scholarly journals Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties

2010 ◽  
Vol 9 (10) ◽  
pp. 1455-1465 ◽  
Author(s):  
Laura A. Jones ◽  
Peter E. Sudbery
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Fluorescence Recovery After Photobleaching ◽  
Dynamic Properties ◽  
Secretory Vesicles ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Actin Cables ◽  
Cytochalasin A ◽  
Subapical Region

ABSTRACT During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.

scholarly journals Candida albicans CUG Mistranslation Is a Mechanism To Create Cell Surface Variation

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Isabel Miranda ◽  
Ana Silva-Dias ◽  
Rita Rocha ◽  
Rita Teixeira-Santos ◽  
Carolina Coelho ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Fungal Pathogen ◽  
Single Gene ◽  
Yeast Cells ◽  
Phenotypic Switching ◽  
Leucine Incorporation ◽  
Host Immune System ◽  
Content Type ◽  
Human Fungal Pathogen

ABSTRACT In the human fungal pathogen Candida albicans, the CUG codon is translated 97% of the time as serine and 3% of the time as leucine, which potentially originates an array of proteins resulting from the translation of a single gene. Genes encoding cell surface proteins are enriched in CUG codons; thus, CUG mistranslation may influence the interactions of the organism with the host. To investigate this, we compared a C. albicans strain that misincorporates 28% of leucine at CUGs with a wild-type parental strain. The first strain displayed increased adherence to inert and host molecules. In addition, it was less susceptible to phagocytosis by murine macrophages, probably due to reduced exposure of cell surface β-glucans. To prove that these phenotypes occurred due to serine/leucine exchange, the C. albicans adhesin and invasin ALS3 was expressed in Saccharomyces cerevisiae in its two natural isoforms (Als3p-Leu and Als3p-Ser). The cells with heterologous expression of Als3p-Leu showed increased adherence to host substrates and flocculation. We propose that CUG mistranslation has been maintained during the evolution of C. albicans due to its potential to generate cell surface variability, which significantly alters fungus-host interactions. IMPORTANCE The translation of genetic information into proteins is a highly accurate cellular process. In the human fungal pathogen Candida albicans, a unique mistranslation event involving the CUG codon occurs. The CUG codon is mainly translated as serine but can also be translated as leucine. Leucine and serine are two biochemically distinct amino acids, hydrophobic and hydrophilic, respectively. The increased rate of leucine incorporation at CUG decoding triggers C. albicans virulence attributes, such as morphogenesis, phenotypic switching, and adhesion. Here, we show that CUG mistranslation masks the fungal cell wall molecule β-glucan that is normally recognized by the host immune system, delaying its response. Furthermore, we demonstrate that two different proteins of the adhesin Als3 generated by CUG mistranslation confer increased hydrophobicity and adhesion ability on yeast cells. Thus, CUG mistranslation functions as a mechanism to create protein diversity with differential activities, constituting an advantage for a mainly asexual microorganism. This could explain its preservation during evolution.

scholarly journals An Amyloid Core Sequence in the Major Candida albicans Adhesin Als1p Mediates Cell-Cell Adhesion

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Vida Ho ◽  
Philippe Herman-Bausier ◽  
Christopher Shaw ◽  
Karen A. Conrad ◽  
Melissa C. Garcia-Sherman ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Force Spectroscopy ◽  
Cell Aggregation ◽  
Analysis Tool ◽  
Content Type ◽  
Core Sequence ◽  
Cell Cell

ABSTRACT The human fungal commensal Candida albicans can become a serious opportunistic pathogen in immunocompromised hosts. The C. albicans cell adhesion protein Als1p is a highly expressed member of a large family of paralogous adhesins. Als1p can mediate binding to epithelial and endothelial cells, is upregulated in infections, and is important for biofilm formation. Als1p includes an amyloid-forming sequence at amino acids 325 to 331, identical to the sequence in the paralogs Als5p and Als3p. Therefore, we mutated Val326 to test whether this sequence is important for activity. Wild-type Als1p (Als1pWT) and Als1p with the V326N mutation (Als1pV326N) were expressed at similar levels in a Saccharomyces cerevisiae surface display model. Als1pV326N cells adhered to bovine serum albumin (BSA)-coated beads similarly to Als1pWT cells. However, cells displaying Als1pV326N showed visibly smaller aggregates and did not fluoresce in the presence of the amyloid-binding dye Thioflavin-T. A new analysis tool for single-molecule force spectroscopy-derived surface mapping showed that statistically significant force-dependent Als1p clustering occurred in Als1pWT cells but was absent in Als1pV326N cells. In single-cell force spectroscopy experiments, strong cell-cell adhesion was dependent on an intact amyloid core sequence on both interacting cells. Thus, the major adhesin Als1p interacts through amyloid-like β-aggregation to cluster adhesin molecules in cis on the cell surface as well as in trans to form cell-cell bonds. IMPORTANCE Microbial cell surface adhesins control essential processes such as adhesion, colonization, and biofilm formation. In the opportunistic fungal pathogen Candida albicans, the agglutinin-like sequence (ALS) gene family encodes eight cell surface glycoproteins that mediate adherence to biotic and abiotic surfaces and cell-cell aggregation. Als proteins are critical for commensalism and virulence. Their activities include attachment and invasion of endothelial and epithelial cells, morphogenesis, and formation of biofilms on host tissue and indwelling medical catheters. At the molecular level, Als5p-mediated cell-cell aggregation is dependent on the formation of amyloid-like nanodomains between Als5p-expressing cells. A single-site mutation to valine 326 abolishes cellular aggregation and amyloid formation. Our results show that the binding characteristics of Als1p follow a mechanistic model similar to Als5p, despite its differential expression and biological roles.

scholarly journals Clathrin- and Arp2/3-Independent Endocytosis in the Fungal Pathogen Candida albicans

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Elias Epp ◽  
Elena Nazarova ◽  
Hannah Regan ◽  
Lois M. Douglas ◽  
James B. Konopka ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Membrane Proteins ◽  
Fungal Pathogen ◽  
Mammalian Cells ◽  
Fluid Phase ◽  
Fungal Species ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Content Type ◽  
Actin Cables

ABSTRACT Clathrin-mediated endocytosis (CME) is conserved among eukaryotes and has been extensively analyzed at a molecular level. Here, we present an analysis of CME in the human fungal pathogen Candida albicans that shows the same modular structure as those in other fungi and mammalian cells. Intriguingly, C. albicans is perfectly viable in the absence of Arp2/3, an essential component of CME in other systems. In C. albicans, Arp2/3 function remains essential for CME as all 15 proteins tested that participate in CME, including clathrin, lose their characteristic dynamics observed in wild-type (WT) cells. However, since arp2/3 cells are still able to endocytose lipids and fluid-phase markers, but not the Ste2 and Mup1 plasma membrane proteins, there must be an alternate clathrin-independent pathway we term Arp2/3-independent endocytosis (AIE). Characterization of AIE shows that endocytosis in arp2 mutants relies on actin cables and other Arp2/3-independent actin structures, as inhibition of actin functions prevented cargo uptake in arp2/3 mutants. Transmission electron microscopy (TEM) showed that arp2/3 mutants still formed invaginating tubules, cell structures whose proper functions are believed to heavily rely on Arp2/3. Finally, Prk1 and Sjl2, two proteins involved in patch disassembly during CME, were not correctly localized to sites of endocytosis in arp2 mutants, implying a role of Arp2/3 in CME patch disassembly. Overall, C. albicans contains an alternative endocytic pathway (AIE) that relies on actin cable function to permit clathrin-independent endocytosis (CIE) and provides a system to further explore alternate endocytic routes that likely exist in fungal species. IMPORTANCE There is a well-established process of endocytosis that is generally used by eukaryotic cells termed clathrin-mediated endocytosis (CME). Although the details are somewhat different between lower and higher eukaryotes, CME appears to be the dominant endocytic process in all eukaryotes. While fungi such as Saccharomyces cerevisiae have proven excellent models for dissecting the molecular details of endocytosis, loss of CME is so detrimental that it has been difficult to study alternate pathways functioning in its absence. Although the fungal pathogen Candida albicans has a CME pathway that functions similarly to that of S. cerevisiae, inactivation of this pathway does not compromise growth of yeast-form C. albicans. In these cells, lipids and fluid-phase molecules are still endocytosed in an actin-dependent manner, but membrane proteins are not. Thus, C. albicans provides a powerful model for the analysis of CME-independent endocytosis in lower eukaryotes.

scholarly journals Adaptations of the Secretome of Candida albicans in Response to Host-Related Environmental Conditions

2015 ◽  
Vol 14 (12) ◽  
pp. 1165-1172 ◽  
Author(s):  
Frans M. Klis ◽  
Stanley Brul
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Human Body ◽  
Environmental Conditions ◽  
Surface Stress ◽  
Hyphal Growth ◽  
Content Type ◽  
General Response ◽  
Culture Supernatants

ABSTRACTThe wall proteome and the secretome of the fungal pathogenCandida albicanshelp it to thrive in multiple niches of the human body. Mass spectrometry has allowed researchers to study the dynamics of both subproteomes. Here, we discuss some major responses of the secretome to host-related environmental conditions. Three β-1,3-glucan-modifying enzymes, Mp65, Sun41, and Tos1, are consistently found in large amounts in culture supernatants, suggesting that they are needed for construction and expansion of the cell wall β-1,3-glucan layer and thus correlate with growth and might serve as diagnostic biomarkers. The genesENG1,CHT3, andSCW11, which encode an endoglucanase, the major chitinase, and a β-1,3-glucan-modifying enzyme, respectively, are periodically expressed and peak in M/G1. The corresponding protein abundances in the medium correlate with the degree of cell separation during single-yeast-cell, pseudohyphal, and hyphal growth. We also discuss the observation that cells treated with fluconazole, or other agents causing cell surface stress, form pseudohyphal aggregates. Fluconazole-treated cells secrete abundant amounts of the transglucosylase Phr1, which is involved in the accumulation of β-1,3-glucan in biofilms, raising the question whether this is a general response to cell surface stress. Other abundant secretome proteins also contribute to biofilm formation, emphasizing the important role of secretome proteins in this mode of growth. Finally, we discuss the relevance of these observations to therapeutic intervention. Together, these data illustrate thatC. albicansactively adapts its secretome to environmental conditions, thus promoting its survival in widely divergent niches of the human body.

scholarly journals Protection from Systemic Candida albicans Infection by Inactivation of the Sts Phosphatases

2014 ◽  
Vol 83 (2) ◽  
pp. 637-645 ◽  
Author(s):  
Shamoon Naseem ◽  
David Frank ◽  
James B. Konopka ◽  
Nick Carpino
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Fungal Growth ◽  
Systemic Infection ◽  
Content Type ◽  
Host Immune Responses ◽  
Human Fungal Pathogen ◽  
Rapid Induction ◽  
Inflammatory Damage ◽  
Functional Inactivation

The human fungal pathogenCandida albicanscauses invasive candidiasis, characterized by fatal organ failure due to disseminated fungal growth and inflammatory damage. Thesuppressor ofTCRsignaling 1 (Sts-1) and Sts-2 are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic cell lineages, including T lymphocytes, mast cells, and platelets. Functional inactivation of both Sts enzymes leads to profound resistance to systemic infection byC. albicans, such that greater than 80% of mice lacking Sts-1 and -2 survive a dose ofC. albicans(2.5 × 105CFU/mouse) that is uniformly lethal to wild-type mice within 10 days. Restriction of fungal growth within the kidney occurs by 24 h postinfection in the mutant mice. This occurs without induction of a hyperinflammatory response, as evidenced by the decreased presence of leukocytes and inflammatory cytokines that normally accompany the antifungal immune response. Instead, the absence of the Sts phosphatases leads to the rapid induction of a unique immunological environment within the kidney, as indicated by the early induction of a proinflammatory cytokine (CXL10). Mice lacking either Sts enzyme individually display an intermediate lethality phenotype. These observations identify an opportunity to optimize host immune responses toward a deadly fungal pathogen.

scholarly journals Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Henry Ng ◽  
Neta Dean
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Virulence Factors ◽  
Fungal Pathogen ◽  
Fungal Virulence ◽  
Important Conclusion ◽  
Content Type ◽  
Guide Rna ◽  
Improve Efficiency ◽  
Human Fungal Pathogen

ABSTRACT Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans. The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5′ and 3′ ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5′ tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.

scholarly journals Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
Arielle Butts ◽  
Christian DeJarnette ◽  
Tracy L. Peters ◽  
Josie E. Parker ◽  
Morgan E. Kerns ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Second Generation ◽  
Active Compounds ◽  
Whole Cell ◽  
Competitive Fitness ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Conventional Drug ◽  
Physiologically Active Compounds

ABSTRACT Conventional drug screening typically employs either target-based or cell-based approaches. The first group rely on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound. Traditional approaches to drug discovery are frustratingly inefficient and have several key limitations that severely constrain our capacity to rapidly identify and develop novel experimental therapeutics. To address this, we have devised a second-generation target-based whole-cell screening assay based on the principles of competitive fitness, which can rapidly identify target-specific and physiologically active compounds. Briefly, strains expressing high, intermediate, and low levels of a preselected target protein are constructed, tagged with spectrally distinct fluorescent proteins (FPs), and pooled. The pooled strains are then grown in the presence of various small molecules, and the relative growth of each strain within the mixed culture is compared by measuring the intensity of the corresponding FP tags. Chemical-induced population shifts indicate that the bioactivity of a small molecule is dependent upon the target protein’s abundance and thus establish a specific functional interaction. Here, we describe the molecular tools required to apply this technique in the prevalent human fungal pathogen Candida albicans and validate the approach using two well-characterized drug targets—lanosterol demethylase and dihydrofolate reductase. However, our approach, which we have termed target abundance-based fitness screening (TAFiS), should be applicable to a wide array of molecular targets and in essentially any genetically tractable microbe. IMPORTANCE Conventional drug screening typically employs either target-based or cell-based approaches. The first group relies on biochemical assays to detect modulators of a purified target. However, hits frequently lack drug-like characteristics such as membrane permeability and target specificity. Cell-based screens identify compounds that induce a desired phenotype, but the target is unknown, which severely restricts further development and optimization. To address these issues, we have developed a second-generation target-based whole-cell screening approach that incorporates the principles of both chemical genetics and competitive fitness, which enables the identification of target-specific and physiologically active compounds from a single screen. We have chosen to validate this approach using the important human fungal pathogen Candida albicans with the intention of pursuing novel antifungal targets. However, this approach is broadly applicable and is expected to dramatically reduce the time and resources required to progress from screening hit to lead compound.

scholarly journals The 5′ Untranslated Region of theEFG1Transcript Promotes Its Translation To Regulate Hyphal Morphogenesis inCandida albicans

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Prashant R. Desai ◽  
Klaus Lengeler ◽  
Mario Kapitan ◽  
Silas Matthias Janßen ◽  
Paula Alepuz ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Untranslated Regions ◽  
Regulatory Sequence ◽  
Transcriptional Level ◽  
Content Type ◽  
Reading Frame ◽  
Human Fungal Pathogen ◽  
Hyphal Morphogenesis ◽  
Incandida Albicans

ABSTRACTExtensive 5′ untranslated regions (UTR) are a hallmark of transcripts determining hyphal morphogenesis inCandida albicans. The major transcripts of theEFG1gene, which are responsible for cellular morphogenesis and metabolism, contain a 5′ UTR of up to 1,170 nucleotides (nt). Deletion analyses of the 5′ UTR revealed a 218-nt sequence that is required for production of the Efg1 protein and its functions in filamentation, without lowering the level and integrity of theEFG1transcript. Polysomal analyses revealed that the 218-nt 5′ UTR sequence is required for efficient translation of the Efg1 protein. Replacement of theEFG1open reading frame (ORF) by the heterologous reporter geneCaCBGlucconfirmed the positive regulatory importance of the identified 5′ UTR sequence. In contrast to other reported transcripts containing extensive 5′ UTR sequences, these results indicate the positive translational function of the 5′ UTR sequence in theEFG1transcript, which is observed in the context of the nativeEFG1promoter. It is proposed that the 5′ UTR recruits regulatory factors, possibly during emergence of the native transcript, which aid in translation of theEFG1transcript.IMPORTANCEMany of the virulence traits that makeCandida albicansan important human fungal pathogen are regulated on a transcriptional level. Here, we report an important regulatory contribution of translation, which is exerted by the extensive 5′ untranslated regulatory sequence (5′ UTR) of the transcript for the protein Efg1, which determines growth, metabolism, and filamentation in the fungus. The presence of the 5′ UTR is required for efficient translation of Efg1, to promote filamentation. Because transcripts for many relevant regulators contain extensive 5′ UTR sequences, it appears that the virulence ofC. albicansdepends on the combination of transcriptional and translational regulatory mechanisms.

scholarly journals Aneuploidy Underlies Tolerance and Cross-Tolerance to Drugs in Candida parapsilosis

2021 ◽  
Author(s):  
Feng Yang ◽  
Hui Lu ◽  
Hao Wu ◽  
Ting Fang ◽  
Judith Berman ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Candida Parapsilosis ◽  
Cross Tolerance ◽  
Content Type ◽  
Human Fungal Pathogen

Candida parapsilosis is an emerging major human fungal pathogen, especially in neonates. Aneuploidy, having uneven numbers of chromosomes, is a well-known mechanism for adapting to stress in Candida albicans , the most common human fungal pathogen.

scholarly journals Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

2010 ◽  
Vol 9 (6) ◽  
pp. 960-970 ◽  
Author(s):  
Luiz R. Basso ◽  
Charles E. Gast ◽  
Yuxin Mao ◽  
Brian Wong
Keyword(s):  
Candida Albicans ◽  
Plasma Membrane ◽  
Dominant Negative ◽  
Secretory Vesicles ◽  
Content Type ◽  
Empty Vector ◽  
Carbonyl Cyanide ◽  
Regulated Expression ◽  
Cytoplasmic Accumulation ◽  
Expression Plasmids

ABSTRACT A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [3H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [3H]fluconazole than did PGVs from empty-vector controls. The Km s (expressed in micromolar concentrations) and V maxs (expressed in picomoles per milligram of protein per minute), respectively, for [3H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [3H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [3H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [3H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC50s; expressed as proportions of the [3H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi.

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