Impact of micafungin on Candida auris ß-glucan masking and neutrophil interactions.

Invasive fungal pathogen Candida auris has become a public health threat causing outbreaks of high mortality infections. Drug resistance often limits treatment options. For Candida albicans, subinhibitory concentrations of echinocandins unmask immunostimulatory ß-glucan, augmenting immunity. Here we analyze the impact of echinocandin treatment of C. auris on ß-glucan exposure and human neutrophil interactions. We show subinhibitory concentrations lead to minimal glucan unmasking and only subtle influences on neutrophil functions for the isolates belonging to circulating clades. The data suggest that echinocandin treatment will not largely alter phagocytic responses. Glucan masking pathways appear to differ between C. auris and C. albicans.

Candida albicans extracellular vesicles trigger type I IFN signalling via cGAS and STING.

The host type I interferon (IFN) pathway is a major signature of inflammation induced by the human fungal pathogen, Candida albicans. However, the molecular mechanism for activating this pathway in the host defence against C. albicans remains unknown. Here we reveal that mice lacking cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway components had improved survival following an intravenous challenge by C. albicans. Biofilm-associated C. albicans DNA packaged in extracellular vesicles triggers the cGAS-STING pathway as determined by induction of interferon-stimulated genes, IFNß production, and phosphorylation of IFN regulatory factor 3 and TANK-binding kinase 1. Extracellular vesicle-induced activation of type I IFNs was independent of the Dectin-1/Card9 pathway and did not require toll-like receptor 9. Single nucleotide polymorphisms in cGAS and STING potently altered inflammatory cytokine production in human monocytes challenged by C. albicans. These studies provide insights into the early innate immune response induced by a clinically significant fungal pathogen.

Liposomal formulation of a new antifungal hybrid compound provides protection against Candida auris in the ex vivo skin colonization model.

The newly emerged pathogen, Candida auris, presents a serious threat to public health worldwide. This multidrug-resistant yeast often colonizes and persists on the skin of patients, can easily spread from person to person, and can cause life-threatening systemic infections. New antifungal therapies are therefore urgently needed to limit and control both superficial and systemic C. auris infections. In this study, we designed a novel antifungal agent, PQA-Az-13, that contains a combination of indazole, pyrrolidine, and arylpiperazine scaffolds substituted with a trifluoromethyl moiety. PQA-Az-13 demonstrated antifungal activity against biofilms of a set of 10 different C. auris clinical isolates, representing all four geographical clades distinguished within this species. This compound showed strong activity, with MIC values between 0.67 and 1.25 µg/mL. Cellular proteomics indicated that PQA-Az-13 partially or completely inhibited numerous enzymatic proteins in C. auris biofilms, particularly those involved in both amino acid biosynthesis and metabolism processes, as well as in general energy-producing processes. Due to its hydrophobic nature and limited aqueous solubility, PQA-Az-13 was encapsulated in cationic liposomes composed of soybean phosphatidylcholine (SPC), 1,2-dioleoyloxy-3-trimethylammonium-propane chloride (DOTAP), and N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (DSPE-PEG 2000), and characterized by biophysical and spectral techniques. These PQA-Az-13-loaded liposomes displayed a mean size of 76.4 nm, a positive charge of +45.0 mV, a high encapsulation efficiency of 97.2%, excellent stability, and no toxicity to normal human dermal fibroblasts. PQA-Az-13 liposomes demonstrated enhanced antifungal activity levels against both C. auris in in vitro biofilms and ex vivo skin colonization models. These initial results suggest that molecules like PQA-Az-13 warrant further study and development.

Mechanisms of pathogenicity for the emerging fungus Candida auris.

Candida auris recently emerged as an urgent public health threat, causing outbreaks of invasive infections in healthcare settings throughout the world. This fungal pathogen persists on the skin of patients and on abiotic surfaces despite antiseptic and decolonization attempts. The heightened capacity for skin colonization and environmental persistence promotes rapid nosocomial spread. Following skin colonization, C. auris can gain entrance to the bloodstream and deeper tissues, often through a wound or an inserted medical device, such as a catheter. C. auris possesses a variety of virulence traits, including the capacity for biofilm formation, production of adhesins and proteases, and evasion of innate immune responses. In this review, we highlight the interactions of C. auris with the host, emphasizing the intersection of laboratory studies and clinical observations.

The Candida albicans ζ-crystallin homolog Zta1 promotes resistance to oxidative stress.

IMPORTANCE: Candida albicans is an important human pathogen that can cause lethal systemic infections. The ability of C. albicans to colonize and establish infections is closely tied to its highly adaptable nature and capacity to resist various types of stress, including oxidative stress. Previous studies showed that four C. albicans proteins belonging to the flavodoxin-like protein family of quinone reductases are needed for resistance to quinones and virulence. Therefore, in this study, we examined the role of a distinct type of quinone reductase, Zta1, and found that it acts in conjunction with the flavodoxin-like proteins to protect against oxidative stress.

Differential skin immune responses in mice intradermally infected with Candida auris and Candida albicans.

IMPORTANCE: Candida auris is a globally emerging fungal pathogen that transmits among individuals in hospitals and nursing home residents. Unlike other Candida species, C. auris predominantly colonizes and persists in skin tissue, resulting in outbreaks of nosocomial infections. Understanding the factors that regulate C. auris skin colonization is critical to develop novel preventive and therapeutic approaches against this emerging pathogen. We established a model of intradermal C. auris inoculation in mice and found that mice infected with C. auris elicit less potent innate and adaptive immune responses in the infected skin compared to C. albicans. These findings help explain the clinical observation of persistent C. auris colonization in skin tissue.

A Candida auris-specific adhesin, Scf1, governs surface association, colonization, and virulence.

Candida auris is an emerging fungal pathogen responsible for health care-associated outbreaks that arise from persistent surface and skin colonization. We characterized the arsenal of adhesins used by C. auris and discovered an uncharacterized adhesin, Surface Colonization Factor (Scf1), and a conserved adhesin, Iff4109, that are essential for the colonization of inert surfaces and mammalian hosts. SCF1 is apparently specific to C. auris, and its expression mediates adhesion to inert and biological surfaces across isolates from all five clades. Unlike canonical fungal adhesins, which function through hydrophobic interactions, Scf1 relies on exposed cationic residues for surface association. SCF1 is required for C. auris biofilm formation, skin colonization, virulence in systemic infection, and colonization of inserted medical devices.

The Candida albicans ζ-crystallin homolog Zta1 promotes resistance to oxidative stress.

The fungal pathogen Candida albicans is capable of causing lethal infections in humans. Its pathogenic potential is due in part to the ability to resist various stress conditions in the host, including oxidative stress. Recent studies showed that a family of four flavodoxin-like proteins (Pst1, Pst2, Pst3, Ycp4) that function as quinone reductases promotes resistance to oxidation and is needed for virulence. Therefore, in this study Zta1 was examined because it belongs to a structurally distinct family of quinone reductases that are highly conserved in eukaryotes and have been called the ζ-crystallins. The levels of Zta1 in C. albicans rapidly increased after exposure to oxidants, consistent with a role in resisting oxidative stress. Accumulation of reactive oxygen species was significantly higher in cells lacking ZTA1 upon exposure to quinones and other oxidants. Furthermore, deletion of ZTA1 in a mutant lacking the four flavodoxin-like proteins, resulted in further increased susceptibility to quinones, indicating that these distinct quinone reductases work in combination. These results demonstrate that Zta1 contributes to C. albicans survival after exposure to oxidative conditions, which increases the understanding of how C. albicans resists stressful conditions in the host.

Novel Keto-Alkyl-Pyridinium Antifungal Molecules Active in Models of In Vivo Candida albicans Vascular Catheter Infection and Ex Vivo Candida auris Skin Colonization.

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. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2 to 16 µg/mL) against medically important yeasts and molds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus. Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce Candida albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial preclinical data suggest that molecules of this class may warrant further study and development for nonsystemic applications.

Analysis of Candida Antifungal Resistance Using Animal Infection Models.

Candida frequently produces three general disease states, including mucosal candidiasis, disseminated candidiasis, and biofilm infection (which can be present with either of the other disease states). Antifungal drug resistance is intrinsic to biofilm growth and has emerged in other disease states. Mechanistic studies have uncovered the genetic pathways governing resistance to a number of antifungal agents. However, analyzing the clinical relevance of distinct mechanisms is fundamental for broadening our knowledge of antifungal drug resistance and for delineating the potential impact of targeting these pathways medically. Also, as drug-resistant strains and biofilms represent important nosocomial problems, preclinical animal models to assess the activity of novel antifungals are of great interest. Here we describe two rodent models that mimic the most common biofilm device and disseminated candidiasis states in patients. The model systems incorporate the anatomical site, immune components, and antifungal exposures relevant for the study of antifungal resistance. The models can be used to analyze mutant strains, assess the extent of drug resistance, examine biofilm formation, test new antimicrobials, and help determine drug exposures that may be linked with clinical failure.

Novel keto-alkyl-pyridinium antifungal molecules active in models of in vivo Candida albicans vascular catheter infection and ex vivo Candida auris skin colonization.

New antifungal therapies are needed for both systemic, invasive infections as well as 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 non-systemic infections. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2-16 µg/mL) against medically important yeasts and moulds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus . Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce C. albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial pre-clinical data suggest that molecules of this class may warrant further study and development.

A common vesicle proteome drives fungal biofilm development.

Extracellular vesicles mediate community interactions among cells ranging from unicellular microbes to complex vertebrates. Extracellular vesicles of the fungal pathogen Candida albicans are vital for biofilm communities to produce matrix, which confers environmental protection and modulates community dispersion. Infections are increasingly due to diverse Candida species, such as the emerging pathogen Candida auris, as well as mixed Candida communities. Here, we define the composition and function of biofilm-associated vesicles among five species across the Candida genus. We find similarities in vesicle size and release over the biofilm lifespan. Whereas overall cargo proteomes differ dramatically among species, a group of 36 common proteins is enriched for orthologs of C. albicans biofilm mediators. To understand the function of this set of proteins, we asked whether mutants in select components were important for key biofilm processes, including drug tolerance and dispersion. We found that the majority of these cargo components impact one or both biofilm processes across all five species. Exogenous delivery of wild-type vesicle cargo returned mutant phenotypes toward wild type. To assess the impact of vesicle cargo on interspecies interactions, we performed cross-species vesicle addition and observed functional complementation for both biofilm phenotypes. We explored the biologic relevance of this cross-species biofilm interaction in mixed species and mutant studies examining the drug-resistance phenotype. We found a majority of biofilm interactions among species restored the community's wild-type behavior. Our studies indicate that vesicles influence the development of protective monomicrobial and mixed microbial biofilm communities.

Examining Neutrophil-Candida auris Interactions with Human Neutrophils Ex Vivo.

Neutrophils play a key role in controlling invasive fungal infections. These phagocytes engage and kill fungal pathogens through a variety of effector mechanisms. Here, we describe how to isolate human neutrophils for ex vivo study of neutrophil-Candida auris interactions. We detail assays to measure fungal killing, phagocytosis, and reactive oxygen species production.

Modeling Candida auris Colonization on Porcine Skin Ex Vivo.

Candida auris spreads person to person in hospitals and other healthcare facilities. The heightened capacity for C. auris to colonize skin contributes to the difficulty in eradicating this drug-resistant and deadly pathogen in nosocomial settings. Models for the study of C. auris skin colonization are critical for understanding this virulence trait. In light of the similarities between the skin properties of humans and pigs, pigs represent an ideal model for the investigation of skin-C. auris interactions. Here, we describe how to utilize porcine skin for ex vivo studies of C. auris colonization.

Ex Vivo Human and Porcine Skin Effectively Model Candida auris Colonization, Differentiating Robust and Poor Fungal Colonizers.

Candida auris proliferates and persists on the skin of patients, often leading to health care-associated infections with high mortality. Here, we describe 2 clinically relevant skin models and show that C. auris grows similarly on human and porcine skin. Additionally, we demonstrate that other Candida spp., including those with phylogenetic similarity to C. auris, do not display high growth in the skin microenvironment. These studies highlight the utility of 2 ex vivo models of C. auris colonization that allow reproducible differentiation among Candida spp., which should be a useful tool for comparison of C. auris clinical isolates and genetically mutated strains.

Augmenting the Activity of Chlorhexidine for Decolonization of Candida auris from Porcine skin.

Candida auris readily colonizes skin and efficiently spreads among patients in healthcare settings worldwide. Given the capacity of this drug-resistant fungal pathogen to cause invasive disease with high mortality, hospitals frequently employ chlorhexidine bathing to reduce skin colonization. Using an ex vivo skin model, we show only a mild reduction in C. auris following chlorhexidine application. This finding helps explain why chlorhexidine bathing may have failures clinically, despite potent in vitro activity. We further show that isopropanol augments the activity of chlorhexidine against C. auris on skin. Additionally, we find both tea tree (Melaleuca alternifolia) oil and lemongrass (Cymbopogon flexuosus) oil to further enhance the activity of chlorhexidine/isopropanol for decolonization. We link this antifungal activity to individual oil components and show how some of these components act synergistically with chlorhexidine/isopropanol. Together, the studies provide strategies to improve C. auris skin decolonization through the incorporation of commonly used topical compounds.

Coordination of fungal biofilm development by extracellular vesicle cargo.

The fungal pathogen Candida albicans can form biofilms that protect it from drugs and the immune system. The biofilm cells release extracellular vesicles (EVs) that promote extracellular matrix formation and resistance to antifungal drugs. Here, we define functions for numerous EV cargo proteins in biofilm matrix assembly and drug resistance, as well as in fungal cell adhesion and dissemination. We use a machine-learning analysis of cargo proteomic data from mutants with EV production defects to identify 63 candidate gene products for which we construct mutant and complemented strains for study. Among these, 17 mutants display reduced biofilm matrix accumulation and antifungal drug resistance. An additional subset of 8 cargo mutants exhibit defects in adhesion and/or dispersion. Representative cargo proteins are shown to function as EV cargo through the ability of exogenous wild-type EVs to complement mutant phenotypic defects. Most functionally assigned cargo proteins have roles in two or more of the biofilm phases. Our results support that EVs provide community coordination throughout biofilm development in C. albicans.

Candida auris Cell Wall Mannosylation Contributes to Neutrophil Evasion through Pathways Divergent from Candida albicans and Candida glabrata.

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.