Multilocus sequence typing (MLST) analysis of Candida albicans isolates colonizing acrylic dentures before and after denture replacement.

Yeast, in particular Candida albicans, are the principal fungal cause of denture stomatitis, and can also be present as a commensal in many individuals. Few studies, however, have examined oral retention of yeast strains over time. We analyzed the yeast present in saliva samples and from the dentures of 10 individuals colonized with yeast but with no signs of stomatitis, before new complete maxillary dentures were fitted and also at 1, 3, and 6 months after denture replacement. Yeast species were presumptively identified on selective agar plates and were present in nine individuals before denture replacement and in six at the 6-month time point. C. albicans was detected in seven individuals pre-replacement, and in three by 6 months post-replacement. Sixty-two isolates (up to five from each C. albicans-positive sample) were analyzed by multilocus sequence typing (MLST) (33 from saliva and 29 from dentures). Six MLST allele profiles were identified that were common to several individuals. These profiles included three previously reported diploid sequence types (DSTs) and three novel DSTs. Two of the novel DSTs were closely related variants of a previously reported DST, and both showed loss of heterozygosity polymorphisms within one of the seven MLST gene sequences. For three individuals, at least one DST that was present before denture replacement was still detected in either saliva or on dentures at subsequent sampling times. Our results indicate that denture replacement reduces but does not remove, colonising yeast and confirm previous observations of C. albicans strain microevolution.

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.

Adhesion of Yeast and Bacteria to Oral Surfaces.

Colonization of surfaces in the human body by microorganisms is an early, essential, step in the initiation of infectious disease. We have developed in vitro assays to investigate interactions between yeast or bacterial cells and human tissues, fluids, or prostheses. Such assays can be used to identify the adhesins, ligands, and receptors involved in these interactions, for example, by determining which components of the microbe or human tissue/fluid interfere with adherence in the assay. The assays can also be applied to find ways of preventing adhesion, and subsequent disease, by investigating the effects of different conditions and added compounds on adherence in the in vitro assays. Here we describe assays for measuring adhesion of the oral yeast Candida albicans, a common commensal and opportunistic pathogen, or the bacterium Staphylococcus epidermidis, which is not normally pathogenic but is known to form biofilms on medical prostheses. The assays described belong to two approaches to investigating adhesion and biofilm formation: (i) retention at a fixed time point following liquid washes, and (ii) retention against a continuous flow of medium.

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.

The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.

Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains.

N-acetylglucosamine increases symptoms and fungal burden in a murine model of oral candidiasis.

The amino sugar N-acetylglucosamine (GlcNAc) is an in vitro inducer of the hyphal mode of growth of the opportunistic pathogen Candida albicans. The development of hyphae by C. albicans is considered to contribute to the pathogenesis of mucosal oral candidiasis. GlcNAc is also a commonly used nutritional supplement for the self-treatment of conditions such as arthritis. To date, no study has investigated whether ingestion of GlcNAc has an effect on the in vivo growth of C. albicans or the pathogenesis of a C. albicans infection. Using a murine model of oral candidiasis, we have found that administration of GlcNAc, but not glucose, increased oral symptoms of candidiasis and fungal burden. Groups of mice were given GlcNAc in either water or in a viscous carrier, i.e., 1% methylcellulose. There was a dose-dependent relationship between GlcNAc concentration and the severity of oral symptoms. Mice given the highest dose of GlcNAc, 45.2 mM, also showed a significant increase in fungal burden, and increased histological evidence of infection compared to controls given water alone. We propose that ingestion of GlcNAc, as a nutritional supplement, may have an impact on oral health in people susceptible to oral candidiasis.

Yeast colonization of voice prostheses: pilot study investigating effect of a bovine milk product containing anti-Candida albicans immunoglobulin A antibodies on yeast colonization and valve leakage.

OBJECTIVES: Our goals were to determine whether a bovine milk product containing anti-Candida albicans immunoglobulin A antibodies ("immune milk") could reduce the adherence of C albicans to voice prosthesis silicone in vitro, and whether administration of the milk could reduce C albicans colonization and voice prosthesis damage in vivo. METHODS: An in vitro assay of C albicans attachment to silicone was developed with radiolabeled C albicans. A pilot crossover in vivo trial, over 3 periods of 3 months, was also undertaken for 4 patients with voice prostheses, comparing daily administrations of immune milk and a control milk product. The prosthesis valves were replaced at each changeover and were assessed for wet weight of removable biofilm, yeast numbers in removable biofilm, valve leakage, and valve damage. RESULTS: Immune milk inhibited C albicans adherence to silicone in vitro. However, in a small clinical pilot study, this effect was not replicated. CONCLUSIONS: There is scope to further investigate the topical use of immune milk for management of voice prosthesis biofilms.

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.

Fungal PDR transporters: Phylogeny, topology, motifs and function.

The overexpression of pleiotropic drug resistance (PDR) efflux pumps of the ATP-binding cassette (ABC) transporter superfamily frequently correlates with multidrug resistance. Phylogenetic analysis of 349 full-size ( approximately 160kDa) PDR proteins (Pdrps) from 55 fungal species, including major fungal pathogens, identified nine separate protein clusters (A-G, H1a/H1b and H2). Fungal, plant and human ABCG-family Pdrps possess a nucleotide-binding domain [NBD] and a transmembrane domain [TMD] in a family-defining 'reverse' ABC transporter topology [NBD-TMD] that is duplicated [NBD-TMD](2) in full-size fungal and plant Pdrps. Although full-size Pdrps have similar halves indicating early gene duplication/fusion, they show asymmetry of their NBDs and extracellular loops (ELs). Members of cluster F are most symmetric and may be closely related to the evolutionary ancestor of Pdrps. Unique structural elements are predicted, new PDR-specific motifs identified, and the significance of these and other structural features discussed.

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 Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p.

Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile red as a new fluorescent substrate for CaCdr1p, CaCdr2p, and CaMdr1p. Nile red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressing these transporters. Enniatin selectively inhibited the efflux of Nile red from S. cerevisiae cells expressing CaCdr1p or CaMdr1p but not from cells expressing CaCdr2p. This indicates that Nile red can be used for the identification of inhibitors specific for particular transporters mediating antifungal resistance in pathogenic yeast.

ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates.

Fluconazole (FLC) remains the antifungal drug of choice for non-life-threatening Candida infections, but drug-resistant strains have been isolated during long-term therapy with azoles. Drug efflux, mediated by plasma membrane transporters, is a major resistance mechanism, and clinically significant resistance in Candida albicans is accompanied by increased transcription of the genes CDR1 and CDR2, encoding plasma membrane ABC-type transporters Cdr1p and Cdr2p. The relative importance of each transporter protein for efflux-mediated resistance in C. albicans, however, is unknown; neither the relative amounts of each polypeptide in resistant isolates nor their contributions to efflux function have been determined. We have exploited the pump-specific properties of two antibody preparations, and specific pump inhibitors, to determine the relative expression and functions of Cdr1p and Cdr2p in 18 clinical C. albicans isolates. The antibodies and inhibitors were standardized using recombinant Saccharomyces cerevisiae strains that hyper-express either protein in a host strain with a reduced endogenous pump background. In all 18 C. albicans strains, including 13 strains with reduced FLC susceptibilities, Cdr1p was present in greater amounts (2- to 20-fold) than Cdr2p. Compounds that inhibited Cdr1p-mediated function, but had no effect on Cdr2p efflux activity, significantly decreased the resistance to FLC of seven representative C. albicans isolates, whereas three other compounds that inhibited both pumps did not cause increased chemosensitization of these strains to FLC. We conclude that Cdr1p expression makes a greater functional contribution than does Cdr2p to FLC resistance in C. albicans.

Adhesion of Yeast and Bacteria to Oral Surfaces.

Colonization of surfaces in the human body by microorganisms is an early, essential, step in the initiation of infectious disease. We have developed in vitro assays to investigate interactions between yeast or bacterial cells and human tissues, fluids, or prostheses. Such assays can be used to identify the adhesins, ligands, and receptors involved in these interactions, for example, by determining which components of the microbe or human tissue/fluid interfere with adherence in the assay. The assays can also be applied to finding ways of preventing adhesion, and subsequent disease, by investigating the effects of different conditions and added compounds on adherence in the in vitro assays.Here we describe assays for measuring adhesion of the oral yeast Candida albicans, a common commensal and opportunistic pathogen, or the bacterium Staphylococcus epidermidis, which is not normally pathogenic but is known to form biofilms on medical prostheses. The assays described belong to two approaches to investigating adhesion and biofilm formation: (1) retention at a fixed time point following liquid washes and (2) retention against a continuous flow of medium.

Amino acid residues affecting drug pump function in Candida albicans--C. albicans drug pump function.

Membrane-located drug transporters are important components in the multidrug resistance of microbial cells and human tissues. In fungi, clinically important resistance to antifungal drugs most often results from the over-expression of efflux pump proteins in the plasma membrane of the resistant cell. This review describes studies of the ATP binding cassette (ABC) family of membrane efflux pumps in the opportunistic human pathogen Candida albicans and, in particular, examines how changes in the polypeptide sequence can affect pump function. The identification of amino acid residues affecting pump function can provide new insights into efflux pump mechanisms and the relationship between structure and function. Such information will be important for the design of pump inhibitors which could supplement existing antifungal drugs.

Targeting efflux pumps to overcome antifungal drug resistance.

Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance of Candida species to azole drugs. One approach to overcome this resistance is to inhibit the pumps and chemosensitize resistant strains to azole drugs. Drug discovery targeting fungal efflux pumps could thus result in the development of azole-enhancing combination therapy. Heterologous expression of fungal efflux pumps in Saccharomyces cerevisiae provides a versatile system for screening for pump inhibitors. Fungal efflux pumps transport a range of xenobiotics including fluorescent compounds. This enables the use of fluorescence-based detection, as well as growth inhibition assays, in screens to discover compounds targeting efflux-mediated antifungal drug resistance. A variety of medium- and high-throughput screens have been used to identify a number of chemical entities that inhibit fungal efflux pumps.

Functional analysis of fungal drug efflux transporters by heterologous expression in Saccharomyces cerevisiae.

Clinically important resistance of fungal pathogens to azole antifungal drugs is most frequently caused by the over-expression of energy-dependent drug efflux pumps. These pumps usually belong to either the ATP-binding cassette (ABC) family or the Major Facilitator Superfamily (MFS) class of membrane transporter. Little is known about how these pumps work and there is an urgent need to develop pump antagonists that circumvent azole resistance. We have developed a protein hyper-expression system to facilitate functional analysis of efflux pumps based on a Saccharomyces cerevisiae host which has been deleted in seven major ABC transporters to reduce the background of endogenous efflux activity. Plasmid pABC3 was engineered to allow functional hyper-expression of foreign proteins in this host. The main advantages of the system include its ease of directional cloning and the use of homologous recombination to stably integrate single copy constructs into the host genome under the control of a highly active transcriptional regulator. The system has been used to clone and functionally hyper-express genes encoding drug efflux pumps from several pathogenic fungi. Furthermore, the protein hyper-expression system has been used to screen for pump inhibitors and study the structure and function of heterologous membrane proteins.

[ABC transporters of pathogenic fungi: recent advances in functional analyses].

ABC (ATP binding cassette) transporters consist of transmembrane domains which confer specificity, and structurally conserved nucleotide binding domains that contain highly conserved amino acid motifs. They act not only as transporters but also as receptors or channels that use energy generated by ATP hydrolysis. ABC transporters are widely dispersed in nature. They are found in cells ranging from prokaryotes (bacteria) to eukaryotes (including humans) and several are considered to play crucial roles in cellular homeostasis. Defects in ABC transporters in humans are associated with severe diseases such as type 2 diabetes and cystic fibrosis. Some ABC transporters extrude xenobiotics and confer resistance to chemotherapeutics on microbial pathogens and cancer cells. Thus ABC transporters are of considerable medical importance. Structure-function analysis of ABC transporters has begun to elucidate their mechanisms of substrate recognition, the functional regulation of ATP-binding and hydrolysis and to identify intrinsic physiological functions. In pathogenic fungi, ABC transporters contribute to the clinical problem of drug resistance. The application of new technologies to the examination of fungal ABC transporter function is providing new insights into the use of antifungal drugs in medical mycology and contributing to a better understanding of these important membrane proteins.

Selective Advantages of a Parasexual Cycle for the Yeast Candida albicans.

The yeast Candida albicans can mate. However, in the natural environment mating may generate progeny (fusants) fitter than clonal lineages too rarely to render mating biologically significant: C. albicans has never been observed to mate in its natural environment, the human host, and the population structure of the species is largely clonal. It seems incapable of meiosis, and most isolates are diploid and carry both mating-type-like (MTL) locus alleles, preventing mating. Only chromosome loss or localized loss of heterozygosity can generate mating-competent cells, and recombination of parental alleles is limited. To determine if mating is a biologically significant process, we investigated if mating is under selection. The ratio of nonsynonymous to synonymous mutations in mating genes and the frequency of mutations abolishing mating indicated that mating is under selection. The MTL locus is located on chromosome 5, and when we induced chromosome 5 loss in 10 clinical isolates, most of the resulting MTL-homozygotes could mate with each other, producing fusants. In laboratory culture, a novel environment favoring novel genotypes, some fusants grew faster than their parents, in which loss of heterozygosity had reduced growth rates, and also faster than their MTL-heterozygous ancestors-albeit often only after serial propagation. In a small number of experiments in which co-inoculation of an oral colonization model with MTL-homozygotes yielded small numbers of fusants, their numbers declined over time relative to those of the parents. Overall, our results indicate that mating generates genotypes superior to existing MTL-heterozygotes often enough to be under selection.

Antifungal drug susceptibilities of commensal Candida isolates.

Two hundred and forty-two oral commensal yeast isolates were obtained from a convenience sample of 134 healthy 7- and 8-year-old children (65 males and 69 females). The isolates were initially tested for their susceptibilities to the antifungal azole drug fluconazole, using an agar diffusion method (Etest), which was suitable for screening large numbers of yeast isolates, and confirmed as equivalent to the broth microdilution reference method. Eighteen isolates from 7 children were found to have low fluconazole susceptibility according to guidelines published by the United States National Committee for Clinical Laboratory Standards (NCCLS). The isolates with low susceptibility were identified as either Candida tropicalis (n = 9 of 34 strains tested) or Candida glabrata (n = 9 of 13 strains tested). Selected isolates (6 susceptible and 7 with lower susceptibility to fluconazole) were also tested by a reference broth microdilution method for susceptibility to fluconazole and to a related over-the-counter azole antifungal, miconazole. A positive correlation between susceptibility to fluconazole and to miconazole was observed. The high rate (38 percent) of reduced susceptibility in commensal C tropicalis and C glabrata strains may represent a future treatment problem if the use of over-the-counter azole drugs increase.