Assessment of cAMP-PKA Signaling in Candida glabrata by FRET-Based Biosensors.

Fluorescence or Förster resonance energy transfer (FRET)-based biosensors are used to monitor activity through molecular pathways inside the cell. Binding of secondary metabolites or enzyme-guided modification of protein targets can be assessed by quantifying the rate of energy transfer between two adequate fluorophores. The AKAR3 sensor contains a protein kinase A (PKA) phosphorylation site, which upon phosphorylation interacts with a ligand domain, bringing together FRET donor and acceptor fluorophores and allowing FRET. The EPAC2 sensor contains a cyclic adenosine monophosphate (cAMP)-binding domain. Upon binding of cAMP, donor and acceptor molecules are separated, thereby lowering energy transfer. Since the cAMP-PKA pathway is of great importance for regulation of growth, survival, and virulence in Candida species, monitoring the activity of this pathway in a time- and space-resolved manner allows for detailed investigation of potential drug targets. In this chapter, we describe how these FRET-based biosensors can be used in a practical setup.

The Role of Fatty Acid Metabolites in Vaginal Health and Disease: Application to Candidiasis.

Although the vast majority of women encounters at least one vaginal infection during their life, the amount of microbiome-related research performed in this area lags behind compared to alternative niches such as the intestinal tract. As a result, effective means of diagnosis and treatment, especially of recurrent infections, are limited. The role of the metabolome in vaginal health is largely elusive. It has been shown that lactate produced by the numerous lactobacilli present promotes health by limiting the chance of infection. Short chain fatty acids (SCFA) have been mainly linked to dysbiosis, although the causality of this relationship is still under debate. In this review, we aim to bring together information on the role of the vaginal metabolome and microbiome in infections caused by Candida. Vulvovaginal candidiasis affects near to 70% of all women at least once in their life with a significant proportion of women suffering from the recurrent variant. We assess the role of fatty acid metabolites, mainly SCFA and lactate, in onset of infection and virulence of the fungal pathogen. In addition, we pinpoint where lack of research limits our understanding of the molecular processes involved and restricts the possibility of developing novel treatment strategies.

Investigating the Antifungal Mechanism of Action of Polygodial by Phenotypic Screening in Saccharomyces cerevisiae.

Polygodial is a "hot" peppery-tasting sesquiterpenoid that was first described for its anti-feedant activity against African armyworms. Using the haploid deletion mutant library of Saccharomyces cerevisiae, a genome-wide mutant screen was performed to shed more light on polygodial's antifungal mechanism of action. We identified 66 deletion strains that were hypersensitive and 47 that were highly resistant to polygodial treatment. Among the hypersensitive strains, an enrichment was found for genes required for vacuolar acidification, amino acid biosynthesis, nucleosome mobilization, the transcription mediator complex, autophagy and vesicular trafficking, while the resistant strains were enriched for genes encoding cytoskeleton-binding proteins, ribosomal proteins, mitochondrial matrix proteins, components of the heme activator protein (HAP) complex, and known regulators of the target of rapamycin complex 1 (TORC1) signaling. WE confirm that polygodial triggers a dose-dependent vacuolar alkalinization and that it increases Ca2+ influx and inhibits glucose-induced Ca2+ signaling. Moreover, we provide evidence suggesting that TORC1 signaling and its protective agent ubiquitin play a central role in polygodial resistance, suggesting that they can be targeted by polygodial either directly or via altered Ca2+ homeostasis.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans.

Fluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

Fluorescent toys 'n' tools lighting the way in fungal research.

Although largely overlooked compared to bacterial infections, fungal infections pose a significant threat to the health of humans and other organisms. Many pathogenic fungi, especially Candida species, are extremely versatile and flexible in adapting to various host niches and stressful situations. This leads to high pathogenicity and increasing resistance to existing drugs. Due to the high level of conservation between fungi and mammalian cells, it is hard to find fungus-specific drug targets for novel therapy development. In this respect, it is vital to understand how these fungi function on a molecular, cellular as well as organismal level. Fluorescence imaging allows for detailed analysis of molecular mechanisms, cellular structures and interactions on different levels. In this manuscript, we provide researchers with an elaborate and contemporary overview of fluorescence techniques that can be used to study fungal pathogens. We focus on the available fluorescent labelling techniques and guide our readers through the different relevant applications of fluorescent imaging, from subcellular events to multispecies interactions and diagnostics. As well as cautioning researchers for potential challenges and obstacles, we offer hands-on tips and tricks for efficient experimentation and share our expert-view on future developments and possible improvements.

The involvement of the Candida glabrata trehalase enzymes in stress resistance and gut colonization.

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.

Molecular Elucidation of Riboflavin Production and Regulation in Candida albicans, toward a Novel Antifungal Drug Target.

Candida albicans is a major cause of fungal infections, both superficial and invasive. The economic costs as well as consequences for patient welfare are substantial. Only a few treatment options are available due to the high resemblance between fungal targets and host molecules, as both are eukaryotes. Riboflavin is a yellow pigment, also termed vitamin B2 Unlike animals, fungi can synthesize this essential component themselves, thereby leading us to appreciate that targeting riboflavin production is a promising novel strategy against fungal infections. Here, we report that the GTP cyclohydrolase encoded by C. albicansRIB1 (CaRIB1) is essential and rate-limiting for production of riboflavin in the fungal pathogen. We confirm the high potential of CaRib1 as an antifungal drug target, as its deletion completely impairs in vivo infectibility by C. albicans in model systems. Furthermore, the stimulating effect of iron deprivation and PKA activation on riboflavin production seems to involve CaRib1 and the upstream transcription factor CaSef1. Gathering insights in the synthesis mechanism of riboflavin in pathogenic fungi, like C. albicans, will allow us to design a novel strategy and specifically target this process to combat fungal infections.IMPORTANCECandida albicans is an important fungal pathogen causing common superficial infections as well as invasive diseases with an extremely high morbidity and mortality. Antifungal therapies are limited in efficiency and availability. In this research, we describe the regulation of riboflavin production in C. albicans Since riboflavin biosynthesis is essential to this organism, we can appreciate that targeting it would be a promising new strategy to combat these fungal infections. We provide evidence that one particular enzyme in the production process, CaRib1, would be most promising as an antifungal drug target, as it plays a central role in regulation and proves to be essential in a mouse model of systemic infection.

Presenting a codon-optimized palette of fluorescent proteins for use in Candida albicans.

Fluorescent proteins with varying colors are indispensable tools for the life sciences research community. These fluorophores are often developed for use in mammalian systems, with incremental enhancements or new versions published frequently. However, the successful application of these labels in other organisms in the tree of life, such as the fungus Candida albicans, can be difficult to achieve due to the difficulty in engineering constructs for good expression in these organisms. In this contribution, we present a palette of Candida-optimized fluorescent proteins ranging from cyan to red and assess their application potential. We also compare a range of reported expression optimization techniques, and find that none of these strategies is generally applicable, and that even very closely related proteins require the application of different strategies to achieve good expression. In addition to reporting new fluorescent protein variants for applications in Candida albicans, our work highlights the ongoing challenges in optimizing protein expression in heterologous systems.

Transcriptional responses of Candida glabrata biofilm cells to fluconazole are modulated by the carbon source.

Candida glabrata is an important human fungal pathogen known to trigger serious infections in immune-compromised individuals. Its ability to form biofilms, which exhibit high tolerance to antifungal treatments, has been considered as an important virulence factor. However, the mechanisms involving antifungal resistance in biofilms and the impact of host niche environments on these processes are still poorly defined. In this study, we performed a whole-transcriptome analysis of C. glabrata biofilm cells exposed to different environmental conditions and constraints in order to identify the molecular pathways involved in fluconazole resistance and understand how acidic pH niches, associated with the presence of acetic acid, are able to modulate these responses. We show that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner. This is particularly relevant for a set of genes involved in DNA replication, ergosterol, and ubiquinone biosynthesis. We also provide additional evidence that the loss of mitochondrial function is associated with fluconazole resistance, independently of the growth condition. Lastly, we propose that C. glabrata Mge1, a cochaperone involved in iron metabolism and protein import into the mitochondria, is a key regulator of fluconazole susceptibility during carbon and pH adaptation by reducing the metabolic flux towards toxic sterol formation. These new findings suggest that different host microenvironments influence directly the physiology of C. glabrata, with implications on how this pathogen responds to antifungal treatment. Our analyses identify several pathways that can be targeted and will potentially prove to be useful for developing new antifungals to treat biofilm-based infections.

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

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.

Adhesion of Staphylococcus aureus to Candida albicans During Co-Infection Promotes Bacterial Dissemination Through the Host Immune Response.

Interspecies interactions greatly influence the virulence, drug tolerance and ultimately the outcome of polymicrobial biofilm infections. A synergistic interaction is observed between the fungus Candida albicans and the bacterium Staphylococcus aureus. These species are both normal commensals of most healthy humans and co-exist in several niches of the host. However, under certain circumstances, they can cause hospital-acquired infections with high morbidity and mortality rates. Using a mouse model of oral co-infection, we previously showed that an oral infection with C. albicans predisposes to a secondary systemic infection with S. aureus. Here, we unraveled this intriguing mechanism of bacterial dissemination. Using static and dynamic adhesion assays in combination with single-cell force spectroscopy, we identified C. albicans Als1 and Als3 adhesins as the molecular players involved in the interaction with S. aureus and in subsequent bacterial dissemination. Remarkably, we identified the host immune response as a key element required for bacterial dissemination. We found that the level of immunosuppression of the host plays a critical yet paradoxical role in this process. In addition, secretion of candidalysin, the C. albicans peptide responsible for immune activation and cell damage, is required for C. albicans colonization and subsequent bacterial dissemination. The physical interaction with C. albicans enhances bacterial uptake by phagocytic immune cells, thereby enabling an opportunity to disseminate.

Can Saccharomyces cerevisiae keep up as a model system in fungal azole susceptibility research?

The difficulty of manipulation and limited availability of genetic tools for use in many pathogenic fungi hamper fast and adequate investigation of cellular metabolism and consequent possibilities for antifungal therapies. S. cerevisiae is a model organism that is used to study many eukaryotic systems. In this review, we analyse the potency and relevance of this model system in investigating fungal susceptibility to azole drugs. Although many of the concepts apply to multiple pathogenic fungi, for the sake of simplicity, we will focus on the validity of using S. cerevisiae as a model organism for two Candida species, C. albicans and C. glabrata. Apart from the general benefits, we explore how S. cerevisiae can specifically be used to improve our knowledge on azole drug resistance and enables fast and efficient screening for novel drug targets in combinatorial therapy. We consider the shortcomings of the model system, yet conclude that it is still opportune to use S. cerevisiae as a model system for pathogenic fungi in this era.

Inhibition of Vesicular Transport Influences Fungal Susceptibility to Fluconazole.

Fungal infections pose a substantial threat to the human population. They can cause either mild and relatively harmless infections or invasive and often lethal diseases in patients with a weakened immune system. The majority of these human fungal infections are caused by Candida species. The limited amount of available therapies, together with the development of resistance against these drugs, strongly emphasizes the need for novel therapeutic strategies. As it is quite time-consuming to introduce completely new drugs to the market, potentiating the efficacy of existing drugs would be a better strategy. Therefore, it is important to identify cellular pathways involved in the development of drug resistance. We found that vesicular transport is involved in fungal susceptibility to the most widely used antifungal drug, fluconazole. We identified specific complexes in the vesicular transport pathway which contribute to fluconazole resistance or tolerance in the model organism Saccharomyces cerevisiae Furthermore, we confirmed our findings in the clinically relevant fungi Candida albicans and Candida glabrata Finally, we show that the combination of fluconazole with a specific inhibitor of the vesicular transport pathway increases the susceptibility of Candida species, indicating the potential of using vesicular transport as a target in combination therapy.

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

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP-PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP-PKA activity in this pathogen, we here present the usage of two FRET-based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time-resolved manner, as we exemplify by glucose-induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP-PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization.

HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP's). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP's is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1's internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin.

A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in Candida albicans.

Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as its alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) assay in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida-optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding our knowledge of molecular mechanisms of protein functions.

Mitochondrial Cochaperone Mge1 Is Involved in Regulating Susceptibility to Fluconazole in Saccharomyces cerevisiae and Candida Species.

MGE1 encodes a yeast chaperone involved in Fe-S cluster metabolism and protein import into the mitochondria. In this study, we identified MGE1 as a multicopy suppressor of susceptibility to the antifungal fluconazole in the model yeast Saccharomyces cerevisiae We demonstrate that this phenomenon is not exclusively dependent on the integrity of the mitochondrial DNA or on the presence of the drug efflux pump Pdr5. Instead, we show that the increased dosage of Mge1 plays a protective role by retaining increased amounts of ergosterol upon fluconazole treatment. Iron metabolism and, more particularly, Fe-S cluster formation are involved in regulating this process, since the responsible Hsp70 chaperone, Ssq1, is required. Additionally, we show the necessity but, by itself, insufficiency of activating the iron regulon in establishing the Mge1-related effect on drug susceptibility. Finally, we confirm a similar role for Mge1 in fluconazole susceptibility in the pathogenic fungi Candida glabrata and Candida albicansIMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker's yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans.

Microbial cell surface proteins and secreted metabolites involved in multispecies biofilms.

A considerable number of infectious diseases involve multiple microbial species coexisting and interacting in a host. Only recently however the impact of these polymicrobial diseases has been appreciated and investigated. Often, the causative microbial species are embedded in an extracellular matrix forming biofilms, a form of existence that offers protection against chemotherapeutic agents and host immune defenses. Therefore, recent efforts have focused on developing novel therapeutic strategies targeting biofilm-associated polymicrobial infections, a task that has proved to be challenging. One promising approach to inhibit the development of such complex infections is to impede the interactions between the microbial species via inhibition of adhesion. To that end, studies have focused on identifying specific cell wall adhesins and receptors involved in the interactions between the various bacterial species and the most pathogenic human fungal species Candida albicans. This review highlights the important findings from these studies and describes the available tools and techniques that have provided insights into the role of secreted molecules orchestrating microbial interactions in biofilms. Specifically, we focus on the interactions that take place in oral biofilms and the implications of these interactions on oral health and therapeutic strategies.