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OBJECTIVES: Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. MATERIALS AND METHODS: Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. RESULTS: Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. CONCLUSION: These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers. [ABSTRACT FROM AUTHOR]

Primary cutaneous gamma/delta (γδ) T-cell lymphoma (PCGDTCL) is a rare, aggressive malignant neoplasm of γδ T lymphocytes arising within the skin and subcutis. We present a challenging case of PCGDTCL diagnosed in a 35-year-old male soldier who presented with constitutional symptoms, pancytopenia, hemophagocytic lymphohistiocytosis (HLH), disseminated lymphadenopathy, and cutaneous lesions on his extremities and back following a deployment to Iraq and Syria. Histopathologic evaluation of an excisional biopsy showed that PCGDTCL can be focal, localized to the subcutaneous adipose tissue, and obscured by predominant HLH in the surrounding tissues. Pathologists should recognize that the diagnosis of PCGDTCL may be confounded by florid HLH and require multiple biopsies and a comprehensive immunohistochemical panel., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Hemoglobin Korle-Bu (Hb KB) is a rare and likely under-reported hemoglobin (Hb) variant resulting from an unusual point mutation on the beta-globin chain. Hb KB is typically clinically silent, and there are limited reports of Hb KB heterozygosity compounded with other hemoglobinopathies that can present with varying clinical phenotypes. Here, we report a case of compound Hb KB heterozygosity with Hb S in an asymptomatic military trainee with a positive sickle cell screening test. Hb capillary and gel electrophoresis predicted a compound Hb S/D-Punjab overlap, which foretells a severe clinical phenotype. Sequencing of the Hb beta gene HBB demonstrated Hb KB, allowing for a diagnosis that fit his asymptomatic clinical phenotype and allowed for retention in the military., Competing Interests: Authors have no conflict of interest to disclose., (Copyright 2024, Bowling et al.)

Myeloid/lymphoid neoplasm with fibroblast growth factor 1 rearrangements (MLN-FGFR1) represents a rare group of hematologic neoplasms, with approximately 100 cases reported to date. A 69-year-old woman with a history of polycythemia and leukocytosis, with negative molecular testing for JAK2, CALR, and MPL, presented with diffuse adenopathy. A lymph node (LN) biopsy revealed effacement by T-lymphoblasts, consistent with T-cell acute lymphoblastic lymphoma (T-ALL). A staging bone marrow (BM) biopsy demonstrated trilineage hyperplasia, which, taken together with the patient's elevated hemoglobin and low serum erythropoietin level, fulfilled diagnostic criteria for polycythemia vera. Karyotype and fluorescence in situ hybridization on both the BM and LN demonstrated a FGFR1 rearrangement due to t(8;13), consistent with MLN-FGFR1. Whole genome sequencing on the LN additionally identified a pathogenic frameshift mutation of ASXL1 NC_000020.11:g32434646dup NM_015338.6(ASXL1):c.1934dup p.(Gly646Trpfs) predicted to result in loss of protein function, a finding also observed in 8.1% of BM reads. Both the BM and LN harbored missense variants in HDAC4 NM_001378414.1(HDAC4):c.[2763G>A]; [2763=] p.(Met921Ile) and CHEK2 NM_007194.4(CHEK2):c.[538C>T];[538=] p.(Arg180Cys), with an unknown significance. Despite initial response to Mini-CVD + venetoclax, the patient subsequently experienced rapid clinical deterioration and death. We report the second case of MLN-FGFR1 with an ASXL1 mutation and the first case with HDAC4 and CHEK2 variants., Competing Interests: Declaration of Competing Interest Dr. Joshua Romain discloses that this work was supported by the 59th Medical Wing Clinical Investigation Program (DHA/Clinical Program Office), (Copyright © 2023. Published by Elsevier Inc.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background and Objectives: The publication of MSLT-II shifted recommendations for management of sentinel lymph node biopsy positive (SLNB+) melanoma to favor active surveillance. We examined trends in immediate completion lymph node dissection (CLND) following publication of MSLT-II., Methods: Using a prospective melanoma database at a high-volume center, we identified a cohort of consecutive SLNB+ patients from July 2016 to April 2019. Patient and disease characteristics were analyzed with multivariate logistic regression to examine factors associated with CLND., Results: Two hundred and thirty-five patients were included for analysis. CLND rates were 67%, 33%, and 26% for the year before, year after, and second-year following MSLT-II. Factors associated with undergoing CLND included primary located in the head and neck (59% vs 33%, P = .003 and odds ratio [OR], 5.22, P = .002) and higher sentinel node tumor burden (43% vs 10% for tumor burden ≥0.1 mm, P < .001 and OR, 8.64, P = .002)., Conclusions: Rates of CLND in SLNB+ melanoma decreased dramatically, albeit not uniformly, following MSLT-II. Factors that increased the likelihood of immediate CLND were primary tumor located in the head and neck and high sentinel node tumor burden. These groups were underrepresented in MSLT-II, suggesting that clinicians are wary of implementing active surveillance recommendations for patients perceived as higher risk., (© 2020 Wiley Periodicals LLC.)

Pulmonary vein thrombosis (PVT) is a rare condition seen almost exclusively in the first two weeks after lung transplantation or lobectomy. Subsequent embolic phenomena are uncommon. Herein, a 47-year-old male with a history of bilateral lung transplantation presented with transient episodes of acute dysphasia and right arm weakness. Brain MRI revealed cortical infarcts in the territory of the left middle cerebral artery. Transesophageal echocardiogram demonstrated a thrombus in the left lower pulmonary vein. This represents the latest manifestation of a PVT reported in the literature-6 years after redo transplantation and 13 years after the original surgery. Investigation for PVT should be considered in any patient with previous lung transplantation that presents with systemic emboli.

Boer × Spanish kid goats (n = 48) were used to evaluate effects of using ground woody products in feedlot diets on growth performance and blood serum chemistry. A completely randomized study design was used with 2 feeding periods (Period 1 = 70% concentrate, days 0 to 26; Period 2 = 86% concentrate, days 27 to 64). Goats were individually fed 1 of 6 diets that differed only by roughage source (n = 4 wether males and 4 females/treatment; initial BW = 22 ± 2 kg): cottonseed hulls (CSH; control) or ground wood consisting of redberry (RED), blueberry (BLUE), one-seed (ONE), or eastern red cedar (ERC) Juniperus spp., or Prosopis glandulosa (MESQ). Ground woody diets were individually compared with CSH. During Period 1, goats fed CSH had greater (P < 0.05) average daily DMI (DMI), ADG, and G:F than goats fed MESQ and tended to have greater (P < 0.10) ADG and G:F than goats fed BLUE. A Treatment × d interaction (P = 0.008) was observed for goat BW during Period 1 and goats fed CSH tended (P < 0.09) to have greater BW on day 27 than goats fed BLUE or MESQ. During Period 2, Treatment × d interactions were not observed (P > 0.29) for DMI, ADG, G:F, or BW and no differences were observed between goats fed CSH and goats fed any of the treatment diets. Various blood serum variables were different between CSH and goats fed diets containing woody plants (mainly during Period 1); however, blood serum profiles did not indicate hepatotoxicity or any other health issue. Collectively, results suggested that ground Juniperus pinchotii, Juniperus ashei, or Juniperus monosperma can completely replace CSH in goat feedlot diets without negatively affecting growth performance or animal health. During Period 1, feeding diets to goats that contain 30% Juniperus virginiana (ERC) or P. glandulosa (MESQ) may not be economically justifiable in most scenarios, even though goat health, assessed by blood serum profiles, was not negatively affected. However, using 14% J. virginiana (ERC) or P. glandulosa (MESQ) in finishing diets is warranted.

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., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

Microscopic polyangiitis and granulomatosis with polyangiitis are rare anti-neutrophilic cytoplasmic antibody-associated systemic vasculitides that predominantly affect small to medium sized vessels of the lungs and kidneys. These syndromes are largely confined to older adults and often present sub-acutely following weeks to months of nonspecific prodromal symptoms. While both diseases often manifest within multiple organ systems concurrently, the disease spectrum of microscopic polyangiitis almost always includes the kidneys, while granulomatosis with polyangiitis is most commonly associated with pulmonary disease. We present two cases of rapid onset respiratory failure secondary to diffuse alveolar hemorrhage in young active duty military personnel. After serological testing and surgical lung biopsy, both patients were diagnosed with microscopic polyangiitis with isolated pulmonary involvement.

ATP-binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy-dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high-level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (Copyright © 2015 by the Genetics Society of America.)

Background: Citizen science, scientific research conducted by non-specialists, has the potential to facilitate biomedical research using available large-scale data, however validating the results is challenging. The Cell Slider is a citizen science project that intends to share images from tumors with the general public, enabling them to score tumor markers independently through an internet-based interface., Methods: From October 2012 to June 2014, 98,293 Citizen Scientists accessed the Cell Slider web page and scored 180,172 sub-images derived from images of 12,326 tissue microarray cores labeled for estrogen receptor (ER). We evaluated the accuracy of Citizen Scientist's ER classification, and the association between ER status and prognosis by comparing their test performance against trained pathologists., Findings: The area under ROC curve was 0.95 (95% CI 0.94 to 0.96) for cancer cell identification and 0.97 (95% CI 0.96 to 0.97) for ER status. ER positive tumors scored by Citizen Scientists were associated with survival in a similar way to that scored by trained pathologists. Survival probability at 15 years were 0.78 (95% CI 0.76 to 0.80) for ER-positive and 0.72 (95% CI 0.68 to 0.77) for ER-negative tumors based on Citizen Scientists classification. Based on pathologist classification, survival probability was 0.79 (95% CI 0.77 to 0.81) for ER-positive and 0.71 (95% CI 0.67 to 0.74) for ER-negative tumors. The hazard ratio for death was 0.26 (95% CI 0.18 to 0.37) at diagnosis and became greater than one after 6.5 years of follow-up for ER scored by Citizen Scientists, and 0.24 (95% CI 0.18 to 0.33) at diagnosis increasing thereafter to one after 6.7 (95% CI 4.1 to 10.9) years of follow-up for ER scored by pathologists., Interpretation: Crowdsourcing of the general public to classify cancer pathology data for research is viable, engages the public and provides accurate ER data. Crowdsourced classification of research data may offer a valid solution to problems of throughput requiring human input.

Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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-β 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.

Objectives: The aim of this study was to investigate the relationship between expression of Candida albicans alcohol dehydrogenases (ADH) genes in archival formalin-fixed paraffin-embedded (FFPE) samples from biopsies of leukoplakia., Materials and Methods: Archival FFPE samples were obtained from four sample groups: normal oral mucosa, non-dysplastic leukoplakia, chronic hyperplastic candidosis (CHC), and non-CHC dysplastic leukoplakia. The presence of C. albicans was determined by periodic acid Schiff staining and by immunocytochemistry. C. albicans ADH1 and ADH2 mRNAs were detected using reverse transcription PCR., Results: Candida albicans was detected in FFPE samples diagnosed as CHC (the histological diagnoses had been made by specialist oral pathologists, using uniform criteria), but not in any other sample group, including the non-dysplastic leukoplakias. RT-PCR confirmed a significant correlation between the expression of CaADH1 mRNA (P = 0.000), but not for CaADH2 mRNA (P = 0.056) in archival FFPE samples (n = 31) from biopsies of leukoplakia., Conclusions: Candida albicans was the predominant species in the lesions diagnosed as CHC, and the presence of C. albicans in CHC lesions was associated with a high expression of C. albicans ADH1 mRNA. There was no association between the presence of Candida and malignant transformation in the cases examined; however, the number of cases was limited and further studies are needed to further elucidate the role of C. albicans ADH1 in the pathogenesis of oral squamous cell carcinoma., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Interactions between Candida albicans, saliva and saliva-coated oral surfaces are initial events in the colonization of the oral cavity by this commensal yeast, which can cause oral diseases such as candidiasis and denture stomatitis. Candida albicans also colonizes silicone voice prostheses, and the microbial biofilm formed can impair valve function, necessitating frequent prosthesis replacement. We have previously shown that saliva promoted binding of C. albicans cells to silicone in vitro, and that the selective binding of specific salivary proteins to voice prosthesis silicone mediated attachment of C. albicans cells. The C. albicans cells adhered to a polypeptide (or polypeptides) of ~36 kDa eluted from saliva-treated silicone. We show here that a protein of similar size was identified in replicate blots of the eluate from saliva-treated silicone when the blots were probed with antibodies to human SPLUNC2, a salivary protein with reported microbial agglutination properties. In addition, SPLUNC2 was depleted from saliva that had been incubated with silicone coupons. To determine whether SPLUNC2 is a yeast-binding protein, SPLUNC2 cDNA was expressed in Escherichia coli. Purified recombinant His-tagged protein (SPLUNC2r) bound to silicone as demonstrated by immunoblot analysis of an eluate from SPLUNC2r-treated silicone coupons and (35) S-radiolabelled C. albicans cells adhered in a dose-dependent manner to SPLUNC2r-coated silicone. We conclude that SPLUNC2 binds to silicone and acts as a receptor for C. albicans adherence to, and subsequent colonization of, voice prosthesis silicone., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A PCR-denaturing gradient gel electrophoresis (DGGE) method was established for the simultaneous presumptive identification of multiple yeast species commonly present in the oral cavity. Published primer sets targeting different regions of the Saccharomyces cerevisiae 26-28S rRNA gene (denoted primer sets N and U) and the 18S rRNA gene (primer set E) were evaluated with ten Candida and four non-Candida yeast species, and twenty Candida albicans isolates. Optimized PCR-DGGE conditions using primer set N were applied to presumptively identify, by band matching, yeasts in the saliva of 25 individuals. Identities were confirmed by DNA sequencing and compared with those using CHROMagar Candida culture. All primer sets yielded detectable DGGE bands for all species tested. Primer set N yielded mainly single bands and could distinguish all species examined, including differentiating Candida dubliniensis from C. albicans. Primer set U was less discriminatory among species but yielded multiple bands that distinguished subspecies groups within C. albicans. Primer set E gave poor yeast discrimination. DGGE analysis identified yeasts in 17 of the 25 saliva samples. Six saliva samples contained two yeast species: three contained C. albicans and three C. dubliniensis. C. dubliniensis was present alone in one saliva sample (total prevalence 16 %). CHROMagar culture detected yeasts in 16 of the yeast-containing saliva samples and did not enable identification of 7 yeast species identified by DGGE. In conclusion, DGGE identification of oral yeast species with primer set N is a relatively fast and reliable method for the simultaneous presumptive identification of mixed yeasts in oral saliva samples.

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 β9 have modes of action similar to RC21v3., (© 2012 Blackwell Publishing Ltd.)

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.

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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.

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.

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., (© 2011 Blackwell Publishing Ltd.)

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., (Copyright 2009 Elsevier Inc. All rights reserved.)

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. We describe six 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 represent two approaches to investigating adhesion; retention at a fixed time point following liquid washes; and retention against a continuous flow of medium.

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.

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.

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.

In his presidential address to the Belfast meeting of the British Association for the Advancement of Science in 1874, John Tyndall launched what David Livingstone has called a 'frontal assault on teleology and Christian theism'. Using Tyndall's intervention as a starting point, this paper seeks to understand the attitudes of Presbyterians in the north of Ireland to science in the first three-quarters of the nineteenth century. The first section outlines some background, including the attitude of Presbyterians to science in the eighteenth century, the development of educational facilities in Ireland for the training of Presbyterian ministers, and the specific cultural and political circumstances in Ireland that influenced Presbyterian responses to science more generally. The next two sections examine two specific applications by Irish Presbyterians of the term 'science': first, the emergence of a distinctive Presbyterian theology of nature and the application of inductive scientific methodology to the study of theology, and second, the Presbyterian conviction that mind had ascendancy over matter which underpinned their commitment to the development of a science of the mind. The final two sections examine, in turn, the relationship between science and an eschatological reading of the signs of the times, and attitudes to Darwinian evolution in the fifteen years between the publication of The Origin of Species in 1859 and Tyndall's speech in 1874.

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.

There are relatively few classes of antifungal drugs. This restricts clinicians' therapeutic choices and these choices are further reduced by the emergence of drug resistance. Exposure to antifungal drugs represents an environmental stress for the fungal pathogen Candida albicans. The immediate response of C. albicans to antifungals may be drug tolerance, which can lead to drug resistance. This article examines C. albicans drug resistance from the perspective of it being a stress response and investigates how commonality with other stress-response pathways gives insights into the prospects for overcoming, or preventing, drug resistance.

Bovine milk antibodies directed against human pathogenic organisms have potential as prophylactic or therapeutic treatments of disorders affecting mucosal surfaces. The cow, however, does not naturally secrete high levels of IgA in milk, the predominant immunoglobulin of the mucosal immune system. We have patented an immunisation protocol that results in increased production of IgA in ruminant milk and in this study established that our protocol can be used on a scale sufficient to produce semi-industrial quantities of milk for processing. Cows were immunised with a common pathogenic yeast, Candida albicans and responded with high levels of antigen-specific IgA antibodies in their milk. The spray-dried milk-protein concentrate (85% protein) powder was shown to reduce adherence of Cand. albicans cells in in vitro adherence assays, demonstrating an ability to retain efficacy through the processing. These results suggest that this milk product may be of therapeutic value if the reduction in Cand. albicans adhesion observed in vitro translates to reduced colonisation in vivo.

The study of eukaryotic membrane proteins has been hampered by a paucity of systems that achieve consistent high-level functional protein expression. We report the use of a modified membrane protein hyperexpression system to characterize three classes of fungal membrane proteins (ABC transporters Pdr5p, CaCdr1p, CaCdr2p, CgCdr1p, CgPdh1p, CkAbc1p, and CneMdr1p, the major facilitator superfamily transporter CaMdr1p, and the cytochrome P450 enzyme CaErg11p) that contribute to the drug resistance phenotypes of five pathogenic fungi and to express human P glycoprotein (HsAbcb1p). The hyperexpression system consists of a set of plasmids that direct the stable integration of a single copy of the expression cassette at the chromosomal PDR5 locus of a modified host Saccharomyces cerevisiae strain, ADDelta. Overexpression of heterologous proteins at levels of up to 29% of plasma membrane protein was achieved. Membrane proteins were expressed with or without green fluorescent protein (GFP), monomeric red fluorescent protein, His, FLAG/His, Cys, or His/Cys tags. Most GFP-tagged proteins tested were correctly trafficked within the cell, and His-tagged proteins could be affinity purified. Kinetic analysis of ABC transporters indicated that the apparent K(m) value and the V(max) value of ATPase activities were not significantly affected by the addition of His tags. The efflux properties of seven fungal drug pumps were characterized by their substrate specificities and their unique patterns of inhibition by eight xenobiotics that chemosensitized S. cerevisiae strains overexpressing ABC drug pumps to fluconazole. The modified hyperexpression system has wide application for the study of eukaryotic membrane proteins and could also be used in the pharmaceutical industry for drug screening.

The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.

Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non-synonymous single nucleotide polymorphisms (NS-SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site-directed mutagenesis showed that the TM-12 NS-SNPs, and principally the G1473A NS-SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele-specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy-Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.

Explanted voice prostheses obtained from 5 patients at the time of prosthesis replacement were consistently colonized by yeast, in particular Candida albicans. A simple, reproducible, in vitro model of C. albicans adherence to saliva-coated voice prosthesis silicone was developed. Whole saliva promoted adherence of C. albicans to silicone in a dose-dependent manner. Saliva rinses from voice prosthesis patients also promoted binding of C. albicans to silicone in vitro (mean adherence 14.9% +/- 2.8% of input C. albicans cells). This was significantly higher than C. albicans adherence to silicone in the absence of saliva (P < .001) or adherence promoted by saliva rinses from healthy volunteers (P < .005). Polyacrylamide gel electrophoresis analysis and a blot overlay adherence assay revealed that certain salivary proteins were selectively adsorbed to silicone and that C. albicans yeast cells adhered specifically to the adsorbed salivary proteins.

The micafungin and caspofungin susceptibilities of Candida albicans laboratory and clinical isolates and of Saccharomyces cerevisiae strains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter from C. albicans gave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressing CDR2 showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressing CDR1 or the null parent deleted of seven ABC transporters. The strains hyperexpressing CDR1 showed slightly decreased susceptibility to micafungin in these assays. A C. albicans clinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.

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.