigate possible mechanisms of azole resistance for future treatment failure. We identified 177 putative MFS transporters and classified into 17 subfamilies. Among those, members of the Drug:H+ antiporter 1 subfamily are known to confer resistance to antifungals. Out of 39 paralogs, three (encoded by PmMDR1, PmMDR2, and PmMDR3) were heterologously overexpressed in S. cerevisiae AD∆ conferred resistance to various drugs and compounds including azoles, albeit to different degrees. PmMDR1-expressing strain showed resistance to the broadest range of drugs, followed by the PmMDR3, and PmMDR2 conferred weak resistance to a limited range of drugs. We conclude that PmMDR1 and PmMDR3, may be able to serve as multidrug efflux pumps.

FK506 Resistance of Saccharomyces cerevisiae Pdr5 and Candida albicans Cdr1 Involves Mutations in the Transmembrane Domains and Extracellular Loops.

The 23-membered-ring macrolide tacrolimus, a commonly used immunosuppressant, also known as FK506, is a broad-spectrum inhibitor and an efflux pump substrate of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters. Little, however, is known about the molecular mechanism by which FK506 inhibits PDR transporter drug efflux. Thus, to obtain further insights we searched for FK506-resistant mutants of Saccharomyces cerevisiae cells overexpressing either the endogenous multidrug efflux pump Pdr5 or its Candida albicans orthologue, Cdr1. A simple but powerful screen gave 69 FK506-resistant mutants with, between them, 72 mutations in either Pdr5 or Cdr1. Twenty mutations were in just three Pdr5/Cdr1 equivalent amino acid positions, T550/T540 and T552/S542 of extracellular loop 1 (EL1) and A723/A713 of EL3. Sixty of the 72 mutations were either in the ELs or the extracellular halves of individual transmembrane spans (TMSs), while 11 mutations were found near the center of individual TMSs, mostly in predicted TMS-TMS contact points, and only two mutations were in the cytosolic nucleotide-binding domains of Pdr5. We propose that FK506 inhibits Pdr5 and Cdr1 drug efflux by slowing transporter opening and/or substrate release, and that FK506 resistance of Pdr5/Cdr1 drug efflux is achieved by modifying critical intramolecular contact points that, when mutated, enable the cotransport of FK506 with other pump substrates. This may also explain why the 35 Cdr1 mutations that caused FK506 insensitivity of fluconazole efflux differed from the 13 Cdr1 mutations that caused FK506 insensitivity of cycloheximide efflux.

Identification and characterization of Candida utilis multidrug efflux transporter CuCdr1p.

The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag.

Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.

Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.

Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-ß mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor.

Overexpression of the Candida albicans ATP-binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ~1.89 × 10(6) member D-octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative of the D-octapeptide D-NH(2) -FFKWQRRR-CONH(2) , as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization-resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug-like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin ß9 have modes of action similar to RC21v3.

Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

BACKGROUND: A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). RESULTS: Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%. CONCLUSION: We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.

The mannan of Candida albicans lacking ß-1,2-linked oligomannosides increases the production of inflammatory cytokines by dendritic cells.

Mannans are mannose polymers attached to cell wall proteins in all Candida species, including the pathogenic fungus Candida albicans. Mannans are sensed by pattern recognition receptors expressed on innate immune cells. However, the detailed structural patterns affecting immune sensing are not fully understood because mannans have a complex structure that includes α- and ß-mannosyl linkages. In this study, we focused on the ß-1,2-mannosides of N-linked mannan in C. albicans because this moiety is not present in the non-pathogenic yeast Saccharomyces cerevisiae. To investigate the impact of ß-1,2-mannosides on immune sensing, we constructed a C. albicans ∆mnn4/∆bmt1 double deletant. Thin-layer chromatography and nuclear magnetic resonance analyses revealed that the deletant lacked ß-1,2-mannosides in N-linked mannan. Mannans lacking the ß-1,2-mannosides induced the production of higher levels of inflammatory cytokines, including IL-6, IL-12p40 and TNF-α, in mice dendritic cells compared to wild-type mannan. Our data show that ß-1,2-mannosides in N-linked mannan reduce the production of inflammatory cytokines by dendritic cells.

Chimeras of Candida albicans Cdr1p and Cdr2p reveal features of pleiotropic drug resistance transporter structure and function.

Members of the pleiotropic drug resistance (PDR) family of ATP binding cassette (ABC) transporters consist of two homologous halves, each containing a nucleotide binding domain (NBD) and a transmembrane domain (TMD). The PDR transporters efflux a variety of hydrophobic xenobiotics and despite the frequent association of their overexpression with the multidrug resistance of fungal pathogens, the transport mechanism of these transporters is poorly understood. Twenty-eight chimeric constructs between Candida albicans Cdr1p (CaCdr1p) and Cdr2p (CaCdr2p), two closely related but functionally distinguishable PDR transporters, were expressed in Saccharomyces cerevisiae. All chimeras expressed equally well, localized properly at the plasma membrane, retained their transport ability, but their substrate and inhibitor specificities differed significantly between individual constructs. A detailed characterization of these proteins revealed structural features that contribute to their substrate specificities and their transport mechanism. It appears that most transmembrane spans of CaCdr1p and CaCdr2p provide or affect multiple, probably overlapping, substrate and inhibitor binding site(s) similar to mammalian ABC transporters. The NBDs, in particular NBD1 and/or the ∼150 amino acids N-terminal to NBD1, can also modulate the substrate specificities of CaCdr1p and CaCdr2p.

Transcription factors CgUPC2A and CgUPC2B regulate ergosterol biosynthetic genes in Candida glabrata.

Zn[2]-Cys[6] binuclear transcription factors Upc2p and Ecm22p regulate the expression of genes involved in ergosterol biosynthesis and exogenous sterol uptake in Saccharomyces cerevisiae. We identified two UPC2/ECM22 homologues in the pathogenic fungus Candida glabrata which we designated CgUPC2A and CgUPC2B. The contribution of these two genes to sterol homeostasis was investigated. Cells that lack CgUPC2A (upc2AΔ) exhibited enhanced susceptibility to the sterol biosynthesis inhibitors, fluconazole and lovastatin, whereas upc2BΔ-mutant cells were as susceptible to the drugs as wild-type cells. The growth of upc2AΔ cells was also severely attenuated under anaerobic conditions. Lovastatin treatment enhanced the expression of ergosterol biosynthetic genes, ERG2 and ERG3 in wild-type and upc2BΔ but not in upc2AΔ cells. Similarly, serum-induced expression of ERG2 and ERG3 was completely impaired in upc2AΔ cells but was unaffected in upc2BΔ cells, whereas serum-induced expression of the sterol transporter gene CgAUS1 was impaired in both upc2AΔ and upc2BΔ cells. These results suggest that in C. glabrata CgUPC2A but not in CgUPC2B is the main transcriptional regulator of the genes responsible for maintaining sterol homeostasis as well as susceptibility to sterol inhibitors.

Reconstitution of high-level micafungin resistance detected in a clinical isolate of Candida glabrata identifies functional homozygosity in glucan synthase gene expression.

OBJECTIVES: A mechanism for the acquisition of high-level echinocandin resistance in Candida glabrata was investigated. FKS mutants were constructed to: determine whether clinically significant micafungin resistance requires a hot-spot mutation in FKS1 and a premature stop codon in FKS2, as was observed in a clinical isolate; select for variants with reduced susceptibility and locate mutations in FKS genes; and assess the roles of FKS1 and FKS2. METHODS: A panel of FKS mutants was constructed using micafungin-susceptible parents by site-directed mutagenesis. Drug susceptibility, gene expression and glucan synthase activities were compared between mutants. Mutations acquired by selection were identified by DNA sequence analysis of FKS genes from selected variants. Single FKS deletants were constructed and their phenotypes examined. RESULTS: Introduction of the hot-spot mutation in FKS1 alone conferred an intermediate reduction in susceptibility, and the premature stop codon in FKS2 alone had no effect on susceptibility, while severely reduced susceptibility equivalent to that of the clinical isolate required both mutations. Exposure of susceptible strains to micafungin yielded variants with an intermediate reduction in susceptibility that possessed a hot-spot mutation in FKS1. Further exposure to micafungin yielded variants with severely reduced susceptibility that acquired various single mutations in FKS2. The phenotypes of Δfks1 and Δfks2 mutants indicate that the two FKS genes are functionally redundant, while deletion of both FKS1 and FKS2 conferred synthetic lethality. CONCLUSIONS: In the laboratory mutants of C. glabrata, clinically significant reduced susceptibility to micafungin required single nucleotide changes in both FKS1 and FKS2, and both genes encoded ß-1,3-glucan synthase catalytic subunits.

Efflux-mediated antifungal drug resistance.

Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis.

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae ADΔΔ is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae ADΔΔ, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Vph1 is associated with the copper homeostasis of Cryptococcus neoformans serotype D.

We clarified the roles of VPH1 in Cryptococcus neoformans serotype D by examining the detailed phenotypes of VPH1-deficient cells (Δvph1) in terms of their capability to grow in acidic and alkaline pH, at a high temperature, and under high osmotic conditions, in addition to the involvement of VPH1 in copper (Cu) homeostasis and the expression of some C. neoformans virulence factors. Δvph1 could grow well on minimal medium (YNB) but exhibited hypersensitivity to 20 µM Cu due to the failure to induce Cu-detoxifying metallothionein genes (CMT1 and CMT2). In contrast, Δvph1 exhibited defective growth on rich medium (YPD), and the induction of Cu transporter genes (CTR1 and CTR4) did not occur in this medium, implying that this strain was incapable of the uptake of Cu ions for growth. However, the addition of excess Cu promoted CTR gene expression and supported Δvph1 growth. These results suggested that the lack of the VPH1 gene disturbed Cu homeostasis in C. neoformans. Moreover, the loss of Vph1 function influenced the urease activity of C. neoformans.

Candida albicans Bgl2p, Ecm33p, and Als1p proteins are involved in adhesion to saliva-coated hydroxyapatite.

Introduction: Candida albicans is an opportunistic pathogen that causes oral candidiasis. A previous study showed that Bgl2p and Ecm33p may mediate the interaction between the yeast and saliva-coated hydroxyapatite (SHA; a model for the tooth surface). This study investigated the roles of these cell wall proteins in the adherence of C. albicans to SHA beads. Methods: C. albicans BGL2 and ECM33 null mutants were generated from wild-type strain SC5314 by using the SAT1-flipper gene disruption method. A novel method based on labelling the yeast with Nile red, was used to investigate the adherence. Results: Adhesion of bgl2Δ and ecm33Δ null mutants to SHA beads was 76.4% and 64.8% of the wild-type strain, respectively. Interestingly, the adhesion of the bgl2Δ, ecm33Δ double mutant (87.7%) was higher than that of both single mutants. qRT-PCR analysis indicated that the ALS1 gene was over-expressed in the bgl2Δ, ecm33Δ strain. The triple null mutant showed a significantly reduced adherence to the beads, (37.6%), compared to the wild-type  strain. Conclusion: Bgl2p and Ecm33p contributed to the interaction between C. albicans and SHA beads. Deletion of these genes triggered overexpression of the ALS1 gene in the bgl2Δ/ecm33Δ mutant strain, and deletion of all three genes caused a significant decrease in adhesion.

Antifungal drug resistance of oral fungi.

Fungi comprise a minor component of the oral microbiota but give rise to oral disease in a significant proportion of the population. The most common form of oral fungal disease is oral candidiasis, which has a number of presentations. The mainstay for the treatment of oral candidiasis is the use of polyenes, such as nystatin and amphotericin B, and azoles including miconazole, fluconazole, and itraconazole. Resistance of fungi to polyenes is rare, but some Candida species, such as Candida glabrata and C. krusei, are innately less susceptible to azoles, and C. albicans can acquire azole resistance. The main mechanism of high-level fungal azole resistance, measured in vitro, is energy-dependent drug efflux. Most fungi in the oral cavity, however, are present in multispecies biofilms that typically demonstrate an antifungal resistance phenotype. This resistance is the result of multiple factors including the expression of efflux pumps in the fungal cell membrane, biofilm matrix permeability, and a stress response in the fungal cell. Removal of dental biofilms, or treatments to prevent biofilm development in combination with antifungal drugs, may enable better treatment and prevention of oral fungal disease.

Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei.

Most Candida krusei strains are innately resistant to fluconazole (FLC) and can cause breakthrough candidemia in immunocompromised individuals receiving long-term prophylactic FLC treatment. Although the azole drug target, Erg11p, of C. krusei has a relatively low affinity for FLC, drug efflux pumps are also believed to be involved in its innate FLC resistance. We describe here the isolation and characterization of Abc1p, a constitutively expressed multidrug efflux pump, and investigate ERG11 and ABC1 expression in C. krusei. Examination of the ERG11 promoter revealed a conserved azole responsive element that has been shown to be necessary for the transcription factor Upc2p mediated upregulation by azoles in related yeast. Extensive cloning and sequencing identified three distinct ERG11 alleles in one of two C. krusei strains. Functional overexpression of ERG11 and ABC1 in Saccharomyces cerevisiae conferred high levels of resistance to azoles and a range of unrelated Abc1p pump substrates, while small molecule inhibitors of Abc1p chemosensitized C. krusei to azole antifungals. Our data show that despite the presence of multiple alleles of ERG11 in some, likely aneuploid, C. krusei strains, it is mainly the low affinity of Erg11p for FLC, together with the constitutive but low level of expression of the multidrug efflux pump Abc1p, that are responsible for the innate FLC resistance of C. krusei.

Identification of the putative protein phosphatase gene PTC1 as a virulence-related gene using a silkworm model of Candida albicans infection.

Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Delta, yvh1Delta, sit4Delta, and ptc1Delta) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Delta, yvh1Delta, and sit4Delta mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Delta revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.

Serum or bile affects the in vitro azole susceptibilities of Candida spp.

Drug susceptibility tests were performed with a series of Candida spp. in media supplemented with serum or bile. The azole susceptibilities of several medically important Candida spp., including C. albicans but not C. parapsilosis, were significantly reduced in supplemented media. These findings have important implications for the mechanisms of acquired azole resistance in pathogenic fungi.