scholarly journals A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

2015 ◽  
Vol 14 (3) ◽  
pp. 311-322 ◽  
Author(s):  
Raphaël Loll-Krippleber ◽  
Adeline Feri ◽  
Marie Nguyen ◽  
Corinne Maufrais ◽  
Jennifer Yansouni ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cancer Progression ◽  
Genome Stability ◽  
Fluorescent Protein ◽  
Temperature Treatment ◽  
Protein Markers ◽  
Heterozygous Locus ◽  
Content Type ◽  
Genome Dynamics ◽  
Green Fluorescent

ABSTRACTLoss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogenCandida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH inC. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched forC. albicansgenes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability inC. albicansand more generally in eukaryotic cells.

scholarly journals Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

2011 ◽  
Vol 77 (22) ◽  
pp. 8193-8196 ◽  
Author(s):  
Lucja M. Jarosz ◽  
Bastiaan P. Krom
Keyword(s):  
Candida Albicans ◽  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Rapid Screening ◽  
Content Type ◽  
Wide Range ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

scholarly journals Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

2014 ◽  
Vol 14 (2) ◽  
pp. 182-193 ◽  
Author(s):  
Areti Gkourtsa ◽  
Janny van den Burg ◽  
Karin Strijbis ◽  
Teja Avula ◽  
Sietske Bijvoets ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Genetic Interaction ◽  
Phenotypic Analysis ◽  
Content Type ◽  
Associated Functions ◽  
Increased Sensitivity ◽  
Green Fluorescent ◽  
And Function

ABSTRACT Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro . A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3 Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167 Δ/Δ and rvs161 Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans : the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.

scholarly journals TheRTA3Gene, Encoding a Putative Lipid Translocase, Influences the Susceptibility of Candida albicans to Fluconazole

2016 ◽  
Vol 60 (10) ◽  
pp. 6060-6066 ◽  
Author(s):  
Sarah G. Whaley ◽  
Sarah Tsao ◽  
Sandra Weber ◽  
Qing Zhang ◽  
Katherine S. Barker ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Clinical Isolate ◽  
Fluorescent Protein ◽  
Microtiter Plate ◽  
Microtiter Plate Assay ◽  
Content Type ◽  
Activating Mutations ◽  
Gene Coding ◽  
Activating Mutation ◽  
Green Fluorescent

ABSTRACTTheRTA3gene, coding for a member of the Rta1p-like lipid-translocating exporter family, is coordinately upregulated with the ATP-binding cassette transporter genesCDR1andCDR2in azole-resistant clinical isolates ofCandida albicansthat carry activating mutations in the transcription factor Tac1p. We show here that deletingRTA3in an azole-resistant clinical isolate carrying a Tac1p-activating mutation lowered fluconazole resistance by 2-fold, while overexpressingRTA3in an azole-susceptible clinical isolate resulted in enhanced fluconazole tolerance associated with trailing growth in a liquid microtiter plate assay. We also demonstrate that an Rta3p-green fluorescent protein (GFP) fusion protein localizes predominantly to the plasma membrane, consistent with a putative function for Rta3p as a lipid translocase.

scholarly journals Motor Protein Myo5p Is Required To Maintain the Regulatory Circuit ControllingWOR1Expression in Candida albicans

2012 ◽  
Vol 11 (5) ◽  
pp. 626-637 ◽  
Author(s):  
Nadezda Kachurina ◽  
Bernard Turcotte ◽  
Malcolm Whiteway
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Content Type ◽  
Bud Emergence ◽  
Regulatory Circuit ◽  
Myosin I ◽  
Bud Growth ◽  
Green Fluorescent ◽  
Germ Tubes

ABSTRACTTheCandida albicans MYO5gene encodes myosin I, a protein required for the formation of germ tubes and true hyphae. Because the polarized growth of opaque-phase cells in response to pheromone results in mating projections that can resemble germ tubes, we examined the role of Myo5p in this process. We localized green fluorescent protein (GFP)-tagged Myo5p in opaque-phase cells ofC. albicansduring both bud and shmoo formation. In vegetatively growing opaque cells, Myo5p is found at sites of bud emergence and bud growth, while in pheromone-stimulated cells, Myo5p localizes at the growing tips of shmoos. Intriguingly, cells homozygous forMTLain which theMYO5gene was deleted failed to switch efficiently from the white phase to the opaque phase, although ectopic expression ofWOR1from theMET3promoter can convertmyo5mutants into mating-competent opaque cells. However, whenWOR1expression was shut off, themyo5-defective cells rapidly lost both their opaque phenotype and mating competence, suggesting that Myo5p is involved in the maintenance of the opaque state. WhenMYO5is expressed conditionally in opaque cells, the opaque phenotype, as well as the mating ability of the cells, becomes unstable under repressive conditions, and quantitative real-time PCR demonstrated that the shutoff ofMYO5expression correlates with a dramatic reduction inWOR1expression. It appears that while myosin I is not directly required for mating inC. albicans, it is involved inWOR1expression and the white-opaque transition and thus is indirectly implicated in mating.

scholarly journals Rab14 Regulates Maturation of Macrophage Phagosomes Containing the Fungal Pathogen Candida albicans and Outcome of the Host-Pathogen Interaction

2015 ◽  
Vol 83 (4) ◽  
pp. 1523-1535 ◽  
Author(s):  
Blessing Okai ◽  
Natalie Lyall ◽  
Neil A. R. Gow ◽  
Judith M. Bain ◽  
Lars-Peter Erwig
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Fluorescent Protein ◽  
Temporal Dynamics ◽  
Immune Defense ◽  
Dominant Negative ◽  
Phagosome Maturation ◽  
Hyphal Length ◽  
Content Type ◽  
Green Fluorescent

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogenCandida albicansundergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated byC. albicanshyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment ofC. albicanscells. Phagosomes containing liveC. albicanscells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages towardC. albicanscells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing byC. albicans. Therefore, Rab14 activity promotes phagosome maturation duringC. albicansinfection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

scholarly journals Insight into the Antiadhesive Effect of Yeast Wall Protein 1 of Candida albicans

2012 ◽  
Vol 11 (6) ◽  
pp. 795-805 ◽  
Author(s):  
Bruce L. Granger
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Surrounding Medium ◽  
Yeast Cells ◽  
Content Type ◽  
A Cell ◽  
Opportunistic Fungal Pathogen ◽  
Green Fluorescent

ABSTRACTYwp1 is a prominent glycosylphosphatidylinositol (GPI)-anchored glycoprotein of the cell wall ofCandida albicans; it is present in the yeast form of this opportunistic fungal pathogen but absent from filamentous forms and chlamydospores. Yeast cells that lack Ywp1 are more adhesive and form thicker biofilms, implying an antiadhesive activity for Ywp1, with a possible role in yeast dispersal. The antiadhesive effect of Ywp1 is transplantable from yeast to hyphae, as hyphae that are forced to expressYWP1lose adhesion in anin vitroassay. Deletion of the GPI anchor results in loss of Ywp1 to the surrounding medium and reduction of the antiadhesive effect, implying an importance of time-dependent residency in the cell wall. Anchor-negative versions of Ywp1 possessing or lacking a C-terminal green fluorescent protein (GFP) tag were created inC. albicansand harvested from culture supernatants; in addition to serving as quantifiable markers for Ywp1 secretion, they revealed that the cleaved 11-kDa propeptide of Ywp1 remains strongly but noncovalently associated with the Ywp1 core. This association is resistant to highly acidic and basic solutions, 8 M urea, and 1% SDS (below 45°C). Above 50°C, SDS dissociates the isolated complex, but even higher temperatures are required to dissociate the propeptide from native Ywp1 that is anchored in a cell wall. This property has permitted detection, for the first time, of orthologs of Ywp1 in other members of theCandidaclade. The cleaved propeptide, which carries the sole N-glycan of Ywp1, must participate in the antiadhesive effect of Ywp1.

scholarly journals Rapid Redistribution of Phosphatidylinositol-(4,5)-Bisphosphate and Septins during the Candida albicans Response to Caspofungin

2012 ◽  
Vol 56 (9) ◽  
pp. 4614-4624 ◽  
Author(s):  
Hassan Badrane ◽  
M. Hong Nguyen ◽  
Jill R. Blankenship ◽  
Shaoji Cheng ◽  
Binghua Hao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Regulatory Protein ◽  
Wall Material ◽  
Pleckstrin Homology Domain ◽  
Calcofluor White ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTWe previously showed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and septin regulation play major roles in maintainingCandida albicanscell wall integrity in response to caspofungin and other stressors. Here, we establish a link between PI(4,5)P2 signaling and septin localization and demonstrate that rapid redistribution of PI(4,5)P2 and septins is part of the natural response ofC. albicansto caspofungin. First, we studied caspofungin-hypersusceptibleC. albicans irs4andinp51mutants, which have elevated PI(4,5)P2 levels due to loss of PI(4,5)P2-specific 5′-phosphatase activity. PI(4,5)P2 accumulated in discrete patches, rather than uniformly, along surfaces of mutants in yeast and filamentous morphologies, as visualized with a green fluorescent protein (GFP)-pleckstrin homology domain. The patches also contained chitin (calcofluor white staining) and cell wall protein Rbt5 (Rbt5-GFP). By transmission electron microscopy, patches corresponded to plasma membrane invaginations that incorporated cell wall material. Fluorescently tagged septins Cdc10 and Sep7 colocalized to these sites, consistent with well-described PI(4,5)P2-septin physical interactions. Based on expression patterns of cell wall damage response genes,irs4andinp51mutants were firmly positioned within a group of caspofungin-hypersusceptible, septin-regulatory protein kinase mutants.irs4andinp51were linked most closely to thegin4mutant by expression profiling, PI(4,5)P2-septin-chitin redistribution and other phenotypes. Finally, sublethal 5-min exposure of wild-typeC. albicansto caspofungin resulted in redistribution of PI(4,5)P2 and septins in a manner similar to those ofirs4,inp51, andgin4mutants. Taken together, our data suggest that theC. albicansIrs4-Inp51 5′-phosphatase complex and Gin4 function upstream of PI(4,5)P2 and septins in a pathway that helps govern responses to caspofungin.

scholarly journals Flow Cytometry-Based Method To Detect Persisters in Candida albicans

2015 ◽  
Vol 59 (8) ◽  
pp. 5044-5048 ◽  
Author(s):  
Wenqiang Chang ◽  
Ming Zhang ◽  
Ying Li ◽  
Hongxiang Lou
Keyword(s):  
Flow Cytometry ◽  
Candida Albicans ◽  
Green Fluorescent Protein ◽  
Amphotericin B ◽  
Propidium Iodide ◽  
Fluorescent Protein ◽  
Fluorescence Microscope ◽  
Gene Encoding ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTCandida albicansbiofilms contain a subpopulation whose members are defined as persisters, displaying great tolerance of fungicides. To directly observe such persisters, an effective method using green fluorescent protein (GFP) strain labeling by mutation of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (TDH3), combined with propidium iodide (PI) staining, was established. Amphotericin B-tolerant persisters harbor the characteristics of both GFP positivity [GFP (+)] and propidium iodide (PI) negativity [PI (−)], which are easily visualized using a fluorescence microscope and measured by flow cytometry.

scholarly journals Highly Dynamic and Specific Phosphatidylinositol 4,5-Bisphosphate, Septin, and Cell Wall Integrity Pathway Responses Correlate with Caspofungin Activity against Candida albicans

2016 ◽  
Vol 60 (6) ◽  
pp. 3591-3600 ◽  
Author(s):  
Hassan Badrane ◽  
M. Hong Nguyen ◽  
Cornelius J. Clancy
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Cell Wall Integrity ◽  
Ph Domain ◽  
Calcofluor White ◽  
Content Type ◽  
Pathway Activation ◽  
Cell Wall Integrity Pathway ◽  
Green Fluorescent

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] activates the yeast cell wall integrity pathway.Candida albicansexposure to caspofungin results in the rapid redistribution of PI(4,5)P2and septins to plasma membrane foci and subsequent fungicidal effects. We studiedC. albicansPI(4,5)P2and septin dynamics and protein kinase C (PKC)-Mkc1 cell wall integrity pathway activation following exposure to caspofungin and other drugs. PI(4,5)P2and septins were visualized by live imaging ofC. albicanscells coexpressing green fluorescent protein (GFP)-pleckstrin homology (PH) domain and red fluorescent protein-Cdc10p, respectively. PI(4,5)P2was also visualized in GFP-PH domain-expressingC. albicans mkc1mutants. Mkc1p phosphorylation was measured as a marker of PKC-Mkc1 pathway activation. Fungicidal activity was assessed using 20-h time-kill assays. Caspofungin immediately induced PI(4,5)P2and Cdc10p colocalization to aberrant foci, a process that was highly dynamic over 3 h. PI(4,5)P2levels increased in a dose-response manner at caspofungin concentrations of ≤4× MIC and progressively decreased at concentrations of ≥8× MIC. Caspofungin exposure resulted in broad-based mother-daughter bud necks and arrested septum-like structures, in which PI(4,5)P2and Cdc10 colocalized. PKC-Mkc1 pathway activation was maximal within 10 min, peaked in response to caspofungin at 4× MIC, and declined at higher concentrations. The caspofungin-induced PI(4,5)P2redistribution remained apparent inmkc1mutants. Caspofungin exerted dose-dependent killing and paradoxical effects at ≤4× and ≥8× MIC, respectively. Fluconazole, amphotericin B, calcofluor white, and H2O2did not impact the PI(4,5)P2or Cdc10p distribution like caspofungin did. Caspofungin exerts rapid PI(4,5)P2-septin and PKC-Mkc1 responses that correlate with the extent ofC. albicanskilling, and the responses are not induced by other antifungal agents. PI(4,5)P2-septin regulation is crucial in early caspofungin responses and PKC-Mkc1 activation.

scholarly journals Identification of GIG1, a GlcNAc-Induced Gene in Candida albicans Needed for Normal Sensitivity to the Chitin Synthase Inhibitor Nikkomycin Z

2010 ◽  
Vol 9 (10) ◽  
pp. 1476-1483 ◽  
Author(s):  
Angelo Gunasekera ◽  
Francisco J. Alvarez ◽  
Lois M. Douglas ◽  
Hong X. Wang ◽  
Adam P. Rosebrock ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Amino Sugar ◽  
Chitin Synthase ◽  
Content Type ◽  
Catabolic Genes ◽  
Nikkomycin Z ◽  
Green Fluorescent ◽  
Synthase Inhibitor ◽  
Normal Sensitivity

ABSTRACT The amino sugar N-acetylglucosamine (GlcNAc) is known to be an important structural component of cells from bacteria to humans, but its roles in cell signaling are less well understood. GlcNAc induces two pathways in the human fungal pathogen Candida albicans. One activates cyclic AMP (cAMP) signaling, which stimulates the formation of hyphal cells and the expression of virulence genes, and the other pathway induces genes needed to catabolize GlcNAc. Microarray analysis of gene expression was carried out under four different conditions in order to characterize the transcriptional changes induced by GlcNAc. The most highly induced genes include those that encode a GlcNAc transporter (NGT1) and the GlcNAc catabolic enzymes (HXK1, DAC1, and NAG1). GlcNAc also activated most of the genes whose expression is increased when cells are triggered with other stimuli to form hyphae. Surprisingly, GlcNAc also induced a subset of genes that are regulated by galactose (GAL1, GAL7, and GAL10), which may be due to cross talk between signaling pathways. A novel GlcNAc-induced gene, GIG1, which is not essential for GlcNAc catabolism or the induction of hyphae, was identified. However, a Gig1-green fluorescent protein (GFP) fusion protein was specifically induced by GlcNAc, and not by other sugars. Gig1-GFP localized to the cytoplasm, where GlcNAc metabolism occurs. Significantly, a gig1Δ mutant displayed increased resistance to nikkomycin Z, which inhibits chitin synthase from converting UDP-GlcNAc into cell wall chitin. Gig1 is highly conserved in fungi, especially those that contain GlcNAc catabolic genes. These results implicate Gig1 in GlcNAc metabolism.

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