Urinary tract infections (UTIs) are quite common and mainly caused by bacteria such as Escherichia coli. However, when patients have urinary catheters, fungal infections comprise up to 15% of these ...types of infections. Moreover, fungal UTIs have a high mortality, due to rapid spreading of the fungi to the kidneys. Most fungal UTIs are caused by Candida species, among which Candida albicans and Candida glabrata are the most common. C. glabrata is an opportunistic pathogenic yeast, phylogenetically quite close to Saccharomyces cerevisiae. Even though it is commonly isolated from the urinary tract and rapidly acquires resistance to antifungals, its pathogenesis has not been studied extensively in vivo. In vivo studies require high numbers of animals, which can be overcome by the use of non-invasive imaging tools. One such tool, bioluminescence imaging, has been used successfully to study different types of C. albicans infections. For C. glabrata, only biofilms on subcutaneously implanted catheters have been imaged using this tool. In this work, we investigated the progression of C. glabrata UTIs from the bladder to the kidneys and the spleen. Furthermore, we optimized expression of a red-shifted firefly luciferase in C. glabrata for in vivo use. We propose the first animal model using bioluminescence imaging to visualize C. glabrata in mouse tissues. Additionally, this UTI model can be used to monitor antifungal activity in vivo over time.
Candida species are the most commonly isolated opportunistic fungal pathogens in humans. Candida albicans causes most of the diagnosed infections, closely followed by Candida glabrata. C. albicans is ...well studied, and many genes have been shown to be important for infection and colonization of the host. It is however less clear how C. glabrata infects the host. With the help of fungal RNA enrichment, we here investigated for the first time the transcriptomic profile of C. glabrata during urinary tract infection (UTI) in mice. In the UTI model, bladders and kidneys are major target organs and therefore fungal transcriptomes were addressed in these organs. Our results showed that, next to adhesins and proteases, nitrogen metabolism and regulation play a vital role during C. glabrata UTI. Genes involved in nitrogen metabolism were upregulated and among them we show that DUR1,2 (urea amidolyase) and GAP1 (amino acid permease) were important for virulence. Furthermore, we confirmed the importance of the glyoxylate cycle in the host and identified MLS1 (malate synthase) as an important gene necessary for C. glabrata virulence. In conclusion, our study shows with the support of in vivo transcriptomics how C. glabrata adapts to host conditions.
Summary
Candida albicans is a major human fungal pathogen, causing superficial, as well as life‐threatening invasive infections. Therefore, it has to adequately sense and respond to the host defense ...by expressing appropriate virulence attributes. The most important virulence factor of C. albicans is the yeast‐to‐hyphae morphogenetic switch, which can be induced by numerous environmental cues, including the amino acid methionine. Here, we show an essential role for methionine permease Mup1 in methionine‐induced morphogenesis, biofilm formation, survival inside macrophages and virulence. Furthermore, we demonstrate that this process requires conversion of methionine into S‐adenosyl methionine (SAM) and its decarboxylation by Spe2. The resulting amino‐propyl group is then used for biosynthesis of polyamines, which have been shown to activate adenylate cyclase. Inhibition of the SPE2 SAM decarboxylase gene strongly impairs methionine‐induced morphogenesis on specific media and significantly delays virulence in the mouse systemic infection model system. Further proof of the connection between methionine uptake and initial metabolism and the cAMP‐PKA pathway was obtained by showing that both Mup1 and Spe2 are required for cAMP production in response to methionine. Our results suggest that amino acid transport and further metabolism are interesting therapeutic targets as inhibitors of this may prevent the morphogenetic switch, thereby preventing virulence.
l‐Methionine is a strong inducer of morphogenesis in Candida albicans. After its transport by Mup1, it is metabolized to S‐adenosyl methionine and then decarboxylated. The resulting aminopropyl group is used for the biosynthesis of polyamines, which activate adenylate cyclase. This ultimately results in the induction of morphogenesis.
The pathogenic fungus
is able to adapt to extremely variable environmental conditions. The
genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important ...regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the
Fus3 ortholog in
, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of
and
, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when
germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade.
is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of
is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.
Amino acids are key sources of nitrogen for growth of
. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus ...must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six
putative general amino acid permeases based on their homology to the
Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to
Gap1, with broad substrate specificity. Expression analysis showed that expression of all
genes is under control of the Csy1 amino acid sensor, which is different from the situation in
, where the expression of
is not regulated by Ssy1. We show that Gap4 is the functional orthologue of
Sam3, the only
-adenosylmethionine (SAM) transporter in
, and we report that Gap4 is required for SAM-induced morphogenesis.
is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the
genome encodes 6 orthologues of the
general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that
Gap2 is the functional orthologue of
Gap1 and that
Gap4 is an orthologue of
Sam3, an
-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of
.
The human fungal pathogen Candida glabrata is less virulent compared to the most isolated Candida species including Candida albicans. Its reduced metabolic flexibility, haploidy, and auxotrophies ...contribute to a "stealth and evasion" infection strategy, resulting in minimal tissue damage in the host. C. glabrata seems to be optimally adapted to infection of the urinary tract (UTI), especially in catheterized patients. UTIs are not well studied and often difficult to treat, given that not all antifungals penetrate in the bladder and that treatments through the catheter are not always possible since maintained catheterization increases the infection risk.In the recent effort to reduce the amount of animals used during scientific experiments, bioluminescence imaging is a useful tool. In this protocol, C. glabrata urinary tract infections were imaged in mice, thus facilitating the testing of possible new antifungals and novel treatment strategies.
G-protein signalling pathways are involved in sensing the environment, enabling fungi to coordinate cell function, metabolism and development with their surroundings, thereby promoting their ...survival, propagation and virulence. G-protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in fungi. Despite the apparent importance of GPCR signalling to fungal biology and virulence, relatively few GPCR-G-protein interactions, and even fewer receptor-binding ligands, have been identified. Approximately 40% of current pharmaceuticals target human GPCRs, due to their cell surface location and central role in cell signalling. Fungal GPCRs do not belong to any of the mammalian receptor classes, making them druggable targets for antifungal development. This Review Article evaluates developments in our understanding of fungal GPCR-mediated signalling, while substantiating the rationale for considering these receptors as potential antifungal targets. The need for insights into the structure-function relationship of receptor-ligand interactions is highlighted, which could facilitate the development of receptor-interfering compounds that could be used in disease control.
Summary
Candida albicans is a major human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. Biofilms have a ...huge impact on public health, as fungal biofilms are highly resistant against most antimycotics. Animal models of biofilm formation are indispensable for improving our understanding of biofilm development inside the host, their antifungal resistance and their interaction with the host immune defence system. In currently used models, evaluation of biofilm development or the efficacy of antifungal treatment is limited to ex vivo analyses, requiring host sacrifice, which excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. In this study, we have demonstrated for the first time that non‐invasive, dynamic imaging and quantification of in vitro and in vivo C. albicans biofilm formation including morphogenesis from the yeast to hyphae state is feasible by using growth‐phase dependent bioluminescent C. albicans strains in a subcutaneous catheter model in rodents. We have shown the defect in biofilm formation of a bioluminescent bcr1 mutant strain. This approach has immediate applications for the screening and validation ofantimycotics under in vivo conditions, for studying host–biofilm interactions in different transgenic mouse models and for testing the virulence of luminescent C. albicans mutants, hereby contributing to a better understanding of the pathogenesis of biofilm‐associated yeast infections.
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