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Host cell damage is a key parameter for research in infection biology, drug testing, and substance safety screening. In this study, we introduce a luciferase reporter system as a new and reliable assay to measure cell damage and validate it with the pathogenic yeast, Candida albicans , as a test case. We transduced human epithelial cell lines with a lentiviral vector to stably express an optimized luciferase enzyme, Nanoluc. Upon cell damage, the release of cytoplasmic luciferase into the extracellular space can be easily detected by a luminometer. We used the luciferase reporter system to investigate the damage caused by C. albicans to different newly generated epithelial reporter cell lines. We found that fungus-induced cell damage, as determined by established methods, correlated tightly with the release of the luciferase. The new luciferase reporter system is a simple, sensitive, robust, and inexpensive method for measuring host cell damage and has a sensitivity comparable to the standard assay, release of lactate dehydrogenase. It is suitable for high-throughput studies of pathogenesis mechanisms of any microbe, for antimicrobial drug screening, and many other applications.IMPORTANCEWe present a quick, easy, inexpensive, and reliable assay to measure damage to mammalian cells. To this end, we created reporter cell lines which artificially express luciferase, an enzyme that can be easily detected in the supernatant when these cells are damaged. We used infections with the well-investigated fungal pathogen of humans, Candida albicans , as a test case of our system. Using our reporter, we were able to recapitulate the known effects of strain variability, gene deletions, and antifungal treatments on host cell damage. This easily adaptable reporter system can be used to screen for damage in infection models with different microbial species, assay cell-damaging potential of substances, discover new non-toxic antibiotics, and many other damage-based applications., Competing Interests: Work from this paper has been used to register the patent "Cytotoxicity assay for detecting cellular damage" (L31002DE) with the Deutsches Patent-und Markenamt authors R.V., M.T., V.T., S.B., and B.H. and Leibniz-HKI as beneficiaries.

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans , and the related species C. dubliniensis , and C tropicalis , suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C . albicans isolates, 10 C . dubliniensis isolates, and 78 C . tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species., Importance: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis , thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans , C. dubliniensis , and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity., Competing Interests: The authors declare no conflict of interest.

SUMMARYA significant increase in the incidence of Candida -mediated infections has been observed in the last decade, mainly due to rising numbers of susceptible individuals. Recently, the World Health Organization published its first fungal pathogen priority list, with Candida species listed in medium, high, and critical priority categories. This review is a synthesis of information and recent advances in our understanding of two of these species -Candida albicans and Candida glabrata . Of these, C. albicans is the most common cause of candidemia around the world and is categorized as a critical priority pathogen. C. glabrata is considered a high-priority pathogen and has become an increasingly important cause of candidemia in recent years. It is now the second most common causative agent of candidemia in many geographical regions. Despite their differences and phylogenetic divergence, they are successful as pathogens and commensals of humans. Both species can cause a broad variety of infections, ranging from superficial to potentially lethal systemic infections. While they share similarities in certain infection strategies, including tissue adhesion and invasion, they differ significantly in key aspects of their biology, interaction with immune cells, host damage strategies, and metabolic adaptations. Here we provide insights on key aspects of their biology, epidemiology, commensal and pathogenic lifestyles, interactions with the immune system, and antifungal resistance., Competing Interests: The authors declare no conflict of interest.

Candida albicans can cause mucosal infections in humans. This includes oropharyngeal candidiasis, which is commonly observed in human immunodeficiency virus infected patients, and vulvovaginal candidiasis (VVC), which is the most frequent manifestation of candidiasis. Epithelial cell invasion by C. albicans hyphae is accompanied by the secretion of candidalysin, a peptide toxin that causes epithelial cell cytotoxicity. During vaginal infections, candidalysin-driven tissue damage triggers epithelial signaling pathways, leading to hyperinflammatory responses and immunopathology, a hallmark of VVC. Therefore, we proposed blocking candidalysin activity using nanobodies to reduce epithelial damage and inflammation as a therapeutic strategy for VVC. Anti-candidalysin nanobodies were confirmed to localize around epithelial-invading C. albicans hyphae, even within the invasion pocket where candidalysin is secreted. The nanobodies reduced candidalysin-induced damage to epithelial cells and downstream proinflammatory responses. Accordingly, the nanobodies also decreased neutrophil activation and recruitment. In silico mathematical modeling enabled the quantification of epithelial damage caused by candidalysin under various nanobody dosing strategies. Thus, nanobody-mediated neutralization of candidalysin offers a novel therapeutic approach to block immunopathogenic events during VVC and alleviate symptoms.IMPORTANCEWorldwide, vaginal infections caused by Candida albicans (VVC) annually affect millions of women, with symptoms significantly impacting quality of life. Current treatments are based on anti-fungals and probiotics that target the fungus. However, in some cases, infections are recurrent, called recurrent VVC, which often fails to respond to treatment. Vaginal mucosal tissue damage caused by the C. albicans peptide toxin candidalysin is a key driver in the induction of hyperinflammatory responses that fail to clear the infection and contribute to immunopathology and disease severity. In this pre-clinical evaluation, we show that nanobody-mediated candidalysin neutralization reduces tissue damage and thereby limits inflammation. Implementation of candidalysin-neutralizing nanobodies may prove an attractive strategy to alleviate symptoms in complicated VVC cases., Competing Interests: E.D. is the CEO of Q.V.Q who produced the anti-candidalysin nanobodies.

Importance: Candida albicans is a leading human fungal pathogen that often causes life-threatening infections in immunocompromised individuals. The ability of C. albicans to transition between yeast and filamentous forms is key to its virulence, and this occurs in response to many host-relevant cues, including engulfment by host macrophages. While previous efforts identified C. albicans genes required for filamentation in other conditions, the genes important for this morphological transition upon internalization by macrophages remained largely enigmatic. Here, we employed a functional genomic approach to identify genes that enable C. albicans filamentation within macrophages and uncovered a role for the mitochondrial ribosome, respiration, and the SNF1 AMP-activated kinase complex. Additionally, we showed that glucose uptake and glycolysis by macrophages support C. albicans filamentation. This work provides insights into the metabolic dueling that occurs during the interaction of C. albicans with macrophages and identifies vulnerabilities in C. albicans that could serve as promising therapeutic targets., Competing Interests: L.E.C. and L.W. are co-founders and shareholders in Bright Angel Therapeutics, a platform company for the development of novel antifungal therapeutics. L.E.C. is a science advisor for Kapoose Creek, a company that harnesses the therapeutic potential of fungi. S.D.L. is currently employed by Bright Angel Therapeutics. All other authors have no competing interests to report.

Candidalysin is the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is secreted by Candida albicans and is critical for driving infection and host immune responses in several model systems. However, Candida infections are also caused by non-C. albicans species. Here, we identify and characterize orthologs of C. albicans candidalysin in C. dubliniensis and C. tropicalis. The candidalysins have different amino acid sequences, are amphipathic, and adopt a predominantly α-helical secondary structure in solution. Comparative functional analysis demonstrates that each candidalysin causes epithelial damage and calcium influx and activates intracellular signaling pathways and cytokine secretion. Importantly, C. dubliniensis and C. tropicalis candidalysins have higher damaging and activation potential than C. albicans candidalysin and exhibit more rapid membrane binding and disruption, although both fungal species cause less damage to epithelial cells than C. albicans. This study identifies the first family of peptide cytolysins in human-pathogenic fungi. IMPORTANCE Pathogenic fungi kill an estimated 1.5 million people every year. Recently, we discovered that the fungal pathogen Candida albicans secretes a peptide toxin called candidalysin during mucosal infection. Candidalysin causes damage to host cells, a process that supports disease progression. However, fungal infections are also caused by Candida species other than C. albicans. In this work, we identify and characterize two additional candidalysin toxins present in the related fungal pathogens C. dubliniensis and C. tropicalis. While the three candidalysins have different amino acid sequences, all three toxins are α-helical and amphipathic. Notably, the candidalysins from C. dubliniensis and C. tropicalis are more potent at inducing cell damage, calcium influx, mitogen-activated protein kinase signaling, and cytokine responses than C. albicans candidalysin, with the C. dubliniensis candidalysin having the most rapid membrane binding kinetics. These observations identify the candidalysins as the first family of peptide toxins in human-pathogenic fungi.

Albumin is abundant in serum but is also excreted at mucosal surfaces and enters tissues when inflammation increases vascular permeability. Host-associated opportunistic pathogens encounter albumin during commensalism and when causing infections. Considering the ubiquitous presence of albumin, we investigated its role in the pathogenesis of infections with the model human fungal pathogen, Candida albicans. Albumin was introduced in various in vitro models that mimic different stages of systemic or mucosal candidiasis, where it reduced the ability of C. albicans to damage host cells. The amphipathic toxin candidalysin mediates necrotic host cell damage induced by C. albicans. Using cellular and biophysical assays, we determined that albumin functions by neutralizing candidalysin through hydrophobic interactions. We discovered that albumin, similarly, can neutralize a variety of fungal (α-amanitin), bacterial (streptolysin O and staurosporin), and insect (melittin) hydrophobic toxins. These data suggest albumin as a defense mechanism against toxins, which can play a role in the pathogenesis of microbial infections. IMPORTANCE Albumin is the most abundant serum protein in humans. During inflammation, serum albumin levels decrease drastically, and low albumin levels are associated with poor patient outcome. Thus, albumin may have specific functions during infection. Here, we describe the ability of albumin to neutralize hydrophobic microbial toxins. We show that albumin can protect against damage induced by the pathogenic yeast C. albicans by neutralizing its cytolytic toxin candidalysin. These findings suggest that albumin is a toxin-neutralizing protein that may play a role during infections with toxin-producing microorganisms.

Loss or inactivation of antivirulence genes is an adaptive strategy in pathogen evolution. Candida glabrata is an important opportunistic pathogen related to baker's yeast, with the ability to both quickly increase its intrinsic high level of azole resistance and persist within phagocytes. During C. glabrata's evolution as a pathogen, the mitochondrial DNA polymerase CgMip1 has been under positive selection. We show that CgMIP1 deletion not only triggers loss of mitochondrial function and a petite phenotype, but increases C. glabrata's azole and endoplasmic reticulum (ER) stress resistance and, importantly, its survival in phagocytes. The same phenotype is induced by fluconazole and by exposure to macrophages, conferring a cross-resistance between antifungals and immune cells, and can be found in clinical isolates despite a slow growth of petite strains. This suggests that petite constitutes a bet-hedging strategy of C. glabrata and, potentially, a relevant cause of azole resistance. Mitochondrial function may therefore be considered a potential antivirulence factor. IMPORTANCE Candida glabrata is an opportunistic pathogen whose incidence has been increasing in the last 40 years. It has risen to become the most prominent non-Candida albicans Candida (NCAC) species to cause candidemia, constituting about one-third of isolates in the United States, and steadily increasing in European countries and in Australia. Despite its clinical importance, C. glabrata's pathogenicity strategies remain poorly understood. Our research shows that loss of mitochondrial function and the resulting petite phenotype is advantageous for C. glabrata to cope with infection-related stressors, such as antifungals and host immune defenses. The (cross-)resistance against both these factors may have major implications in the clinical outcome of infections with this major fungal pathogen.

Only four species, Candida albicans , C. glabrata , C. parapsilosis , and C. tropicalis , together account for about 90% of all Candida bloodstream infections and are among the most common causes of invasive fungal infections of humans. However, virulence potential varies among these species, and the phylogenetic tree reveals that their pathogenicity may have emerged several times independently during evolution. We therefore tested these four species in a human whole-blood infection model to determine, via comprehensive dual-species RNA-sequencing analyses, which fungal infection strategies are conserved and which are recent evolutionary developments. The ex vivo infection progressed from initial immune cell interactions to nearly complete killing of all fungal cells. During the course of infection, we characterized important parameters of pathogen-host interactions, such as fungal survival, types of interacting immune cells, and cytokine release. On the transcriptional level, we obtained a predominantly uniform and species-independent human response governed by a strong upregulation of proinflammatory processes, which was downregulated at later time points after most of the fungal cells were killed. In stark contrast, we observed that the different fungal species pursued predominantly individual strategies and showed significantly different global transcriptome patterns. Among other findings, our functional analyses revealed that the fungal species relied on different metabolic pathways and virulence factors to survive the host-imposed stress. These data show that adaptation of Candida species as a response to the host is not a phylogenetic trait, but rather has likely evolved independently as a prerequisite to cause human infections. IMPORTANCE To ensure their survival, pathogens have to adapt immediately to new environments in their hosts, for example, during the transition from the gut to the bloodstream. Here, we investigated the basis of this adaptation in a group of fungal species which are among the most common causes of hospital-acquired infections, the Candida species. On the basis of a human whole-blood infection model, we studied which genes and processes are active over the course of an infection in both the host and four different Candida pathogens. Remarkably, we found that, while the human host response during the early phase of infection is predominantly uniform, the pathogens pursue largely individual strategies and each one regulates genes involved in largely disparate processes in the blood. Our results reveal that C. albicans , C. glabrata , C. parapsilosis , and C. tropicalis all have developed individual strategies for survival in the host. This indicates that their pathogenicity in humans has evolved several times independently and that genes which are central for survival in the host for one species may be irrelevant in another., (Copyright © 2020 Kämmer et al.)

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.)

Jagaricin is a lipopeptide produced by the bacterial mushroom pathogen Janthinobacterium agaricidamnosum , the causative agent of mushroom soft rot disease. Apart from causing lesions in mushrooms, jagaricin is a potent antifungal active against human-pathogenic fungi. We show that jagaricin acts by impairing membrane integrity, resulting in a rapid flux of ions, including Ca 2+ , into susceptible target cells. Accordingly, the calcineurin pathway is required for jagaricin tolerance in the fungal pathogen Candida albicans Transcriptional profiling of pathogenic yeasts further revealed that jagaricin triggers cell wall strengthening, general shutdown of membrane potential-driven transport, and the upregulation of lipid transporters, linking cell envelope integrity to jagaricin action and resistance. Whereas jagaricin shows hemolytic effects, it exhibited either no or low plant toxicity at concentrations at which the growth of prevalent phytopathogenic fungi is inhibited. Therefore, jagaricin may have potential for agricultural applications. The action of jagaricin as a membrane-disrupting antifungal is promising but would require modifications for use in humans., (Copyright © 2019 American Society for Microbiology.)

Macrophages rely on phagosomal acidity to destroy engulfed microorganisms. To survive this hostile response, opportunistic fungi such as Candida albicans developed strategies to evade the acidic environment. C. albicans is polymorphic and able to convert from yeast to hyphae, and this transition is required to subvert the microbicidal activity of the phagosome. However, the phagosomal lumen, which is acidic and nutrient deprived, is believed to inhibit the yeast-to-hypha transition. To account for this apparent paradox, it was recently proposed that C. albicans produces ammonia that alkalinizes the phagosome, thus facilitating yeast-to-hypha transition. We reexamined the mechanism underlying phagosomal alkalinization by applying dual-wavelength ratiometric pH measurements. The phagosomal membrane was found to be highly permeable to ammonia, which is therefore unlikely to account for the pH elevation. Instead, we find that yeast-to-hypha transition begins within acidic phagosomes and that alkalinization is a consequence of proton leakage induced by excessive membrane distension caused by the expanding hypha. IMPORTANCE C. albicans is the most common cause of nosocomial fungal infection, and over 3 million people acquire life-threatening invasive fungal infections every year. Even if antifungal drugs exist, almost half of these patients will die. Despite this, fungi remain underestimated as pathogens. Our study uses quantitative biophysical approaches to demonstrate that yeast-to-hypha transition occurs within the nutrient-deprived, acidic phagosome and that alkalinization is a consequence, as opposed to the cause, of hyphal growth., (Copyright © 2018 Westman et al.)

Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans , a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans , candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. IMPORTANCE Candida albicans , usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer., (Copyright © 2018 Allert et al.)

Candida albicans is an opportunistic fungal pathogen responsible for superficial and life-threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete candidalysin, a 31-amino-acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein 1 (AP-1) transcription factor c-Fos (via p38-mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]-MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational processing of Ece1p to produce candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature candidalysin. C. albicans strains harboring mutations of Arg61 and/or Arg93 did not secrete candidalysin, were unable to induce epithelial damage and inflammatory responses in vitro , and showed attenuated virulence in vivo in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of C. albicans Ece1p by kexin-like proteinases as crucial steps required for candidalysin production and fungal pathogenicity. IMPORTANCE Candida albicans is an opportunistic fungal pathogen that causes mucosal infection in millions of individuals worldwide. Successful infection requires the secretion of candidalysin, the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is derived from its parent protein Ece1p. Here, we identify two key amino acids within Ece1p vital for processing and production of candidalysin. Mutations of these residues render C. albicans incapable of causing epithelial damage and markedly reduce mucosal infection in vivo Importantly, candidalysin production requires two individual enzymatic events. The first involves processing of Ece1p by Kex2p, yielding immature candidalysin, which is then further processed by Kex1p to produce the mature toxin. These observations identify important steps for C. albicans pathogenicity at mucosal surfaces., (Copyright © 2018 Richardson et al.)

Unlike other forms of candidiasis, vulvovaginal candidiasis, caused primarily by the fungal pathogen Candida albicans , is a disease of immunocompetent and otherwise healthy women. Despite its prevalence, the fungal factors responsible for initiating symptomatic infection remain poorly understood. One of the hallmarks of vaginal candidiasis is the robust recruitment of neutrophils to the site of infection, which seemingly do not clear the fungus, but rather exacerbate disease symptomatology. Candidalysin, a newly discovered peptide toxin secreted by C. albicans hyphae during invasion, drives epithelial damage, immune activation, and phagocyte attraction. Therefore, we hypothesized that Candidalysin is crucial for vulvovaginal candidiasis immunopathology. Anti- Candida immune responses are anatomical-site specific, as effective gastrointestinal, oral, and vaginal immunities are uniquely compartmentalized. Thus, we aimed to identify the immunopathologic role of Candidalysin and downstream signaling events at the vaginal mucosa. Microarray analysis of C. albicans -infected human vaginal epithelium in vitro revealed signaling pathways involved in epithelial damage responses, barrier repair, and leukocyte activation. Moreover, treatment of A431 vaginal epithelial cells with Candidalysin induced dose-dependent proinflammatory cytokine responses (including interleukin 1α [IL-1α], IL-1β, and IL-8), damage, and activation of c-Fos and mitogen-activated protein kinase (MAPK) signaling, consistent with fungal challenge. Mice intravaginally challenged with C. albicans strains deficient in Candidalysin exhibited no differences in colonization compared to isogenic controls. However, significant decreases in neutrophil recruitment, damage, and proinflammatory cytokine expression were observed with these strains. Our findings demonstrate that Candidalysin is a key hypha-associated virulence determinant responsible for the immunopathogenesis of C. albicans vaginitis., (Copyright © 2018 American Society for Microbiology.)

Iron is an essential micronutrient for both pathogens and their hosts, which restrict iron availability during infections in an effort to prevent microbial growth. Successful human pathogens like the yeast Candida glabrata have thus developed effective iron acquisition strategies. Their regulation has been investigated well for some pathogenic fungi and in the model organism Saccharomyces cerevisiae, which employs an evolutionarily derived system. Here, we show that C. glabrata uses a regulation network largely consisting of components of the S. cerevisiae regulon but also of elements of other pathogenic fungi. Specifically, similarly to baker's yeast, Aft1 is the main positive regulator under iron starvation conditions, while Cth2 degrades mRNAs encoding iron-requiring enzymes. However, unlike the case with S. cerevisiae, a Sef1 ortholog is required for full growth under iron limitation conditions, making C. glabrata an evolutionary intermediate to SEF1-dependent fungal pathogens. Therefore, C. glabrata has evolved an iron homeostasis system which seems to be unique within the pathogenic fungi., Importance: The fungus Candida glabrata represents an evolutionarily close relative of the well-studied and benign baker's yeast and model organism Saccharomyces cerevisiae On the other hand, C. glabrata is an important opportunistic human pathogen causing both superficial and systemic infections. The ability to acquire trace metals, in particular, iron, and to tightly regulate this process during infection is considered an important virulence attribute of a variety of pathogens. Importantly, S. cerevisiae uses a highly derivative regulatory system distinct from those of other fungi. Until now, the regulatory mechanism of iron homeostasis in C. glabrata has been mostly unknown. Our study revealed a hybrid iron regulation network that is unique to C. glabrata and is placed at an evolutionary midpoint between those of S. cerevisiae and related fungal pathogens. We thereby show that, in the host, even a successful human pathogen can rely largely on a strategy normally found in nonpathogenic fungi from a terrestrial environment., (Copyright © 2016 Gerwien et al.)

Unlabelled: Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation., Importance: Biofilm formation by the opportunistic fungal pathogen C. albicans is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by C. albicans biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during C. albicans biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from C. albicans and also from additional pathogenic Candida species. Furthermore, SAP5 and SAP6 deletions confirm that both proteases are required for proper biofilm development in vitro and in vivo We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of Candida biofilm-associated infections., (Copyright © 2016 Winter et al.)

The supply and intracellular homeostasis of trace metals are essential for every living organism. Therefore, the struggle for micronutrients between a pathogen and its host is an important determinant in the infection process. In this work, we focus on the acquisition of zinc by Candida dubliniensis, an emerging pathogen closely related to Candida albicans. We show that the transcription factor Csr1 is essential for C. dubliniensis to regulate zinc uptake mechanisms under zinc limitation: it governs the expression of the zinc transporter genes ZRT1, ZRT2, and ZRT3 and of the zincophore gene PRA1. Exclusively, artificial overexpression of ZRT2 partially rescued the growth defect of a csr1Δ/Δ mutant in a zinc-restricted environment. Importantly, we found that, in contrast to what is seen in C. albicans, Csr1 (also called Zap1) is not a major regulator of dimorphism in C. dubliniensis. However, although a csr1Δ/Δ strain showed normal germ tube formation, we detected a clear attenuation in virulence using an embryonated chicken egg infection model. We conclude that, unlike in C. albicans, Csr1 seems to be a virulence factor of C. dubliniensis that is not coupled to filamentation but is strongly linked to zinc acquisition during pathogenesis., (Copyright © 2015, Böttcher et al.)

Unlabelled: Vaginal inflammation (vaginitis) is the most common disease caused by the human-pathogenic fungus Candida albicans. Secretory aspartyl proteinases (Sap) are major virulence traits of C. albicans that have been suggested to play a role in vaginitis. To dissect the mechanisms by which Sap play this role, Sap2, a dominantly expressed member of the Sap family and a putative constituent of an anti-Candida vaccine, was used. Injection of full-length Sap2 into the mouse vagina caused local neutrophil influx and accumulation of the inflammasome-dependent interleukin-1β (IL-1β) but not of inflammasome-independent tumor necrosis factor alpha. Sap2 could be replaced by other Sap, while no inflammation was induced by the vaccine antigen, the N-terminal-truncated, enzymatically inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library, inhibited or abolished the inflammatory response, provided the antibodies were able, like the Sap inhibitor Pepstatin A, to inhibit Sap enzyme activity. The same antibodies and Pepstatin A also inhibited neutrophil influx and cytokine production stimulated by C. albicans intravaginal injection, and a mutant strain lacking SAP1, SAP2, and SAP3 was unable to cause vaginal inflammation. Sap2 induced expression of activated caspase-1 in murine and human vaginal epithelial cells. Caspase-1 inhibition downregulated IL-1β and IL-18 production by vaginal epithelial cells, and blockade of the IL-1β receptor strongly reduced neutrophil influx. Overall, the data suggest that some Sap, particularly Sap2, are proinflammatory proteins in vivo and can mediate the inflammasome-dependent, acute inflammatory response of vaginal epithelial cells to C. albicans. These findings support the notion that vaccine-induced or passively administered anti-Sap antibodies could contribute to control vaginitis., Importance: Candidal vaginitis is an acute inflammatory disease that affects many women of fertile age, with no definitive cure and, in its recurrent forms, causing true devastation of quality of life. Unraveling the fungal factors causing inflammation is important to be able to devise novel tools to fight the disease. In an experimental murine model, we have discovered that aspartyl proteinases, particularly Sap2, may cause the same inflammatory signs of vaginitis caused by the fungus and that anti-Sap antibodies and the protease inhibitor Pepstatin A almost equally inhibit Sap- and C. albicans-induced inflammation. Sap-induced vaginitis is an early event during vaginal infection, is uncoupled from fungal growth, and requires Sap and caspase-1 enzymatic activities to occur, suggesting that Sap or products of Sap activity activate an inflammasome sensor of epithelial cells. Our data support the notion that anti-Sap antibodies could help control the essence of candidal vaginitis, i.e., the inflammatory response., (Copyright © 2015 Pericolini et al.)

We recently demonstrated that the secreted aspartyl proteinases (Saps), Sap2 and Sap6, of Candida albicans have the potential to induce the canonical activation of the NLRP3 inflammasome, leading to the secretion of interleukin-1β (IL-1β) and IL-18 via caspase-1 activation. We also observed that the activation of caspase-1 is partially independent from the NLRP3 activation pathway. In this study, we examined whether Sap2 and Sap6 are also able to activate the noncanonical inflammasome pathway in murine macrophages. Our data show that both Sap2 and Sap6 can activate caspase-11 through type I interferon (IFN) production. Caspase-11 cooperates to activate caspase-1, with a subsequent increase of IL-1β secretion. Endocytosis and internalization of Saps are required for the induction of type I IFN production, which is essential for induction of noncanonical inflammasome activation. Our study indicates a sophisticated interplay between caspase-1 and caspase-11 that connects the canonical and noncanonical pathways of inflammasome activation in response to C. albicans Saps., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Human fungal pathogens are distributed throughout their kingdom, suggesting that pathogenic potential evolved independently. Candida albicans is the most virulent member of the CUG clade of yeasts and a common cause of both superficial and invasive infections. We therefore hypothesized that C. albicans possesses distinct pathogenicity mechanisms. In silico genome subtraction and comparative transcriptional analysis identified a total of 65 C. albicans-specific genes (ASGs) expressed during infection. Phenotypic characterization of six ASG-null mutants demonstrated that these genes are dispensable for in vitro growth but play defined roles in host-pathogen interactions. Based on these analyses, we investigated two ASGs in greater detail. An orf19.6688Δ mutant was found to be fully virulent in a mouse model of disseminated candidiasis and to induce higher levels of the proinflammatory cytokine interleukin-1β (IL-1β) following incubation with murine macrophages. A pga16Δ mutant, on the other hand, exhibited attenuated virulence. Moreover, we provide evidence that secondary filamentation events (multiple hyphae emerging from a mother cell and hyphal branching) contribute to pathogenicity: PGA16 deletion did not influence primary hypha formation or extension following contact with epithelial cells; however, multiple hyphae and hyphal branching were strongly reduced. Significantly, these hyphae failed to damage host cells as effectively as the multiple hypha structures formed by wild-type C. albicans cells. Together, our data show that species-specific genes of a eukaryotic pathogen can play important roles in pathogenicity., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The ability to acquire nutrients during infections is an important attribute in microbial pathogenesis. Amino acids are a valuable source of nitrogen if they can be degraded by the infecting organism. In this work, we analyzed histidine utilization in the fungal pathogen of humans Candida glabrata. Hemiascomycete fungi, like C. glabrata or Saccharomyces cerevisiae, possess no gene coding for a histidine ammonia-lyase, which catalyzes the first step of a major histidine degradation pathway in most other organisms. We show that C. glabrata instead initializes histidine degradation via the aromatic amino acid aminotransferase Aro8. Although ARO8 is also present in S. cerevisiae and is induced by extracellular histidine, the yeast cannot use histidine as its sole nitrogen source, possibly due to growth inhibition by a downstream degradation product. Furthermore, C. glabrata relies only on Aro8 for phenylalanine and tryptophan utilization, since ARO8, but not its homologue ARO9, was transcriptionally activated in the presence of these amino acids. Accordingly, an ARO9 deletion had no effect on growth with aromatic amino acids. In contrast, in S. cerevisiae, ARO9 is strongly induced by tryptophan and is known to support growth on aromatic amino acids. Differences in the genomic structure of the ARO9 gene between C. glabrata and S. cerevisiae indicate a possible disruption in the regulatory upstream region. Thus, we show that, in contrast to S. cerevisiae, C. glabrata has adapted to use histidine as a sole source of nitrogen and that the aromatic amino acid aminotransferase Aro8, but not Aro9, is the enzyme required for this process., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Candida glabrata is both a human fungal commensal and an opportunistic pathogen which can withstand activities of the immune system. For example, C. glabrata can survive phagocytosis and replicates within macrophages. However, the mechanisms underlying intracellular survival remain unclear. In this work, we used a functional genomic approach to identify C. glabrata determinants necessary for survival within human monocyte-derived macrophages by screening a set of 433 deletion mutants. We identified 23 genes which are required to resist killing by macrophages. Based on homologies to Saccharomyces cerevisiae orthologs, these genes are putatively involved in cell wall biosynthesis, calcium homeostasis, nutritional and stress response, protein glycosylation, or iron homeostasis. Mutants were further characterized using a series of in vitro assays to elucidate the genes' functions in survival. We investigated different parameters of C. glabrata-phagocyte interactions: uptake by macrophages, replication within macrophages, phagosomal pH, and recognition of mutant cells by macrophages as indicated by production of reactive oxygen species and tumor necrosis factor alpha (TNF-α). We further studied the cell surface integrity of mutant cells, their ability to grow under nutrient-limited conditions, and their susceptibility to stress conditions mirroring the harsh environment inside a phagosome. Additionally, resistance to killing by neutrophils was analyzed. Our data support the view that immune evasion is a key aspect of C. glabrata virulence and that increased immune recognition causes increased antifungal activities by macrophages. Furthermore, stress resistance and efficient nutrient acquisition, in particular, iron uptake, are crucial for intraphagosomal survival of C. glabrata.

The pathology of vulvovaginal candidiasis (VVC) caused by Candida albicans is associated with a nonprotective inflammatory response and is frequently treated with clotrimazole. We investigated the mechanisms by which clotrimazole resolves VVC. Low levels of clotrimazole, which do not block fungal growth, inhibit expression of a "danger response" transcription factor, c-Fos, block production of proinflammatory cytokines, and inhibit neutrophil infiltration to the site of infection.

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

Clotrimazole and bifonazole are highly effective antifungal agents against mucosal Candida albicans infections. Here we examined the effects of low levels of clotrimazole and bifonazole on the ability of C. albicans to adhere, invade, and damage vaginal epithelial cells. Although adhesion and invasion were not affected, damage was greatly reduced upon azole treatment. This clearly indicates that low levels of azoles influence specific activities of C. albicans during distinct stages of vaginal epithelium infections.

The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.

The secretion of aspartic proteases (Saps) has long been recognized as a virulence-associated trait of the pathogenic yeast Candida albicans. In this study, we report that different recombinant Saps, including Sap1, Sap2, Sap3, and Sap6, have differing abilities to induce secretion of proinflammatory cytokines by human monocytes. In particular Sap1, Sap2, and Sap6 significantly induced interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 production. Sap3 was able to stimulate the secretion of IL-1β and TNF-α. All Saps tested were able to induce Ca(2+) influx in monocytes. Treatment of these Saps with pepstatin A did not have any effect on cytokine secretion, indicating that their stimulatory potential was independent from their proteolytic activity. The capacity of Saps to induce inflammatory cytokine production was also independent from protease-activated receptor (PAR) activation and from the optimal pH for individual Sap activity. The interaction of Saps with monocytes induced Akt activation and phosphorylation of IκBα, which mediates translocation of NF-κB into the nucleus. Overall, these results suggest that individual Sap proteins can induce an inflammatory response and that this phenomenon is independent from the pH of a specific host niche and from Sap enzymatic activity. The inflammatory response is partially dependent on Sap denaturation and is triggered by the Akt/NF-κB activation pathway. Our data suggest a novel, activity-independent aspect of Saps during interactions of C. albicans with the host.

Infection models are essential tools for studying microbial pathogenesis. Murine models are considered the "gold standard" for studying in vivo infections caused by Aspergillus species, such as A. fumigatus. Recently developed molecular protocols allow rapid construction of high numbers of fungal deletion mutants, and alternative infection models based on cell culture or invertebrates are widely used for screening such mutants to reduce the number of rodents in animal experiments. To bridge the gap between invertebrate models and mice, we have developed an alternative, low-cost, and easy-to-use infection model for Aspergillus species based on embryonated eggs. The outcome of infections in the egg model is dose and age dependent and highly reproducible. We show that the age of the embryos affects the susceptibility to A. fumigatus and that increased resistance coincides with altered chemokine production after infection. The progress of disease in the model can be monitored by using egg survival and histology. Based on pathological analyses, we hypothesize that invasion of embryonic membranes and blood vessels leads to embryonic death. Defined deletion mutant strains previously shown to be fully virulent or partially or strongly attenuated in a mouse model of bronchopulmonary aspergillosis showed comparable degrees of attenuation in the egg model. Addition of nutrients restored the reduced virulence of a mutant lacking a biosynthetic gene, and variations of the infectious route can be used to further analyze the role of distinct genes in our model. Our results suggest that embryonated eggs can be a very useful alternative infection model to study A. fumigatus virulence and pathogenicity.

Common iatrogenic procedures can result in translocation of the human pathogenic fungus Candida albicans from mucosal surfaces to the bloodstream. Subsequent disseminated candidiasis and infection of deep-seated organs may occur if the fungus is not eliminated by blood cells. In these cases, fungal cells adhere to the endothelial cells of blood vessels, penetrate through endothelial layers, and invade deeper tissue. In this scenario, endothelial adhesion events must occur during circulation under conditions of physiological blood pressure. To investigate the fungal and host factors which contribute to this essential step of disseminated candidiasis, we have developed an in vitro circulatory C. albicans-endothelium interaction model. We demonstrate that both C. albicans yeast and hyphae can adhere under flow at a pressure similar to capillary blood pressure. Serum factors significantly enhanced the adhesion potential of viable but not killed C. albicans cells to endothelial cells. During circulation, C. albicans cells produced hyphae and the adhesion potential first increased, then decreased with time. We provide evidence that a specific temporal event in the yeast-to-hyphal transition, regulated by the G(1) cyclin Hgc1, is critical for C. albicans-endothelium adhesion during circulation.

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRR) and transduces downstream signaling to activate the host defense. Here we report that in addition to direct PAMP-PRR interactions, live Candida albicans cells can release soluble factors to actively potentiate interleukin-6 (IL-6) and IL-8 production induced in human mononuclear cells by the fungi. Although protease-activated receptor 1 (PAR1) and PAR2 ligation can moderately upregulate Toll-like receptor 4 (TLR4)-mediated IL-8 production, no effect on the C. albicans-induced cytokine was apparent. Similarly, the blockade of PAR signaling did not reverse the potentiation of cytokine production induced by soluble factors released by C. albicans. In conclusion, C. albicans releases soluble factors that potentiate cytokine release in a PAR1/2-independent manner. Thus, human PAR1 and PAR2 have a redundant role in the activation of human cells by C. albicans.

Human polymorphonuclear neutrophils (PMNs) play a major role in the immune defense against invasive Candida albicans infection. This fungal pathogen produces a set of aspartic proteases that directly contributes to virulence properties such as adhesion, tissue invasion, and immune evasion. We show here that, in contrast to other secreted proteases, the cell surface-associated isoform Sap9 has a major impact on the recognition of C. albicans by PMNs. SAP9 is required for the induction of PMN chemotaxis toward C. albicans filaments, an essential prerequisite of effective PMN activation. Furthermore, deletion of SAP9 leads to a mitigated release of reactive oxygen intermediates (ROI) in human PMNs and decreases C. albicans-induced apoptosis triggered by ROI formation. In confrontation assays, killing of a SAP9 deletion mutant is reduced in comparison to wild-type C. albicans. These data clearly implicate Sap9 protease activity in the initiation of protective innate immunity and suggest novel molecular mechanisms in C. albicans-host interaction leading to neutrophil activation.

Candida albicans is a polymorphic opportunistic fungus that can cause life-threatening systemic infections following hematogenous dissemination in patients susceptible to nosocomial infection. Neutrophils form part of the innate immune response, which is the first line of defense against microbes and is particularly important in C. albicans infections. To compare the transcriptional response of leukocytes exposed to C. albicans, we investigated the expression of key cytokine genes in polymorphonuclear and mononuclear leukocytes after incubation with C. albicans for 1 h. Isolated mononuclear cells expressed high levels of genes encoding proinflammatory signaling molecules, whereas neutrophils exhibited much lower levels, similar to those observed in whole blood. The global transcriptional profile of neutrophils was examined by using an immunology-biased human microarray to determine whether different morphological forms or the viability of C. albicans altered the transcriptome. Hyphal cells appeared to have the broadest effect, although the most strongly induced genes were regulated independently of morphology or viability. These genes were involved in proinflammatory cell-cell signaling, cell signal transduction, and cell growth. Generally, genes encoding known components of neutrophil granules showed no upregulation at this time point; however, lactoferrin, a well-known candidacidal peptide, was secreted by neutrophils. Addition to inhibitors of RNA or protein de novo synthesis did not influence the killing activity within 30 min. These results support the general notion that neutrophils do not require gene transcription to mount an immediate and direct attack against microbes. However, neutrophils exposed to C. albicans express genes involved in communication with other immune cells.

Secreted aspartyl proteinases (Saps) are important virulence factors of Candida albicans during mucosal and disseminated infections and may also contribute to the induction of an inflammatory host immune response. We used a model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) to study the epithelial cytokine response induced by C. albicans. In order to study the impact of the overall proteolytic activity and of distinct Sap isoenzymes, we studied the effect of the proteinase inhibitor pepstatin A on the immune response and compared the cytokine expression pattern induced by the wild-type strain SC5314 with the pattern induced by Sap-deficient mutants. Infection of RHVE with the C. albicans wild-type strain induced strong interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha responses in comparison with cytokine expression in noninfected tissue. Addition of the aspartyl proteinase inhibitor pepstatin A strongly reduced the cytokine response of RHVE. Furthermore, SAP-null mutants lacking either SAP1 or SAP2 caused reduced tissue damage and had a significantly reduced potential to stimulate cytokine expression. In contrast, the vaginopathic and cytokine-inducing potential of mutants lacking SAP4 to SAP6 was similar to that of the wild-type strain. These data show that the potential of specific Saps to cause tissue damage correlates with an epithelium-induced proinflammatory cytokine response, which may be crucial in controlling and managing C. albicans infections at the vaginal mucosa in vivo.

Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis.

Secreted aspartyl proteinases (Saps) contribute to the ability of Candida albicans to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these C. albicans proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by C. albicans SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of SAP2, SAP9, and SAP10 was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of SAP1, SAP4, and SAP5. Additional transcripts of SAP6 and SAP7 were detected at a later stage of the artificial infection (24 h). Similar SAP expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by C. albicans was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by C. albicans cells. Furthermore, SAP null mutants lacking either SAP1 or SAP2 had a drastically reduced potential to cause tissue damage even though SAP3, SAP4, and SAP7 were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking SAP3 or SAP4 to SAP6 was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in C. albicans vaginal infections.

The hydroxypyridone ciclopirox olamine belongs to the antimycotic drugs used for the treatment of superficial mycoses. In contrast to the azoles and other antimycotic drugs, its specific mode of action is only poorly understood. To investigate the mode of action of ciclopirox olamine on fungal viability, pathogenicity, and drug resistance, we examined the expression patterns of 47 Candida albicans genes in cells grown in the presence of a subinhibitory concentration (0.6 micro g/ml) of ciclopirox olamine by reverse transcription-PCR. In addition, we used suppression-subtractive hybridization to further identify genes that are up-regulated in the presence of ciclopirox olamine. The expression of essential genes such as ACT1 was not significantly modified in cells exposed to ciclopirox olamine. Most putative and known virulence genes such as genes encoding secreted proteinases or lipases had no or only moderately reduced expression levels. In contrast, exposure of cells to ciclopirox olamine led to a distinct up- or down-regulation of genes encoding iron permeases or transporters (FTR1, FTR2, FTH1), a copper permease (CCC2), an iron reductase (CFL1), and a siderophore transporter (SIT1); these effects resembled those found under iron-limited conditions. Addition of FeCl(3) to ciclopirox olamine-treated cells reversed the effect of the drug. Addition of the iron chelator bipyridine to the growth medium induced similar patterns of expression of distinct iron-regulated genes (FTR1, FTR2). While serum-induced yeast-to-hyphal phase transition of C. albicans was not affected in ciclopirox olamine-treated cells in the presence of subinhibitory conditions, a dramatic increase in sensitivity to oxidative stress was noted, which may indicate the reduced activities of iron-containing gene products responsible for detoxification. Although the Candida drug resistance genes CDR1 and CDR2 were up-regulated, no change in resistance or increased tolerance could be observed even after an incubation period of 6 months. This was in contrast to control experiments with fluconazole, in which the MICs for cells incubated with this drug had noticeably increased after 2 months. These data support the view that the antifungal activity of ciclopirox olamine may at least be partially caused by iron limitation. Furthermore, neither the expression of certain multiple-drug resistance genes nor other resistance mechanisms caused C. albicans resistance to this drug even after long-term exposure.

The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.

Peritonitis with Candida albicans is an important complication of bowel perforation and continuous ambulatory peritoneal dialysis. To define potential virulence factors, we investigated 50 strains of C. albicans in a murine peritonitis model. There was considerable variation in their virulence in this model when virulence was measured as release of organ-specific enzymes into the plasma of infected mice. Alanine aminotransferase (ALT) and alpha-amylase (AM) were used as parameters for damage of the liver and pancreas, respectively. The activities of ALT and AM in the plasma correlated with invasion into the organs measured in histologic sections and the median germ tube length induced with serum in vitro. When the activity of proteinases was inhibited in vivo with pepstatin A, there was a significant reduction of ALT and AM activities. This indicates that proteinases contributed to virulence in this model. Using strains of C. albicans with disruption of secreted aspartyl proteinase gene SAP1, SAP2, SAP3, or SAP4 through SAP6 (collectively referred to as SAP4-6), we showed that only a Deltasap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain. In contrast to the Deltasap1, Deltasap2, and Deltasap3 mutants, the ALT induced by the Deltasap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model.

The effects of therapeutically relevant concentrations of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on the in vitro proteinase activity of Candida albicans were investigated with isolates from HIV-infected and uninfected patients with oral candidiasis. After exposure to the HIV proteinase inhibitors, proteinase activity was significantly reduced in a dose-dependent manner. These inhibitory effects, which were similar to that of pepstatin A, and the reduced virulence phenotype in experimental candidiasis after application of saquinavir indicate the usefulness of these HIV proteinase inhibitors as potential anticandidal agents.

The endothelial cell interactions of homozygous null mutants of Candida albicans that were deficient in secreted aspartyl proteinase 1 (Sap1), Sap2, or Sap3 were investigated. Only Sap2 was found to contribute to the ability of C. albicans to damage endothelial cells and stimulate them to express E-selectin. None of the Saps studied appears to play a role in C. albicans adherence to endothelial cells.