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New antifungal therapies are needed for both systemic, invasive infections in addition to superficial infections of mucosal and skin surfaces as well as biofilms associated with medical devices. The resistance of biofilm and biofilm-like growth phases of fungi contributes to the poor efficacy of systemic therapies to nonsystemic infections.

Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.

Invasive fungal infections constitute a lethal threat, with patient mortality as high as 90%. The incidence of invasive fungal infections is increasing, especially in the setting of patients receiving immunomodulatory agents, chemotherapy, or immunosuppressive medications following solid-organ or bone marrow transplantation. In addition, inhibitors of spleen tyrosine kinase (Syk) have been recently developed for the treatment of patients with refractory autoimmune and hematologic indications. Neutrophils are the initial innate cellular responders to many types of pathogens, including invasive fungi. A central process governing neutrophil recognition of fungi is through lectin binding receptors, many of which rely on Syk for cellular activation. We previously demonstrated that Syk activation is essential for cellular activation, phagosomal maturation, and elimination of phagocytosed fungal pathogens in macrophages. Here, we used combined genetic and chemical inhibitor approaches to evaluate the importance of Syk in the response of neutrophils to Candida species. We took advantage of a Cas9-expressing neutrophil progenitor cell line to generate isogenic wild-type and Syk-deficient neutrophils. Syk-deficient neutrophils are unable to control the human pathogens Candida albicans, Candida glabrata, and Candida auris. Neutrophil responses to Candida species, including the production of reactive oxygen species and of cytokines such as tumor necrosis factor alpha (TNF-α), the formation of neutrophil extracellular traps (NETs), phagocytosis, and neutrophil swarming, appear to be critically dependent on Syk. These results demonstrate an essential role for Syk in neutrophil responses to Candida species and raise concern for increased fungal infections with the development of Syk-modulating therapeutics. IMPORTANCE Neutrophils are recognized to represent significant immune cell mediators for the clearance and elimination of the human-pathogenic fungal pathogen Candida. The sensing of fungi by innate cells is performed, in part, through lectin receptor recognition of cell wall components and downstream cellular activation by signaling components, including spleen tyrosine kinase (Syk). While the essential role of Syk in macrophages and dendritic cells is clear, there remains uncertainty with respect to its contribution in neutrophils. In this study, we demonstrated that Syk is critical for multiple cellular functions in neutrophils responding to major human-pathogenic Candida species. These data not only demonstrate the vital nature of Syk with respect to the control of fungi by neutrophils but also warn of the potential infectious complications arising from the recent clinical development of novel Syk inhibitors for hematologic and autoimmune disorders.

The emerging fungal pathogen Candida auris causes invasive infections and is spreading in hospitals worldwide. Why this species exhibits the capacity to transfer efficiently among patients is unknown. Our findings reveal that C. auris forms high-burden biofilms in conditions mimicking sweat on the skin surface. These adherent biofilm communities persist in environmental conditions expected in the hospital setting. Using a pig skin model, we show that C. auris also forms high-burden biofilm structures on the skin surface. Identification of this mode of growth sheds light on how this recently described pathogen persists in hospital settings and spreads among patients., Emerging pathogen Candida auris causes nosocomial outbreaks of life-threatening invasive candidiasis. It is unclear how this species colonizes skin and spreads in health care facilities. Here, we analyzed C. auris growth in synthetic sweat medium designed to mimic axillary skin conditions. We show that C. auris demonstrates a high capacity for biofilm formation in this milieu, well beyond that observed for the most commonly isolated Candida sp., Candida albicans. The C. auris biofilms persist in environmental conditions expected in the hospital setting. To model C. auris skin colonization, we designed an ex vivo porcine skin model. We show that C. auris proliferates on porcine skin in multilayer biofilms. This capacity to thrive in skin niche conditions helps explain the propensity of C. auris to colonize skin, persist on medical devices, and rapidly spread in hospitals. These studies provide clinically relevant tools to further characterize this important growth modality. IMPORTANCE The emerging fungal pathogen Candida auris causes invasive infections and is spreading in hospitals worldwide. Why this species exhibits the capacity to transfer efficiently among patients is unknown. Our findings reveal that C. auris forms high-burden biofilms in conditions mimicking sweat on the skin surface. These adherent biofilm communities persist in environmental conditions expected in the hospital setting. Using a pig skin model, we show that C. auris also forms high-burden biofilm structures on the skin surface. Identification of this mode of growth sheds light on how this recently described pathogen persists in hospital settings and spreads among patients.

Candida auris has emerged as an outbreak pathogen associated with high mortality. Biofilm formation and linked drug resistance are common among Candida species. Drug sequestration by the biofilm matrix accounts for much of the antifungal tolerance. In this study, we examine the biofilm matrix composition and function for a diverse set of C. auris isolates. We show that matrix sequesters nearly 70% of the available triazole antifungal. Like the biofilms formed by other Candida spp., we find that the matrix of C. auris is rich in mannan-glucan polysaccharides and demonstrate that their hydrolysis reduces drug tolerance. This biofilm matrix resistance mechanism appears conserved among Candida species, including C. auris. IMPORTANCECandida auris is an emerging fungal threat linked to poor patient outcomes. The factors responsible for this apparent increase in pathogenicity remain largely unknown. Biofilm formation has been suggested as an important factor for persistence of this organism in patients and the environment. Our findings reveal one mechanism utilized by C. auris to evade the effect of triazole antifungal therapy during biofilm growth. The conservation of the protective biofilm matrix among Candida spp. suggests that is a promising pan-fungal Candida biofilm drug target.

Indwelling urinary catheters are commonly used in the management of hospitalized patients. Candida can adhere to the device surface and propagate as a biofilm. These Candida biofilm communities differ from free-floating Candida , exhibiting high tolerance to antifungal therapy. The significance of catheter-associated candiduria is often unclear, and treatment may be problematic considering the biofilm drug-resistant phenotype. Here we describe a rodent model for the study of urinary catheter-associated Candida albicans biofilm infection that mimics this common process in patients. In the setting of a functioning, indwelling urinary catheter in a rat, Candida proliferated as a biofilm on the device surface. Characteristic biofilm architecture was observed, including adherent, filamentous cells embedded in an extracellular matrix. Similar to what occurs in human patients, animals with this infection developed candiduria and pyuria. Infection progressed to cystitis, and a biofilmlike covering was observed over the bladder surface. Furthermore, large numbers of C. albicans cells were dispersed into the urine from either the catheter or bladder wall biofilm over the infection period. We successfully utilized the model to test the efficacy of antifungals, analyze transcriptional patterns, and examine the phenotype of a genetic mutant. The model should be useful for future investigations involving the pathogenesis, diagnosis, therapy, prevention, and drug resistance of Candida biofilms in the urinary tract.

Candida spp. infect medical devices, such as venous and urinary catheters, by adhering to the surface and forming a community of drug-resistant cells surrounded by a matrix. The ability to measure drug activity during this biofilm mode of growth is of interest for the investigation of resistance mechanisms and novel antifungal therapies. The tetrazolium salt (XTT) reduction assay is the test most commonly used to estimate viable biofilm growth and to examine the impact of biofilm therapies. The primary goal of the current experiments was to identify assay variables that affect the XTT assay result in order to improve assay reproducibility, sensitivity, and throughput for the study of antifungal activity. The species used in the current studies included Candida albicans , C. parapsilosis , and C. glabrata . The assay variables that were studied included the impact of culture conditions, the duration of biofilm growth, the timing and frequency of drug administration, the XTT source and concentration, and the duration of XTT incubation. The conditions that impacted the assay readout and altered assay sensitivity included the duration of biofilm growth, the frequency of drug dosing, and the duration of XTT incubation. Several factors were found to reduce time and assay expense, including the elimination of washing steps, the shortening of incubation times, and the use of lower XTT concentrations. A description of assay pitfalls and troubleshooting is included. Recognition of these technical variables should allow investigators to better design reproducible biofilm therapeutic studies.

The most common form of oral candidiasis, denture-associated stomatitis, involves biofilm growth on an oral prosthetic surface. Cells in this unique environment are equipped to withstand host defenses and survive antifungal therapy. Studies of the biofilm process on dentures have primarily been limited to in vitro models. We developed a rodent acrylic denture model and characterized the Candida albicans and mixed oral bacterial flora biofilm formation, architecture, and drug resistance in vivo , using time course quantitative culture experiments, confocal microscopy, scanning electron microscopy, and antifungal susceptibility assays. We also examined the utility of the model for measurement of C. albicans gene expression and tested the impact of a specific gene product (Bcr1p) on biofilm formation. Finally, we assessed the mucosal host response to the denture biofilm and found the mucosal histopathology to be consistent with that of acute human denture stomatitis, demonstrating fungal invasion and neutrophil infiltration. This current oral denture model mimics human denture stomatitis and should be useful for testing the impact of gene disruption on biofilm formation, studying the impact of anti-infectives, examining the biology of mixed Candida -oral bacterial flora biofilm infections, and characterizing the host immunologic response to this disease process.

Calcineurin is a Ca 2+ -calmodulin-activated serine/threonine-specific protein phosphatase that governs multiple aspects of fungal physiology, including cation homeostasis, morphogenesis, antifungal drug susceptibility, and virulence. Growth of Candida albicans planktonic cells is sensitive to the calcineurin inhibitors FK506 and cyclosporine A (CsA) in combination with the azole antifungal fluconazole. This drug synergism is attributable to two effects: first, calcineurin inhibitors render fluconazole fungicidal rather than simply fungistatic, and second, membrane perturbation by azole inhibition of ergosterol biosynthesis increases intracellular calcineurin inhibitor concentrations. C. albicans cells in biofilms are up to 1,000-fold more resistant to fluconazole than planktonic cells. In both in vitro experiments and in an in vivo rat catheter model, C. albicans cells in biofilms were resistant to individually delivered fluconazole or calcineurin inhibitors but exquisitely sensitive to the combination of FK506-fluconazole or CsA-fluconazole. C. albicans strains lacking FKBP12 or expressing a dominant FK506-resistant calcineurin mutant subunit (Cnb1-1) formed biofilms that were resistant to FK506-fluconazole but susceptible to CsA-fluconazole, demonstrating that drug synergism is mediated via direct calcineurin inhibition. These findings reveal that calcineurin contributes to fluconazole resistance of biofilms and provide evidence that synergistic drug combinations may prove efficacious as novel therapeutic interventions to treat or prevent biofilms.

Among the most fascinating virulence attributes of Candida is the ability to transition to a biofilm lifestyle. As a biofilm, Candida cells adhere to a surface, such as a vascular catheter, and become encased in an extracellular matrix. During this mode of growth, Candida resists the normal immune response, often causing devastating disease. Based on scanning electron microscopy images, we hypothesized that host cells and proteins become incorporated into clinical biofilms. As a means to gain an understanding of these host-biofilm interactions, we explored biofilm-associated host components by using microscopy and liquid chromatography-mass spectrometry. Here we characterize the host proteins associated with several in vivo rat Candida albicans biofilms, including those from vascular catheter, denture, and urinary catheter models as well as uninfected devices. A conserved group of 14 host proteins were found to be more abundant during infection at each of the niches. The host proteins were leukocyte and erythrocyte associated and included proteins involved in inflammation, such as C-reactive protein, myeloperoxidase, and alarmin S100-A9. A group of 59 proteins were associated with both infected and uninfected devices, and these included matricellular and inflammatory proteins. In addition, site-specific proteins were identified, such as amylase in association with the denture device. Cellular analysis revealed neutrophils as the predominant leukocytes associating with biofilms. These experiments demonstrate that host cells and proteins are key components of in vivo Candida biofilms, likely with one subset associating with the device and another being recruited by the proliferating biofilm.

Antimicrobial drug resistance can limit the ability to effectively treat patients. Numerous factors have been proposed to impact the development of antimicrobial resistance, including those specific to the drug and the dosing regimen. The field of investigation that examines the relationship between dosing regimen and outcome is termed antimicrobial pharmacokinetics and pharmacodynamics. Our prior in vivo investigations examined the relationship between fluconazole pharmacodynamics and the modulation of isogenic resistant and susceptible Candida albicans populations in a mixed-inoculum design (1). The goal of the current studies was to examine the impact of fluconazole pharmacodynamics on resistance emergence from a susceptible parent population over time using a murine systemic-candidiasis model. Both microbiologic and transcriptional endpoints were examined during the evolution of cell populations. As in our previous investigation, the more frequently administered dosing regimen prevented the emergence of a resistant cell phenotype. Conversely, dosing regimens that produced prolonged sub-MIC concentrations were associated with resistance development. The studies also demonstrated a striking relationship between fluconazole pharmacodynamic exposures and the mRNA abundance of drug resistance-associated efflux pumps. Global transcriptional profiling of cell populations during the progressive emergence of a resistance phenotype provides insight into the mechanisms underlying this complex physiologic process.

Virulence of Candida is linked with its ability to form biofilms. Once established, biofilm infections are nearly impossible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we provide a comprehensive analysis of the matrix manufactured by Candida albicans both in vitro and in a clinical niche animal model. We further explore the function of matrix components, including the impact on drug resistance. We uncovered components from each of the macromolecular classes (55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid) in the C. albicans biofilm matrix. Three individual polysaccharides were identified and were suggested to interact physically. Surprisingly, a previously identified polysaccharide of functional importance, β-1,3-glucan, comprised only a small portion of the total matrix carbohydrate. Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched β-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx). Functional matrix proteomic analysis revealed 458 distinct activities. The matrix lipids consisted of neutral glycerolipids (89.1%), polar glycerolipids (10.4%), and sphingolipids (0.5%). Examination of matrix nucleic acid identified DNA, primarily noncoding sequences. Several of the in vitro matrix components, including proteins and each of the polysaccharides, were also present in the matrix of a clinically relevant in vivo biofilm. Nuclear magnetic resonance (NMR) analysis demonstrated interaction of aggregate matrix with the antifungal fluconazole, consistent with a role in drug impedance and contribution of multiple matrix components., IMPORTANCE This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs. The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle.

Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans , Cryptococcus neoformans , and Aspergillus fumigatus . In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans , in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis . We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis . Similar to C. albicans , C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.

Hwp1 is a well-characterized Candida albicans cell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1 expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1 / hwp1 mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1 / HWP1 reconstituted strain. In a biofilm cell retention assay, we find that the hwp1 / hwp1 mutant is defective in retention of nonadherent bcr1 / bcr1 mutant cells. In an in vivo rat venous catheter model, the hwp1 / hwp1 mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1 / hwp1 mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1 in a bcr1 / bcr1 mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo and is thus an excellent therapeutic target.

Biofilms represent a niche for microorganisms where they are protected from both the host immune system and antimicrobial therapies. Biofilm growth serves as an increasing source of clinical infections. Candida infections are difficult to manage due to their persistent nature and associated drug resistance. Observations made in biofilm research have generally been limited to in vitro models. Using a rat central venous catheter model, we characterized in vivo Candida albicans biofilm development. Time-course quantitative culture demonstrated a progressive increase in the burden of viable cells for the first 24 h of development. Fluorescence and scanning electron microscopy revealed a bilayered architecture. Adjacent to the catheter surface, yeast cells were densely embedded in an extracellular matrix. The layer adjacent to the catheter lumen was less dense. The outermost surface of the biofilm contained both yeast and hyphal forms, and the extracellular material in which they were embedded appeared fibrous. These architectural features were similar in many respects to those described for in vitro models. However, scanning electron microscopy also revealed host cells embedded within the biofilm matrix. Drug susceptibility was determined by using two assays and demonstrated a biofilm-associated drug resistance phenotype. The first assay demonstrated continued growth of cells in the presence of supra-MIC antifungal drug concentrations. The second assay demonstrated reduced susceptibility of biofilm-grown cells following removal from the biofilm structure. Lastly, the model provided sufficient nucleic material for study of differential gene expression associated with in vivo biofilm growth. Two fluconazole efflux pumps, CDR1 and CDR2 , were upregulated in the in vivo biofilm-associated cells. Most importantly, the studies described provide a model for further investigation into the molecular mechanisms of C. albicans biofilm biology and drug resistance. In addition, the model provides a means to study novel drug therapies and device technologies targeted to the control of biofilm-associated infections.