Characterization of an autonomously replicating sequence in Candida guilliermondii

Foureau, Emilien; Courdavault, Vincent; Gallón, Sandra M. Navarro; Besseau, Sébastien; Simkin, Andrew J.; Crèche, Joël; Atehortúa, Lucía; Giglioli-Guivarc’h, Nathalie; Clastre, Marc; Papon, Nicolas

doi:10.1016/j.micres.2013.04.006

Cited by 16 publications

(11 citation statements)

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“…A thorough search in the literature and available databases indicates that there are a limited number of disrupted genes in C. guilliermondii , and most of them have been used as markers to develop gene manipulation techniques (Millerioux et al, 2011a,b; Foureau et al, 2013a,c; Papon et al, 2013). Our work now presents the first C. guilliermondii null mutant with defects in protein mannosylation, in the cell wall and interaction with the host.…”

Section: Discussionmentioning

confidence: 99%

“…However, in the mouse model the re-integrant strain failed to colonize and kill as the parental control cells, recuperating similar CFU from organs to those obtained from animals infected with the null mutant strain. The backbone vector used to generate the pmr1 Δ + PMR1 re-integrant strain did not integrate into the C. guilliermondii genome, as it contains an autonomously replicating sequence (Foureau et al, 2013a), so it is tempting to speculate that cells growing in a rich nutrient condition such as that which is present in the mouse milieu tend to lose the plasmid containing PMR1 . Indeed, when this strain was grown in rich medium, like YPD or Sabouraud, the cells tended to show phenotypic traits of the null mutant strain (data not shown).…”

Section: Discussionmentioning

confidence: 99%

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Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

et al. 2016

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The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite the significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1Δ null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O-linked mannans.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

et al. 2016

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“…De acuerdo a Savini y colaboradores [22] C. guilliermondii en su estado telemórfico también recibe el nombre de Pichia guilliermondii [23]. Se encuentra distribuido ampliamente en la naturaleza y puede ser aislado del suelo, plantas, insectos, agua de mar y exudados de varios árboles, siendo además parte la microflora humana.…”

Section: Figuraunclassified

Crecimiento dimórfico y caracterización molecular de Candida guillermondi aislado de Pannicum maximun

Rosales-López

Valerín-Berrocal

Jiménez-Bonilla

2018

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Candida guilliermondii es una levadura ascomicete ampliamente distribuida en el medio ambiente natural, a esta levadura se le atribuyen varias propiedades que le hacen ser de interés biotecnológico, como por ejemplo, para control biológico. En este proyecto C. guillermondii se logró aislar del zacate Panicum maximun, recolectado en plazas de futbol, orillas de calle y en potreros de la zonas de Cartago y Heredia. Se purificaron colonias pequeñas, ovoides y brotantes, de 3 a 5 µm y de color crema, a las 48 h de cultivo a 25 °C en PDA. Se cultivaron en medio líquido, para estimular el crecimiento de biomasa y poder realizar el análisis molecular, se extrajo el ADN y se realizaron las pruebas de PCR con diferentes imprimadores. Las secuencias generadas fueron comparadas mediante un alineamiento utilizando la herramienta Blast del National Center for Biotechnology Information (NCBI) y compararon con la base de datos del Gen Bank, identificando a Candida guillermondii. Se procedió a la inducción de pseudohifas, para ello se probaron diferentes condiciones de cultivo: luz, temperatura y pH, y varios medios de cultivo (fuentes de carbono, nitrógeno, medios pobres en minerales y medio con solo agaragua). Se logró la formación de pseudomicelio bien desarrollado desde las 96 h de incubación. El pH y la luz, medios como el agar agua, suplementado con sacarosa, el que no contenía fuente de nitrógeno y Vogels, fueron determinantes para la aparición de hifas en las levaduras. Concluyendo que el provocar estrés al microorganismo permite un cambio morfológico en el mismo.

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“…For this purpose, we used a recently developed C. guilliermondii expression plasmid, named pURA5-YFP (Courdavault et al, 2011) (Figure 2F), including an autonomously replicating sequence (ARS) (Foureau et al, 2013a;Millerioux et al, 2011b). The coding sequence of URA5 was replaced by the functional MET2 gene in pURA5-YFP to yield pMET2 ARS -YFP ( Figure 2G).…”

Section: Met2 As a Selectable Marker For C Guilliermondii Autonomousmentioning

confidence: 99%

Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter‐selectable marker

Montoya

Mélin

Blanc

et al. 2014

Yeast

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Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the fungal CTG clade. This yeast remains actively studied as a result of its moderate clinical importance and most of all for its potential uses in biotechnology. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both a methionine auxotroph recipient strain and a functional MET gene as selection marker. We first disrupted the MET2 and MET15 genes encoding homoserine-O-acetyltransferase and O-acetylserine O-acetylhomoserine sulphydrylase, respectively. The met2 mutant was shown to be a methionine auxotroph in contrast to met15 which was not. Interestingly, met2 and met15 mutants formed brown colonies when cultured on lead-containing medium, contrary to the wild-type strain, which develop as white colonies on this medium. The MET2 wild-type allele was successfully used to transfer a yellow fluorescent protein (YFP) gene-expressing vector into the met2 recipient strain. In addition, we showed that the loss of the MET2-containing YFPexpressing plasmid can be easily observed on lead-containing medium. The MET2 wildtype allele, flanked by two short repeated sequences, was then used to disrupt the LYS2 gene (encoding the α-aminoadipate reductase) in the C. guilliermondii met2 recipient strain. The resulting lys2 mutants displayed, as expected, auxotrophy for lysine. Unfortunately, all our attempts to pop-out the MET2 marker (following the recombination of the bordering repeat sequences) from a target lys2 locus were unsuccessful using white/brown colony colour screening. Nevertheless, this MET2 transformation/disruption system represents a new versatile genetic tool for C. guilliermondii.

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Characterization of an autonomously replicating sequence in Candida guilliermondii

Cited by 16 publications

References 32 publications

Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

Disruption of Protein Mannosylation Affects Candida guilliermondii Cell Wall, Immune Sensing, and Virulence

Crecimiento dimórfico y caracterización molecular de Candida guillermondi aislado de Pannicum maximun

Disrupting the methionine biosynthetic pathway in Candida guilliermondii: characterization of the MET2 gene as counter‐selectable marker

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