Search

Salivary histatin 5 (Hst 5) kills the fungal pathogen C. albicans via a mechanism that involves binding and subsequent efflux of cellular ATP. Our aims were to identify inorganic ions found in saliva that influence Hst 5 fungicidal activity. Increasing ionic strength with relevant salivary anions (Cl- and CO3-) did not reduce Hst 5 binding or uptake by yeast cells, but reduced the Hst-induced efflux of ATP. Extracellular MgCl2 (25 mM) maximally inhibited 30-40% of Hst 5 killing with 40% reduction in ATP efflux, while pre-treatment of cells with only 2 mM CaCl2 inhibited 80-90% of killing, and prevented ATP efflux. Loss of fungicidal activity by the addition of CaCl2 or MgCl2 was a result of inhibition of binding of Hst 5 to C. albicans cells. Calcium is a potent inhibitor of Hst 5 candidacidal activity at physiological concentrations and may be the primary salivary ion responsible for the masking effect of saliva. [ABSTRACT FROM AUTHOR]

The fungus Candida albicans and the Gram-positive bacterium Enterococcus faecalis share mucosal niches in the human body. As opportunistic pathogens, both are found to expand population size during dysbiosis, and can cause severe systemic infections in susceptible individuals. Here, we show that the presence of C. albicans results in increased host cell damage by E. faecalis . Furthermore, E. faecalis aggravates oropharyngeal candidiasis in mice. Increased damage is mediated by enterococcal cytolysin, and involves both physical interaction and altered glucose availability. Physical interaction promotes accumulation of bacteria on host cells, facilitating contact of cytolysin with host cells. Glucose depletion by the metabolic activity of the fungus sensitized host cells to cytolysin. This work illustrates how a complex interplay between fungi and bacteria can result in detrimental consequences for the host.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis , differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections., Competing Interests: The authors declare no conflict of interest.

Candida albicans is a pathobiont of the gastrointestinal tract. It can contribute to the diversity of the gut microbiome without causing harmful effects. When the immune system is compromised, C. albicans can damage intestinal cells and cause invasive disease. We hypothesize that a therapeutic approach against C. albicans infections can rely on the antimicrobial properties of probiotic bacteria. We investigated the impact of the probiotic strain Escherichia coli Nissle 1917 (EcN) on C. albicans growth and its ability to cause damage to intestinal cells. In co-culture kinetic assays, C. albicans abundance gradually decreased over time compared with C. albicans abundance in the absence of EcN. Quantification of C. albicans survival suggests that EcN exerts a fungicidal activity. Cell-free supernatants (CFS) collected from C. albicans -EcN co-culture mildly altered C. albicans growth, suggesting the involvement of an EcN-released compound. Using a model of co-culture in the presence of human intestinal epithelial cells, we further show that EcN prevents C. albicans from damaging enterocytes both distantly and through direct contact. Consistently, both C. albicans 's filamentous growth and microcolony formation were altered by EcN. Taken together, our study proposes that probiotic-strain EcN can be exploited for future therapeutic approaches against C. albicans infections.

Candida auris, a multidrug-resistant human fungal pathogen that causes outbreaks of invasive infections, emerged as four distinct geographical clades. Previous studies identified genomic and proteomic differences in nutrient utilization on comparison to Candida albicans, suggesting that certain metabolic features may contribute to C. auris emergence. Since no high-throughput clade-specific metabolic characterization has been described yet, we performed a phenotypic screening of C. auris strains from all 4 clades on 664 nutrients, 120 chemicals, and 24 stressors. We identified common and clade- or strain-specific responses, including the preferred utilization of various dipeptides as nitrogen source and the inability of the clade II isolate AR 0381 to withstand chemical stress. Further analysis of the metabolic properties of C. auris isolates showed robust growth on intermediates of the tricarboxylic acid cycle, such as citrate and succinic and malic acids. However, there was reduced or no growth on pyruvate, lactic acid, or acetate, likely due to the lack of the monocarboxylic acid transporter Jen1, which is conserved in most pathogenic Candida species. Comparison of C. auris and C. albicans transcriptomes of cells grown on alternative carbon sources and dipeptides as a nitrogen source revealed common as well as species-unique responses. C. auris induced a significant number of genes with no ortholog in C. albicans, e.g., genes similar to the nicotinic acid transporter TNA1 (alternative carbon sources) and to the oligopeptide transporter ( OPT ) family (dipeptides). Thus, C. auris possesses unique metabolic features which could have contributed to its emergence as a pathogen. IMPORTANCE Four main clades of the emerging, multidrug-resistant human pathogen Candida auris have been identified, and they differ in their susceptibilities to antifungals and disinfectants. Moreover, clade- and strain-specific metabolic differences have been identified, but a comprehensive overview of nutritional characteristics and resistance to various stressors is missing. Here, we performed high-throughput phenotypic characterization of C. auris on various nutrients, stressors, and chemicals and obtained transcriptomes of cells grown on selected nutrients. The generated data sets identified multiple clade- and strain-specific phenotypes and induction of C. auris-specific metabolic genes, showing unique metabolic properties. The presented work provides a large amount of information for further investigations that could explain the role of metabolism in emergence and pathogenicity of this multidrug-resistant fungus., Competing Interests: The authors declare no conflict of interest.

Innate immune responses vary by pathogen and host genetics. We analyze quantitative trait loci (eQTLs) and transcriptomes of monocytes from 215 individuals stimulated by fungal, Gram-negative or Gram-positive bacterial pathogens. We identify conserved monocyte responses to bacterial pathogens and a distinct antifungal response. These include 745 response eQTLs (reQTLs) and corresponding genes with pathogen-specific effects, which we find first in samples of male donors and subsequently confirm for selected reQTLs in females. reQTLs affect predominantly upregulated genes that regulate immune response via e.g., NOD-like, C-type lectin, Toll-like and complement receptor-signaling pathways. Hence, reQTLs provide a functional explanation for individual differences in innate response patterns. Our identified reQTLs are also associated with cancer, autoimmunity, inflammatory and infectious diseases as shown by external genome-wide association studies. Thus, reQTLs help to explain interindividual variation in immune response to infection and provide candidate genes for variants associated with a range of diseases., (© 2023. The Author(s).)

Fungal infections are a major global health burden where Candida albicans is among the most common fungal pathogen in humans and is a common cause of invasive candidiasis. Fungal phenotypes, such as those related to morphology, proliferation and virulence are mainly driven by gene expression, which is primarily regulated by kinase signaling cascades. Serine-arginine (SR) protein kinases are highly conserved among eukaryotes and are involved in major transcriptional processes in human and S. cerevisiae . Candida albicans harbors two SR protein kinases, while Sky2 is important for metabolic adaptation, Sky1 has similar functions as in S. cerevisiae . To investigate the role of these SR kinases for the regulation of transcriptional responses in C. albicans , we performed RNA sequencing of sky1 Δ and sky2 Δ and integrated a comprehensive phosphoproteome dataset of these mutants. Using a Systems Biology approach, we study transcriptional regulation in the context of kinase signaling networks. Transcriptomic enrichment analysis indicates that pathways involved in the regulation of gene expression are downregulated and mitochondrial processes are upregulated in sky1 Δ. In sky2 Δ, primarily metabolic processes are affected, especially for arginine, and we observed that arginine-induced hyphae formation is impaired in sky2 Δ. In addition, our analysis identifies several transcription factors as potential drivers of the transcriptional response. Among these, a core set is shared between both kinase knockouts, but it appears to regulate different subsets of target genes. To elucidate these diverse regulatory patterns, we created network modules by integrating the data of site-specific protein phosphorylation and gene expression with kinase-substrate predictions and protein-protein interactions. These integrated signaling modules reveal shared parts but also highlight specific patterns characteristic for each kinase. Interestingly, the modules contain many proteins involved in fungal morphogenesis and stress response. Accordingly, experimental phenotyping shows a higher resistance to Hygromycin B for sky1 Δ. Thus, our study demonstrates that a combination of computational approaches with integration of experimental data can offer a new systems biological perspective on the complex network of signaling and transcription. With that, the investigation of the interface between signaling and transcriptional regulation in C. albicans provides a deeper insight into how cellular mechanisms can shape the phenotype., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Luther, Brandt, Vylkova, Dandekar, Müller and Dittrich.)

The formation of hyphae is a key virulence attribute of Candida albicans as they are required for adhesion to and invasion of host cells, and ultimately deep-tissue dissemination. Hyphae also secrete the peptide toxin candidalysin, which is crucial for destruction of host cell membranes. The peptide is derived from a precursor protein encoded by the gene ECE1 which is strongly induced during hyphal growth. Previous studies revealed a very complex regulation of this gene involving several transcription factors. However, the promoter of the gene is still not characterized. Here, we present a functional analysis of the intergenic region upstream of the ECE1 gene. Rapid amplification of cDNA ends (RACE)-PCR was performed to identify the 5' untranslated region, which has a size of 49 bp regardless of the hyphae-inducing condition. By using green fluorescent protein (GFP) reporter constructs we further defined a minimal promoter length of 1,500 bp which was verified by RT-qPCR. Finally, we identified the TATA element required for the expression of the gene. It is located 106 to 109 bp upstream of the ECE1 start codon. Our results illustrate that despite a very short 5' UTR, a relatively long promoter is required to secure ECE1 transcription, indicating a complex regulatory machinery tightly controlling the expression of the gene. IMPORTANCE In recent years it was shown that secretion of the toxic peptide candidalysin from hyphae of the major human fungal pathogen Candida albicans contributes heavily to its virulence. The peptide is derived from a precursor protein which is encoded by the ECE1 gene whose transcription is known to be closely associated with formation of hyphae. Here, we used a GFP reporter system to determine the length of the ECE1 promoter and were able to show that it has a minimal size of 1,500 bp. Surprisingly, the gene has a very short 5' UTR of only 49 bp. In accordance with this, the TATA element required for transcription is located 106 to 109 bp upstream of the start codon. This indicates that ECE1 expression is controlled by a very long promoter allowing a complex network of transcription factors to contribute to the gene's regulation.

The yeast-to-hypha transition is a key virulence attribute of the opportunistic human fungal pathogen Candida albicans, since it is closely tied to infection-associated processes such as tissue invasion and escape from phagocytes. While the nature of hypha-associated gene expression required for fungal virulence has been thoroughly investigated, potential morphotype-dependent activity of metabolic pathways remained unclear. Here, we combined global transcriptome and metabolome analyses for the wild-type SC5314 and the hypha-defective hgc1 Δ and cph1 Δ efg1 Δ strains under three hypha-inducing (human serum, N -acetylglucosamine, and alkaline pH) and two yeast-promoting conditions to identify metabolic adaptions that accompany the filamentation process. We identified morphotype-related activities of distinct pathways and a metabolic core signature of 26 metabolites with consistent depletion or enrichment during the yeast-to-hypha transition. Most strikingly, we found a hypha-associated activation of de novo sphingolipid biosynthesis, indicating a connection of this pathway and filamentous growth. Consequently, pharmacological inhibition of this partially fungus-specific pathway resulted in strongly impaired filamentation, verifying the necessity of de novo sphingolipid biosynthesis for proper hypha formation. IMPORTANCE The reversible switch of Candida albicans between unicellular yeast and multicellular hyphal growth is accompanied by a well-studied hypha-associated gene expression, encoding virulence factors like adhesins, toxins, or nutrient scavengers. The investigation of this gene expression consequently led to fundamental insights into the pathogenesis of this fungus. In this study, we applied this concept to hypha-associated metabolic adaptations and identified morphotype-dependent activities of distinct pathways and a stimulus-independent metabolic signature of hyphae. Most strikingly, we found the induction of de novo sphingolipid biosynthesis as hypha associated and essential for the filamentation of C. albicans. These findings verified the presence of morphotype-specific metabolic traits in the fungus, which appear connected to the fungal virulence. Furthermore, the here-provided comprehensive description of the fungal metabolome will help to foster future research and lead to a better understanding of fungal physiology.

Candida albicans biofilm maturation is accompanied by enhanced expression of amino acid acquisition genes. Three state-of-the-art omics techniques were applied to detail the importance of active amino acid uptake during biofilm development. Comparative analyses of normoxic wild-type biofilms were performed under three metabolically challenging conditions: aging, hypoxia, and disabled amino acid uptake using a strain lacking the regulator of amino acid permeases Stp2. Aging-induced amino acid acquisition and stress responses to withstand the increasingly restricted environment. Hypoxia paralyzed overall energy metabolism with delayed amino acid consumption, but following prolonged adaptation, the metabolic fingerprints aligned with aged normoxic biofilms. The extracellular metabolome of stp2Δ biofilms revealed deficient uptake for 11 amino acids, resulting in extensive transcriptional and metabolic changes including induction of amino acid biosynthesis and carbohydrate and micronutrient uptake. Altogether, this study underscores the critical importance of a balanced amino acid homeostasis for C. albicans biofilm development., (© 2022. The Author(s).)

Intestinal microbiota dysbiosis can initiate overgrowth of commensal Candida species - a major predisposing factor for disseminated candidiasis. Commensal bacteria such as Lactobacillus rhamnosus can antagonize Candida albicans pathogenicity. Here, we investigate the interplay between C. albicans, L. rhamnosus, and intestinal epithelial cells by integrating transcriptional and metabolic profiling, and reverse genetics. Untargeted metabolomics and in silico modelling indicate that intestinal epithelial cells foster bacterial growth metabolically, leading to bacterial production of antivirulence compounds. In addition, bacterial growth modifies the metabolic environment, including removal of C. albicans' favoured nutrient sources. This is accompanied by transcriptional and metabolic changes in C. albicans, including altered expression of virulence-related genes. Our results indicate that intestinal colonization with bacteria can antagonize C. albicans by reshaping the metabolic environment, forcing metabolic adaptations that reduce fungal pathogenicity., (© 2022. The Author(s).)

Protein kinases play a crucial role in regulating cellular processes such as growth, proliferation, environmental adaptation and stress responses. Serine-arginine (SR) protein kinases are highly conserved in eukaryotes and regulate fundamental processes such as constitutive and alternative splicing, mRNA processing and ion homeostasis. The Candida albicans genome encodes two (Sky1, Sky2) and the Candida glabrata genome has one homolog (Sky1) of the human SR protein kinase 1, but their functions have not yet been investigated. We used deletion strains of the corresponding genes in both fungi to study their cellular functions. C. glabrata and C. albicans strains lacking SKY1 exhibited higher resistance to osmotic stress and toxic polyamine concentrations, similar to Saccharomyces cerevisiae sky1 Δ mutants. Deletion of SKY2 in C. albicans resulted in impaired utilization of various dipeptides as the sole nitrogen source. Subsequent phosphoproteomic analysis identified the di- and tripeptide transporter Ptr22 as a potential Sky2 substrate. Sky2 seems to be involved in Ptr22 regulation since overexpression of PTR22 in the sky2 Δ mutant restored the ability to grow on dipeptides and made the cells more susceptible to the dipeptide antifungals Polyoxin D and Nikkomycin Z. Altogether, our results demonstrate that C. albicans and C. glabrata Sky1 protein kinases are functionally similar to Sky1 in S. cerevisiae , whereas C. albicans Sky2, a unique kinase of the CTG clade, likely regulates dipeptide uptake via Ptr22., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brandt, Gerwien, Wagner, Krüger, Ramírez-Zavala, Mirhakkak, Schäuble, Kniemeyer, Panagiotou, Brakhage, Morschhäuser and Vylkova.)

The tight association of Candida albicans with the human host has driven the evolution of mechanisms that permit metabolic flexibility. Amino acids, present in a free or peptide-bound form, are abundant carbon and nitrogen sources in many host niches. In C. albicans, the capacity to utilize certain amino acids, like proline, is directly connected to fungal morphogenesis and virulence. Yet the precise nature of proline sensing and uptake in this pathogenic fungus has not been investigated. Since C. albicans encodes 10 putative orthologs of the four Saccharomyces cerevisiae proline transporters, we tested deletion strains of the respective genes and identified Gnp2 (CR_09920W) as the main C. albicans proline permease. In addition, we found that this specialization of Gnp2 was reflected in its transcriptional regulation and further assigned distinct substrate specificities for the other orthologs, indicating functional differences of the C. albicans amino acid permeases compared to the model yeast. The physiological relevance of proline uptake is exemplified by the findings that strains lacking GNP2 were unable to filament in response to extracellular proline and had a reduced capacity to damage macrophages and impaired survival following phagocytosis. Furthermore, GNP2 deletion rendered the cells more sensitive to oxidative stress, illustrating new connections between amino acid uptake and stress adaptation in C. albicans. IMPORTANCE The utilization of various nutrients is of paramount importance for the ability of Candida albicans to successfully colonize and infect diverse host niches. In this context, amino acids are of special interest due to their ubiquitous availability, relevance for fungal growth, and direct influence on virulence traits like filamentation. In this study, we identify a specialized proline transporter in C. albicans encoded by GNP2 . The corresponding amino acid permease is essential for proline-induced filamentation, oxidative stress resistance, and fungal survival following interaction with macrophages. Altogether, this work highlights the importance of amino acid uptake for metabolic and stress adaptation in this fungus.

Candida albicans is a leading cause of life-threatening hospital-acquired infections and can lead to Candidemia with sepsis-like symptoms and high mortality rates. We reconstructed a genome-scale C. albicans metabolic model to investigate bacterial-fungal metabolic interactions in the gut as determinants of fungal abundance. We optimized the predictive capacity of our model using wild type and mutant C. albicans growth data and used it for in silico metabolic interaction predictions. Our analysis of more than 900 paired fungal-bacterial metabolic models predicted key gut bacterial species modulating C. albicans colonization levels. Among the studied microbes, Alistipes putredinis was predicted to negatively affect C. albicans levels. We confirmed these findings by metagenomic sequencing of stool samples from 24 human subjects and by fungal growth experiments in bacterial spent media. Furthermore, our pairwise simulations guided us to specific metabolites with promoting or inhibitory effect to the fungus when exposed in defined media under carbon and nitrogen limitation. Our study demonstrates that in silico metabolic prediction can lead to the identification of gut microbiome features that can significantly affect potentially harmful levels of C. albicans.

Burn wounds are highly susceptible sites for colonization and infection by bacteria and fungi. Large wound surface, impaired local immunity, and broad-spectrum antibiotic therapy support growth of opportunistic fungi such as Candida albicans, which may lead to invasive candidiasis. Currently, it remains unknown whether depressed host defenses or fungal virulence drive the progression of burn wound candidiasis. Here we established an ex vivo burn wound model, where wounds were inflicted by applying preheated soldering iron to human skin explants, resulting in highly reproducible deep second-degree burn wounds. Eschar removal by debridement allowed for deeper C. albicans penetration into the burned tissue associated with prominent filamentation. Active migration of resident tissue neutrophils towards the damaged tissue and release of pro-inflammatory cytokine IL-1β accompanied the burn. The neutrophil recruitment was further increased upon supplementation of the model with fresh immune cells. Wound area and depth decreased over time, indicating healing of the damaged tissue. Importantly, prominent neutrophil presence at the infected site correlated to the limited penetration of C. albicans into the burned tissue. Altogether, we established a reproducible burn wound model of candidiasis using ex vivo human skin explants, where immune responses actively control the progression of infection and promote tissue healing.

We report a case of catheter associated bloodstream infection due to Enterobacter ludwigii with a massive aggregation on the outside surface of a central venous catheter (CVC). The 57 years old patient with a history of spondylodiscitis and Staphylococcus aureus-associated endocarditis was admitted to the intensive care unit for acute cerebral infarction. The patient developed signs of infections and the CVC was removed 11 days after placement. The infectious agent was identified by standard diagnostics to the genus level as belonging to the Enterobacter cloacae complex, and additional molecular testing determined the species as E. ludwigii. The catheter was selected for a study aiming to identify the influence of blood components on the formation of central venous catheter-associated biofilms. In this course a massive biofilm was recognized and is presented here.

Competing Interests: The authors have declared that no competing interests exist.

Typically, established lab strains are widely used to study host-pathogen interactions. However, to better reflect the infection process, the experimental use of clinical isolates has come more into focus. Here, we analyzed the interaction of multiple vaginal isolates of the opportunistic fungal pathogen Candida albicans , the most common cause of vulvovaginal candidiasis in women, with key players of the host immune system: macrophages. We tested several strains isolated from asymptomatic or symptomatic women with acute and recurrent infections. While all clinical strains showed a response similar to the commonly used lab strain SC5314 in various in vitro assays, they displayed remarkable differences during interaction with macrophages. This coincided with significantly reduced β-glucan exposure on the cell surface, which appeared to be a shared property among the tested vaginal strains for yeast extract/peptone/dextrose-grown cells, which is partly lost when the isolates faced vaginal niche-like nutrient conditions. However, macrophage damage, survival of phagocytosis, and filamentation capacities were highly strain-specific. These results highlight the high heterogeneity of C. albicans strains in host-pathogen interactions, which have to be taken into account to bridge the gap between laboratory-gained data and disease-related outcomes in an actual patient. IMPORTANCE Vulvovaginal candidiasis is one of the most common fungal infections in humans with Candida albicans as the major causative agent. This study is the first to compare clinical vaginal isolates of defined patient groups in their interaction with macrophages, highlighting the vastly different outcomes in comparison to a laboratory strain using commonly applied virulence-determining assays., (Copyright © 2020 Gerwien et al.)

The fungal pathogen Candida albicans forms polymorphic biofilms where hyphal morphogenesis and metabolic adaptation are tightly coordinated by a complex intertwined network of transcription factors. The sensing and metabolism of amino acids play important roles during various phases of biofilm development - from adhesion to maturation. Stp2 is a transcription factor that activates the expression of amino acid permease genes and is required for environmental alkalinization and hyphal growth in vitro and during macrophage phagocytosis. While it is well established that Stp2 is activated in response to external amino acids, its role in biofilm formation remains unknown. In addition to widely used techniques, we applied newly developed approaches for automated image analysis to quantify Stp2-regulated filamentation and biofilm growth. Our results show that in the stp2 Δ deletion mutant adherence to abiotic surfaces and initial germ tube formation were strongly impaired, but formed mature biofilms with cell density and morphological structures comparable to the control strains. Stp2-dependent nutrient adaptation appeared to play an important role in biofilm development: stp2 Δ biofilms formed under continuous nutrient flow displayed an overall reduction in biofilm formation, whereas under steady conditions the mutant strain formed biofilms with lower metabolic activity, resulting in increased cell survival and biofilm longevity. A deletion of STP2 led to increased rapamycin susceptibility and transcriptional activation of GCN4 , the transcriptional regulator of the general amino acid control pathway, demonstrating a connection of Stp2 to other nutrient-responsive pathways. In summary, the transcription factor Stp2 is important for C. albicans biofilm formation, where it contributes to adherence and induction of morphogenesis, and mediates nutrient adaption and cell longevity in mature biofilms., (Copyright © 2020 Böttcher, Hoffmann, Garbe, Weise, Cseresnyés, Brandt, Dietrich, Driesch, Figge and Vylkova.)

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1 , which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1 In contrast to NRG1 , overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1 In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies. IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans ., (Copyright © 2020 Ruben et al.)

The interaction of Candida albicans with the innate immune system is the key determinant of the pathogen/commensal balance and has selected for adaptations that facilitate the utilization of nutrients commonly found within the host, including proteins and amino acids; many of the catabolic pathways needed to assimilate these compounds are required for persistence in the host. We have shown that C. albicans co-opts amino acid catabolism to generate and excrete ammonia, which raises the extracellular pH, both in vitro and in vivo and induces hyphal morphogenesis. Mutants defective in the uptake or utilization of amino acids, such as those lacking STP2, a transcription factor that regulates the expression of amino acid permeases, are impaired in multiple aspects of fungus-macrophage interactions resulting from an inability to neutralize the phagosome. Here we identified a novel role in amino acid utilization for Ahr1p, a transcription factor previously implicated in regulation of adherence and hyphal morphogenesis. Mutants lacking AHR1 were defective in growth, alkalinization, and ammonia release on amino acid-rich media, similar to stp2Δ and ahr1Δ stp2Δ cells, and occupied more acidic phagosomes. Notably, ahr1Δ and stp2Δ strains did not induce pyroptosis, as measured by caspase-1-dependent interleukin-1β release, though this phenotype could be suppressed by pharmacological neutralization of the phagosome. Altogether, we show that C. albicans-driven neutralization of the phagosome promotes hyphal morphogenesis, sufficient for induction of caspase-1-mediated macrophage lysis., (Copyright © 2017 American Society for Microbiology.)

The opportunistic fungal pathogen Candida albicans thrives within diverse niches in the mammalian host. Among the adaptations that underlie this fitness is an ability to utilize a wide array of nutrients, especially sources of carbon that are disfavored by many other fungi; this contributes to its ability to survive interactions with the phagocytes that serve as key barriers against disseminated infections. We have reported that C. albicans generates ammonia as a byproduct of amino acid catabolism to neutralize the acidic phagolysosome and promote hyphal morphogenesis in a manner dependent on the Stp2 transcription factor. Here, we report that this species rapidly neutralizes acidic environments when utilizing carboxylic acids like pyruvate, α-ketoglutarate (αKG), or lactate as the primary carbon source. Unlike in cells growing in amino acid-rich medium, this does not result in ammonia release, does not induce hyphal differentiation, and is genetically distinct. While transcript profiling revealed significant similarities in gene expression in cells grown on either carboxylic or amino acids, genetic screens for mutants that fail to neutralize αKG medium identified a nonoverlapping set of genes, including CWT1, encoding a transcription factor responsive to cell wall and nitrosative stresses. Strains lacking CWT1 exhibit retarded αKG-mediated neutralization in vitro, exist in a more acidic phagolysosome, and are more susceptible to macrophage killing, while double cwt1Δ stp2Δ mutants are more impaired than either single mutant. Together, our observations indicate that C. albicans has evolved multiple ways to modulate the pH of host-relevant environments to promote its fitness as a pathogen., Importance: The fungal pathogen Candida albicans is a ubiquitous and usually benign constituent of the human microbial ecosystem. In individuals with weakened immune systems, this organism can cause potentially life-threatening infections and is one of the most common causes of hospital-acquired infections. Understanding the interactions between C. albicans and immune phagocytic cells, such as macrophages and neutrophils, will define the mechanisms of pathogenesis in this species. One such adaptation is an ability to make use of nonstandard nutrients that we predict are plentiful in certain niches within the host, including within these phagocytic cells. We show here that the metabolism of certain organic acids enables C. albicans to neutralize acidic environments, such as those within macrophages. This phenomenon is distinct in several significant ways from previous reports of similar processes, indicating that C. albicans has evolved multiple mechanisms to combat the harmful acidity of phagocytic cells., (Copyright © 2016 Danhof et al.)

Candida albicans, the most important fungal pathogen of humans, has a unique interaction with macrophages in which phagocytosis induces a switch from the yeast to hyphal form, allowing it to escape by rupturing the immune cell. While a variety of factors induce this switch in vitro, including neutral pH, it is not clear what triggers morphogenesis within the macrophage where the acidic environment should inhibit this transition. In vitro, C. albicans grown in similar conditions in which amino acids are the primary carbon source generate large quantities of ammonia to raise the extracellular pH and induce the hyphal switch. We show here that C. albicans cells neutralize the macrophage phagosome and that neutral pH is a key inducer of germination in phagocytosed cells by using a mutant lacking STP2, a transcription factor that regulates the expression of multiple amino acid permeases, that is completely deficient in alkalinization in vitro. Phagocytosed stp2Δ mutant cells showed significant reduction in hypha formation and escaped from macrophages less readily compared to wild type cells; as a result stp2Δ mutant cells were killed at a higher rate and caused less damage to RAW264.7 macrophages. Stp2p-regulated import leads to alkalinization of the phagosome, since the majority of the wild type cells fail to co-localize with acidophilic dyes, whereas the stp2Δ mutant cells were located in acidic phagosomes. Furthermore, stp2Δ mutant cells were able to form hyphae and escape from neutral phagosomes, indicating that the survival defect in these cells was pH dependent. Finally, these defects are reflected in an attenuation of virulence in a mouse model of disseminated candidiasis. Altogether our results suggest that C. albicans utilizes amino acids to promote neutralization of the phagosomal pH, hyphal morphogenesis, and escape from macrophages.

Unlabelled: pH homeostasis is critical for all organisms; in the fungal pathogen Candida albicans, pH adaptation is critical for virulence in distinct host niches. We demonstrate that beyond adaptation, C. albicans actively neutralizes the environment from either acidic or alkaline pHs. Under acidic conditions, this species can raise the pH from 4 to >7 in less than 12 h, resulting in autoinduction of the yeast-hyphal transition, a critical virulence trait. Extracellular alkalinization has been reported to occur in several fungal species, but under the specific conditions that we describe, the phenomenon is more rapid than previously observed. Alkalinization is linked to carbon deprivation, as it occurs in glucose-poor media and requires exogenous amino acids. These conditions are similar to those predicted to exist inside phagocytic cells, and we find a strong correlation between the use of amino acids as a cellular carbon source and the degree of alkalinization. Genetic and genomic approaches indicate an emphasis on amino acid uptake and catabolism in alkalinizing cells. Mutations in four genes, STP2, a transcription factor regulating amino acid permeases, ACH1 (acetyl-coenzyme A [acetyl-CoA] hydrolase), DUR1,2 (urea amidolyase), and ATO5, a putative ammonia transporter, abolish or delay neutralization. The pH changes are the result of the extrusion of ammonia, as observed in other fungi. We propose that nutrient-deprived C. albicans cells catabolize amino acids as a carbon source, excreting the amino nitrogen as ammonia to raise environmental pH and stimulate morphogenesis, thus directly contributing to pathogenesis., Importance: Candida albicans is the most important fungal pathogen of humans, causing disease at multiple body sites. The ability to switch between multiple morphologies, including a rounded yeast cell and an elongated hyphal cell, is a key virulence trait in this species, as this reversible switch is thought to promote dissemination and tissue invasion in the host. We report here that C. albicans can actively alter the pH of its environment and induce its switch to the hyphal form. The change in pH is caused by the release of ammonia from the cells produced during the breakdown of amino acids. This phenomenon is unprecedented in a human pathogen and may substantially impact host physiology by linking morphogenesis, pH adaptation, carbon metabolism, and interactions with host cells, all of which are critical for the ability of C. albicans to cause disease.

TRK proteins - essential potassium (K(+)) transporters in fungi and bacteria, as well as in plants - are generally absent from animal cells, which makes them potential targets for selective drug action. Indeed, in the human pathogen Candida albicans, the single TRK isoform (CaTrk1p) has recently been demonstrated to be required for activity of histidine-rich salivary antimicrobial peptides (histatins). Background for a detailed molecular investigation of TRK-protein design and function is provided here in sequence analysis and quantitative functional comparison of CaTrk1p with its better-known homologues from Saccharomyces cerevisiae. Among C. albicans strains (ATCC 10261, SC5314, WO-1), the DNA sequence is essentially devoid of single nucleotide polymorphisms in regions coding for evolutionarily conserved segments of the protein, meaning the four intramembranal [membrane-pore-membrane (MPM)] segments thought to be involved directly with the conduction of K(+) ions. Among 48 fungal (ascomycete) TRK homologues now described by complete sequences, clades (but not the detailed order within clades) appear conserved for all four MPM segments, independently assessed. The primary function of TRK proteins, 'active' transport of K(+) ions, is quantitatively conserved between C. albicans and S. cerevisiae. However, the secondary function, chloride efflux channeling, is present but poorly conserved between the two species, being highly variant with respect to activation velocity, amplitude, flickering (channel-like) behavior, pH dependence, and inhibitor sensitivity.

Acetyl coenzyme A (acetyl-CoA) is the central intermediate of the pathways required to metabolize nonfermentable carbon sources. Three such pathways, i.e., gluconeogenesis, the glyoxylate cycle, and beta-oxidation, are required for full virulence in the fungal pathogen Candida albicans. These processes are compartmentalized in the cytosol, mitochondria, and peroxosomes, necessitating transport of intermediates across intracellular membranes. Acetyl-CoA is trafficked in the form of acetate by the carnitine shuttle, and we hypothesized that the enzymes that convert acetyl-CoA to/from acetate, i.e., acetyl-CoA hydrolase (ACH1) and acetyl-CoA synthetase (ACS1 and ACS2), would regulate alternative carbon utilization and virulence. We show that C. albicans strains depleted for ACS2 are unviable in the presence of most carbon sources, including glucose, acetate, and ethanol; these strains metabolize only fatty acids and glycerol, a substantially more severe phenotype than that of Saccharomyces cerevisiae acs2 mutants. In contrast, deletion of ACS1 confers no phenotype, though it is highly induced in the presence of fatty acids, perhaps explaining why acs2 mutants can utilize fatty acids. Strains lacking ACH1 have a mild growth defect on some carbon sources but are fully virulent in a mouse model of disseminated candidiasis. Both ACH1 and ACS2 complement mutations in their S. cerevisiae homolog. Together, these results show that acetyl-CoA metabolism and transport are critical for growth of C. albicans on a wide variety of nutrients. Furthermore, the phenotypic differences between mutations in these highly conserved genes in S. cerevisiae and C. albicans support recent findings that significant functional divergence exists even in fundamental metabolic pathways between these related yeasts.

Histatin 5 (Hst 5) is a salivary cationic peptide that has toxicity for Candida albicans by inducing rapid cellular ion imbalance and cell volume loss. Microarray analyses of peptide-treated cells were used to evaluate global gene responses elicited by Hst 5. The major transcriptional response of C. albicans to Hst 5 was expression of genes involved in adaptation to osmotic stress, including production of glycerol (RHR2, SKO1, and PDC11) and the general stress response (CTA1 and HSP70). The oxidative-stress genes AHP1, TRX1, and GPX1 were mildly induced by Hst 5. Cell defense against Hst 5 was dependent on the Hog1 mitogen-activated protein kinase (MAPK) pathway, since C. albicans hog1/hog1 mutants were significantly hypersensitive to Hst 5 but not to Mkc1 MAPK or Cek1 MAPK mutants. Activation of the high-osmolarity glycerol (HOG) pathway was demonstrated by phosphorylation of Hog1 MAPK as well as by glycerol production following Hst 5 treatment in a dose-dependent manner. C. albicans cells prestressed with sorbitol were less sensitive to subsequent Hst 5 treatment; however, cells treated concurrently with osmotic stress and Hst 5 were hypersensitive to Hst 5. In contrast, cells subjected to oxidative stress had no difference in sensitivity to Hst 5. These results suggest a common underlying cellular response to osmotic stress and Hst 5. The HOG stress response pathway likely represents a significant and effective challenge to physiological levels of Hst 5 and other toxic peptides in fungal cells.

A unifying theme common to the action of many cationic peptides that display lethal activities against microbial pathogens is their specific action at microbial membranes that results in selective loss of ions and small nucleotides-chiefly ATP. One model cationic peptide that induces non-lytic release of ATP from the fungal pathogen Candida albicans is salivary histatin 5 (Hst 5). The major characteristic of Hst 5-induced ATP release is that it occurs rapidly while cells are still metabolically active and have polarized membranes, thus precluding cell lysis as the means of release of ATP. Other cationic peptides that induce selective release of ATP from target microbes are lactoferricin, human neutrophil defensins, bactenecin, and cathelicidin peptides. The role of released extracellular ATP induced by cationic peptides is not known, but localized increases in extracellular ATP concentration may serve to potentiate cell killing, facilitate further peptide uptake, or function as an additional signal to activate the host innate immune system at the site of infection.

Human beta-defensin 2 (hBD-2) and hBD-3 have potent fungicidal activity in the micromolar range. Although little is known about their mechanism of action against Candida species, some similarities to the antifungal mechanism of salivary peptide histatin 5 (Hst 5) seem to exist. Since hBD-2 and hBD-3 have been reported to cause direct disruption of target cell membranes, we compared the effects of hBD-2 and hBD-3 on Candida albicans membrane integrity. Incubation of calcein-loaded C. albicans cells with a dose of hBD-2 lethal for 90% of the strains tested (LD(90)) resulted in a maximal dye efflux of only 10.3% +/- 2.8% at 90 min, similar to that induced by Hst 5. In contrast, an LD(90) of hBD-3 more than doubled calcein release from cells yet did not result in more than 24% of total release, showing that neither peptide caused gross membrane damage. As for Hst 5, killing of C. albicans cells by hBD-2 and hBD-3 was salt sensitive; however, Ca(2+) and Mg(2+) inhibited hBD-2 but not hBD-3 fungicidal activity. Pretreatment of C. albicans cells with sodium azide resulted in significantly decreased ATP release and susceptibility of cells to hBD-2 and hBD-3. However, hBD-3 killing was partially restored at concentrations of > or =0.8 microM, showing energy-independent mechanisms at higher doses. C. glabrata resistance to Hst 5, hBD-2, and hBD-3 is not a result of loss of expression of cell wall Ssa proteins. The candidacidal effects of hBD-2-hBD-3 and Hst 5-hBD-2 were additive, while the index of interaction between Hst 5 and hBD-3 was 0.717 (P < 0.05). Thus, the candidacidal action of hBD-2 shows many similarities to that of Hst 5 in terms of salt sensitivity, ion selectivity, and energy requirements while hBD-3 exhibits biphasic concentration-dependent mechanisms of candidacidal action complementary to those of Hst 5.

Salivary histatin 5 (Hst 5) kills the fungal pathogen Candida albicans via a multistep process which includes binding to Ssa1/2 proteins on the cell surface and requires the TRK1 potassium transporter. Hst 5-induced membrane permeability to propidium iodide (PI) was nearly abolished in strain CaTK1 (TRK1/trk1), suggesting that Hst 5-induced influx of PI is via Trk1p. To explore the functional role of Trk1p in the mechanism of other antifungal peptides, we evaluated candidacidal activity and PI uptake in wild-type strain CaTK2 (TRK1/TRK1) and strain CaTK1 following treatment with lactoferricin 11 (LFcn 11), bactenecin 16 (BN 16), and virion-associated protein VPR 12. Strain CaTK1 was resistant to killing with these peptides (VPR 12 > LFcn 11 > BN 16), showing the requirement of Trk1p for fungicidal activity. In contrast, human neutrophil defensin 1 (HNP-1), human beta-defensin 2 (hBD-2), and hBD-3 effects on viability of and membrane permeability to PI were not different between mutant and wild-type strains, clearly showing that their candidacidal mechanism does not involve Trk1p as a functional effector. To test whether defensins require binding to Candida surface Ssa1/2 proteins for their activity, we measured the killing effectiveness in SSA1/2 mutant strains. Both hBD-2 and hBD-3, but not HNP-1, exhibited reduced killing of ssa1Delta and ssa2Delta strains compared to the wild type, showing that Ssa1 and Ssa2 proteins are required for their fungicidal activity. These results demonstrate that (i) Trk1p mediates candidacidal activities of cysteine-free peptides, but not of defensins, and (ii) hBD-2 and hBD-3, but not HNP-1, require Ssa1/2p for antifungal activity.

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K(+), which can be blocked by the anion channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K(+) transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K(+) ((86)Rb(+)) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5-7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.

Salivary histatin 5 (Hst 5), a potent toxin for the human fungal pathogen Candida albicans, induces noncytolytic efflux of cellular ATP, potassium, and magnesium in the absence of cytolysis, implicating these ion movements in the toxin's fungicidal activity. Hst 5 action on Candida resembles, in many respects, the action of the K1 killer toxin on Saccharomyces cerevisiae, and in that system the yeast plasma membrane potassium channel, Tok1p, has recently been reported to be a primary target of toxin action. The question of whether the Candida homologue of Saccharomyces Tok1p might be a primary target of Hst 5 action has now been investigated by disruption of the C. albicans TOK1 gene. The resultant strains (TOK1/tok1) and (tok1/tok1) were compared with wild-type Candida (TOK1/TOK1) for relative ATP leakage and killing in response to Hst 5. Patch-clamp measurements on Candida protoplasts were used to verify the functional deletion of Tok1p and to provide its first description in Candida. Tok1p is an outwardly rectifying, noisily gated, 40-pS channel, very similar to that described in Saccharomyces. Knockout of CaTOK1 (tok1/tok1) completely abolishes the currents and gating events characteristic of Tok1p. Also, knockout (tok1/tok1) increases residual viability of Candida after Hst 5 treatment to 27%, from 7% in the wild type, while the single allele deletion (TOK1/tok1) increases viability to 18%. Comparable results were obtained for Hst-induced ATP efflux, but quantitative features of ATP loss suggest that wild-type TOK1 genes function cooperatively. Overall, very substantial killing and ATP efflux are produced by Hst 5 treatment after complete knockout of wild-type TOK1, making clear that Tok1p channels are not the primary site of Hst 5 action, even though they do play a modulating role.