Azeredo, Joana

Henriques, Mariana

Martins, Margarida Isabel Barros Coelho

Oliveira, Rosário

Universidade do Minho: RepositoriUM

438th ASM Conference on Candida and CandidiasisPOSTER ABSTRACTSPOSTER ABSTRACTSB59NUTRIENT REGULATION OF THE MATINGRESPONSE IN C. ALBICANS AND THEROLE OF THE GPA2 PROTEINR. J. Bennett1, A. D. Johnson2;1Brown University, Providence, RI, 2UCSF, San Francisco, CAMating in C. albicans occurs between diploid cells and is mediated bymating pheromones secreted by a-type and á-type cells. Theresponse to mating pheromone occurs via a conserved MAP kinasecascade that transduces the signal from a pheromone receptor onthe cell surface to a transcriptional response in the nucleus. We haveanalyzed the response of C. albicans a-type cells to á matingpheromone under a number of  different experimental conditions.The morphological and transcriptional response to pheromonevaried greatly depending on the conditions and strains employed. Inparticular, the growth medium had a major effect on the efficiencyof  the response to pheromone, with the most robust pheromoneresponse evident in nutrient-limiting medium. Under these optimalconditions, microarray analysis revealed that 152 genes were up-regulated in response to pheromone and 75 genes were down-regulated. In addition, we demonstrate that the GPA2 gene plays animportant role in the nutrient regulation of  the mating response. Inparticular, Ägpa2 mutants showed increased sensitivity to phero-mone in nutrient-rich media, and, unlike wild-type C. albicans, Ägpa2cells showed efficient cell cycle arrest in response to matingpheromone, including the formation of  characteristic ‘halos’ onsolid medium. These results indicate that C. albicans, like severaldiverse fungi but unlike S. cerevisiae, integrates signals from anutrient-sensing pathway and the MAP kinase pathway to generatethe response to pheromone.C60EFFECT OF BIOFILM EXTRACELLULARMETABOLITES ON CANDIDA ALBICANSAND CANDIDA DUBLINIENSISMORPHOGENESISM. Henriques, M. Martins, J. Azeredo, R. Oliveira;University of  Minho, Braga, PORTUGALCandida dubliniensis has been recovered from the oral cavity ofimmunosupressed patients and is an opportunistic yeast responsiblefor severe Candidiasis. One of  its phenotypic characteristics is thecapacity to switch from yeast to hyphal (pseudohyphal) morphology.This behaviour, characteristic of  both this species and Candidaalbicans, made the mismatch of  these two species for years. Thecapacity of  switching morphology has been related with virulenceand several factors are responsible for it. Quorum sensing moleculesare responsible for community genetic regulation mechanisms,controlling microbiological functions and are considered one ofsuch factors. These molecules are metabolites released by planktonicand biofilm cells acting as cell-cell signaling molecules. Bacterialquorum sensing has been largely studied, but concerning yeasts,namely, Candida species, little is known. Regarding Candida albicans,only few studies have been done in this field mostly reporting thepresence of  farnesol as a quorum sensing molecule, able tomodulate the morphogenesis. Growth and biofilm formation byCandida albicans and Candida dubliniensis were performed in RPMI1640. After 24, 48, 72 and 96h, biofilm supernatants were recoveredand used to grow fresh Candida albicans and Candida dubliniensis cells.After overnight growth, the cells were inspected for alterations inmorphology under contrast light microscopy. Cells grown in RPMI1640 were used as a control. It was interesting to observe thatCandida albicans cells grown in RPMI 1640 presented hyphal forms,while Candida dubliniensis presented pseudo-hyphal forms. The firstresult observed was the alteration of  cell morphology of  bothCandida albicans and Candida dubliniensis grown on supernatants ofbiofilms of  both species. It was observed a time dependent effectof  supernatants collected from biofilms on cell morphology. Thesupernatants of  both species had different effects on morphologyswitching. So, the main conclusion of  this work is that there aresome metabolites excreted by Candida cells that induce changes in itsmorphology and on the morphology of  the other species and thatthese metabolites are released from biofilms. The results clearlydemonstrate that Candida dubliniensis also undergoes morphologicalalterations triggered by quorum sensing molecules, which arealready described and confirmed here, for Candida albicans.A61IDENTIFICATION OF GENES IMPORTANTFOR ENTRY OR MAINTENANCE OF C.ALBICANS STATIONARY PHASE BYSCREENING DELETION MUTANTSP. Uppuluri, W. L. Chaffin; Texas Tech University Health SciencesCenter, Lubbock, TXThe stationary phase of  growth is the most common microbialgrowth phase encountered in the wild. In vitro, C. albicans stationaryphase is induced by nutrient exhaustion such as glucose starvation.This phase confers on the yeast properties of  better adherence,higher virulence and greater resistance to antifungal drugs. Thestudy of  C. albicans stationary phase is still in the nascent stage. Oneapproach we have taken to understand more about this phase isscreening a group of  deletion mutants for strains that weredefective for entry and maintenance of  stationary phase. Wescreened a library of  83 C. albicans transcription factor mutants and22 cell wall mutants obtained from Aaron Mitchell. Exponentiallygrowing cells were inoculated at a concentration of  1x105 into YPDmedium and incubated at 370C. Cells were assayed for viability at10h, 1d, 2d, 4d, 8d, and 11d after inoculation. Serial 10-folddilutions with cell densities ranging from 5 x 105 to 5 x 101 cells/mlwere prepared for mutant and wild type cells for drop culturescreening. Dilutions were spotted onto a YPD plate and incubatedat 37°C. Each spot was checked for growth after 2 days. Moderategrowth defects were identified by slow growth, reduction in colonysize, or marginal reduction in viable counts and severe defects bymajor loss in viability of  cells. Mutant strains grew as well as thewild type strain for the first three days. Between 4 and 11 days, 34and 16 mutants showed moderate and severe growth defectsrespectively. Of  the 16 genes with the most severe defects five werenot annotated: putative cell wall protein gene orf19.4981 andtranscription factor genes 0rf19.1694, Orf19.2612, Orf10.6850, andOrf19.1589. The 11 annotated genes were cell wall protein gene

Effect of biofilm extracellular metabolites on Candida albicans and Candida dubliniensis morphogenesis

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