Introducing fluorescence resonance energy transfer-based biosensors for the analysis of cAMP-PKA signalling in the fungal pathogen<i>Candida glabrata</i>

Demuyser, Liesbeth; Genechten, Wouter Van; Mizuno, Hideaki; Colombo, Sonia; Dijck, Patrick Van

doi:10.1111/cmi.12863

Cited by 13 publications

(6 citation statements)

References 18 publications

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“…Here, it is shown, that after addition of glucose to derepressed cells, both the intracellular cAMP level increases and PKA is activated. These findings suggest that, similar to the situation in S. cerevisiae , glucose stimulates the activation of PKA via cAMP (Demuyser et al, 2018).…”

Section: Glucose Sensing and Signalingsupporting

confidence: 52%

Sugar Sensing and Signaling in Candida albicans and Candida glabrata

2019

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Candida species, such as Candida albicans and Candida glabrata, cause infections at different host sites because they adapt their metabolism depending on the available nutrients. They are able to proliferate under both nutrient-rich and nutrient-poor conditions. This adaptation is what makes these fungi successful pathogens. For both species, sugars are very important nutrients and as the sugar level differs depending on the host niche, different sugar sensing systems must be present. Saccharomyces cerevisiae has been used as a model for the identification of these sugar sensing systems. One of the main carbon sources for yeast is glucose, for which three different pathways have been described. First, two transporter-like proteins, ScSnf3 and ScRgt2, sense glucose levels resulting in the induction of different hexose transporter genes. This situation is comparable in C. albicans and C. glabrata, where sensing of glucose by CaHgt4 and CgSnf3, respectively, also results in hexose transporter gene induction. The second glucose sensing mechanism in S. cerevisiae is via the G-protein coupled receptor ScGpr1, which causes the activation of the cAMP/PKA pathway, resulting in rapid adaptation to the presence of glucose. The main components of this glucose sensing system are also conserved in C. albicans and C. glabrata. However, it seems that the ligand(s) for CaGpr1 are not sugars but lactate and methionine. In C. glabrata, this pathway has not yet been investigated. Finally, the glucose repression pathway ensures repression of respiration and repression of the use of alternative carbon sources. This pathway is not well characterized in Candida species. It is important to note that, apart from glucose, other sugars and sugar-analogs, such as N-acetylglucosamine in the case of C. albicans, are also important carbon sources. In these fungal pathogens, sensing sugars is important for a number of virulence attributes, including adhesion, oxidative stress resistance, biofilm formation, morphogenesis, invasion, and antifungal drug tolerance. In this review, the sugar sensing and signaling mechanisms in these Candida species are compared to S. cerevisiae.

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Section: Glucose Sensing and Signalingsupporting

confidence: 52%

Sugar Sensing and Signaling in Candida albicans and Candida glabrata

2019

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“…It is also possible to use fluorophores and luciferases in a Förster Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET) system, however, BRET has so far not been reported in C. albicans (Chatr-Aryamontri et al, 2017). A FRET biosensor has been shown to work in C. albicans (Jain et al, 2018) and Candida glabrata (Demuyser et al, 2018).…”

Section: Resultsmentioning

confidence: 99%

Protein-Protein Interactions in Candida albicans

Schoeters

Dijck

2019

Front. Microbiol.

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Despite being one of the most important human fungal pathogens, Candida albicans has not been studied extensively at the level of protein-protein interactions (PPIs) and data on PPIs are not readily available in online databases. In January 2018, the database called “Biological General Repository for Interaction Datasets (BioGRID)” that contains the most PPIs for C. albicans , only documented 188 physical or direct PPIs (release 3.4.156) while several more can be found in the literature. Other databases such as the String database, the Molecular INTeraction Database (MINT), and the Database for Interacting Proteins (DIP) database contain even fewer interactions or do not even include C. albicans as a searchable term. Because of the non-canonical codon usage of C. albicans where CUG is translated as serine rather than leucine, it is often problematic to use the yeast two-hybrid system in Saccharomyces cerevisiae to study C. albicans PPIs. However, studying PPIs is crucial to gain a thorough understanding of the function of proteins, biological processes and pathways. PPIs can also be potential drug targets. To aid in creating PPI networks and updating the BioGRID, we performed an exhaustive literature search in order to provide, in an accessible format, a more extensive list of known PPIs in C. albicans .

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“…The FRETR ratios were calculated for an average of 20 cells by dividing the corrected acceptor value by the corrected donor value, as previously described [37]. Correction was achieved by subtracting the background and the autofluorescence signal intensity from the sample signal intensity of every cell.…”

Section: Discussionmentioning

confidence: 99%

“…The CellASIC ONIX2 Microfluidic Platform from Millipore for automated addition of compounds to cells during continuous imaging by microscopy was used, as previously described [37]. The cells were loaded into a chamber of a microwell plate (Y04C, Merck), and liquid was continuously fluxed through the cell chamber.…”

Section: Ratiometric Fret Measurements Microfluidics and Imagingmentioning

confidence: 99%

Nutrient sensing and cAMP signaling in yeast: G-protein coupled receptor versus transceptor activation of PKA

Zeebroeck

Demuyser

Zhang³

et al. 2021

Microb Cell

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A major signal transduction pathway regulating cell growth and many associated physiological properties as a function of nutrient availability in the yeast Saccharomyces cerevisiae is the protein kinase A (PKA) pathway. Glucose activation of PKA is mediated by G-protein coupled receptor (GPCR) Gpr1, and secondary messenger cAMP. Other nutrients, including nitrogen, phosphate and sulfate, activate PKA in accordingly-starved cells through nutrient transceptors, but apparently without cAMP signaling. We have now used an optimized EPAC-based fluorescence resonance energy transfer (FRET) sensor to precisely monitor in vivo cAMP levels after nutrient addition. We show that GPCR-mediated glucose activation of PKA is correlated with a rapid transient increase in the cAMP level in vivo, whereas nutrient transceptor-mediated activation by nitrogen, phosphate or sulfate, is not associated with any significant increase in cAMP in vivo. We also demonstrate direct physical interaction between the Gap1 amino acid transceptor and the catalytic subunits of PKA, Tpk1, 2 and 3. In addition, we reveal a conserved consensus motif in the nutrient transceptors that is also present in Bcy1, the regulatory subunit of PKA. This suggests that nutrient transceptor activation of PKA may be mediated by direct release of bound PKA catalytic subunits, triggered by the conformational changes occurring during transport of the substrate by the transceptor. Our results support a model in which nutrient transceptors are evolutionary ancestors of GPCRs, employing a more primitive direct signaling mechanism compared to the indirect cAMP second-messenger signaling mechanism used by GPCRs for activation of PKA.

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Introducing fluorescence resonance energy transfer-based biosensors for the analysis of cAMP-PKA signalling in the fungal pathogenCandida glabrata

Cited by 13 publications

References 18 publications

Sugar Sensing and Signaling in Candida albicans and Candida glabrata

Sugar Sensing and Signaling in Candida albicans and Candida glabrata

Protein-Protein Interactions in Candida albicans

Nutrient sensing and cAMP signaling in yeast: G-protein coupled receptor versus transceptor activation of PKA

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