Tools and techniques to identify, study, and control Candida auris.

Candida auris, is an emerging fungal pathogen that can cause life-threatening infections in humans. Unlike many other Candida species that colonize the intestine, C. auris most efficiently colonizes the skin. Such colonization contaminates the patient's environment and can result in rapid nosocomial transmission. In addition, this transmission can lead to outbreaks of systemic infections that have mortality rates between 40% and 60%. C. auris isolates resistant to all known classes of antifungals have been identified and as such, understanding the underlying biochemical mechanisms of how skin colonization initiates and progresses is critical to developing better therapeutic options. With this review, we briefly summarize what is known about horizontal transmission and current tools used to identify, understand, and control C. auris infections.

Transcriptional control of hyphal morphogenesis in Candida albicans.

Candida albicans is a multimorphic commensal organism and opportunistic fungal pathogen in humans. A morphological switch between unicellular budding yeast and multicellular filamentous hyphal growth forms plays a vital role in the virulence of C. albicans, and this transition is regulated in response to a range of environmental cues that are encountered in distinct host niches. Many unique transcription factors contribute to the transcriptional regulatory network that integrates these distinct environmental cues and determines which phenotypic state will be expressed. These hyphal morphogenesis regulators have been extensively investigated, and represent an increasingly important focus of study, due to their central role in controlling a key C. albicans virulence attribute. This review provides a succinct summary of the transcriptional regulatory factors and environmental signals that control hyphal morphogenesis in C. albicans.

Dispiropyrrolidine tethered piperidone heterocyclic hybrids with broad-spectrum antifungal activity against Candida albicans and Cryptococcus neoformans.

Invasive fungal infections along with rising incidence of resistance to antifungal drugs pose increasing threat to immunocompromised individuals, including cancer patients. In this study, we examined the antifungal activity of dispiropyrrolidine tethered piperidone heterocyclic hybrids. Results indicate that compounds 5a and 6i have demonstrated a potent antifungal effect on multiple fungal strains, including Candida albicans, without exhibiting cytotoxicity to mammalian cells. Furthermore, these two compounds exhibited significant inhibition on Candida albicans hyphae and biofilm development that surpasses the FDA-approved antifungal drug currently used for treatment. Taken together, our results suggest that 5a and 6i are promising candidates for development into new antifungal drugs.

Broad-spectrum antifungal activity of spirooxindolo-pyrrolidine tethered indole/imidazole hybrid heterocycles against fungal pathogens.

Invasive fungal infections are one of the leading causes of nosocomial bloodstream infections with a limited treatment option. A series of derivatized spirooxindolo-pyrrolidine tethered indole and imidazole heterocyclic hybrids have been synthesized, and their antifungal activity against fungal strains were determined. Here we characterize the antifungal activity of a specific spirooxindolo-pyrrolidine hybrid, dubbed compound 9c, a spirooxindolo-pyrrolidine tethered imidazole synthesized with a 2-chloro and trifluoromethoxy substituent. The compound 9c exhibited no cytotoxicity against mammalian cell line at concentrations that inhibited fungal strains. Compound 9c also significantly inhibited the fungal hyphae and biofilm formation. Our results indicate that spirooxindolo-pyrrolidine heterocyclic hybrids potentially represent a broad class of chemical agents with promising antifungal potential.

Benzimidazole tethered pyrrolo[3,4-b]quinoline with broad-spectrum activity against fungal pathogens.

Fungal infections caused by Candida and Cryptococcus are particularly dangerous for immunocompromised individuals. In this study, we identified that benzimidazole fused pyrrolo[3,4-b]quinoline compounds have potent antifungal activity against several clinical isolates of pathogenic fungal strains. Specifically, the compound 6a did not show cytotoxicity against mammalian cells at a concentration that inhibits the growth of fungal strains. In addition, the compound 6a also significantly reduced the metabolic activity of fungal cells in the Candida albicans biofilms. Collectively, our results indicate that benzimidazole fused quinoline compounds have a potential to develop as an antifungal agents.

Multicomponent Domino Synthesis, Anticancer Activity and Molecular Modeling Simulation of Complex Dispirooxindolopyrrolidines.

A series of spirooxindolopyrrolidine fused N-styrylpiperidone heterocyclic hybrids has been synthesized in excellent yield via a domino multicomponent protocol that involves one-pot three component 1,3-dipolar cycloaddition and concomitant enamine reactions performed in an inexpensive ionic liquid, namely 1-butyl-3-methylimidazolium bromide ([bmim]Br). Compounds thus synthesized were evaluated for their cytotoxicity against U-937 tumor cells. Interestingly; compounds 5i and 5m exhibited a better cytotoxicity than the anticancer drug bleomycin. In addition; the effect of the synthesized compounds on the nuclear morphology of U937 FaDu cells revealed that treatment with compounds 5a⁻m led to their apoptotic cell death.

Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells.

BACKGROUND: Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear. METHODS: The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model. RESULTS: Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2µg/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load. CONCLUSION: Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent. GENERAL SIGNIFICANCE: The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development.

Cutting edge: progesterone directly upregulates vitamin d receptor gene expression for efficient regulation of T cells by calcitriol.

The two nuclear hormone receptor ligands progesterone and vitamin D (vit.D) play important roles in regulating T cells. The mechanism that connects these two hormones in regulating T cells has not been established. In this study, we report that progesterone is a novel inducer of vit.D receptor (VDR) in T cells and makes T cells highly sensitive to calcitriol. At the molecular level, the induction by progesterone is mediated by two progesterone receptor-binding elements in the intron region after the first noncoding exon of the human VDR gene. Increased expression of VDR by progesterone allows highly sensitive regulation of T cells by vit.D even when vit.D levels are suboptimal. This novel regulatory pathway allows enhanced induction of regulatory T cells but suppression of Th1 and Th17 cells by the two nuclear hormones. The results have significant ramifications in effective regulation of T cells to prevent adverse immune responses during pregnancy.

Targeting intracellular bacteria with an extended cationic amphiphilic polyproline helix.

An extended cationic and amphiphilic polyproline helix (CAPH) is described with a dual mode of action: effective cell penetration of human macrophages, and potent antimicrobial activity in vitro against both Gram-positive and negative pathogens, including Acinetobacter baumannii, Escherichia coli O157 and Bacillus anthracis. This dual action was successfully combined to clear pathogenic bacteria (Brucella and Salmonella) residing within macrophages.

Retinoic acid promotes the development of Arg1-expressing dendritic cells for the regulation of T-cell differentiation.

Arginase I (Arg1), an enzyme expressed by many cell types including myeloid cells, can regulate immune responses. Expression of Arg1 in myeloid cells is regulated by a number of cytokines and tissue factors that influence cell development and activation. Retinoic acid, produced from vitamin A, regulates the homing and differentiation of lymphocytes and plays important roles in the regulation of immunity and immune tolerance. We report here that optimal expression of Arg1 in DCs requires retinoic acid. Induction of Arg1 by retinoic acid is directly mediated by retinoic acid-responsive elements in the 5' noncoding region of the Arg1 gene. Arg1, produced by DCs in response to retinoic acid, promotes the generation of FoxP3(+) regulatory T (Treg) cells. Importantly, blocking the retinoic acid receptor makes DCs hypo-responsive to known inducers of Arg1 such as IL-4 and GM-CSF in Arg1 expression. We found that intestinal CD103(+) DCs that are known to produce retinoic acid highly express Arg1. Our results establish retinoic acid as a key signal in expression of Arg1 in DCs.

The adhesin SCF1 mediates Candida auris colonization.

Candida auris is an emerging human fungal pathogen that can rapidly spread and cause outbreaks of invasive infections. Santana et al. discovered that a novel surface colonization factor (SCF1), and a conserved adhesin, Iff4109, mediates C. auris colonization on abiotic surfaces, skin, and virulence in vivo.

Intradermal infection and dissemination of Candida auris in immunocompetent and immunocompromised mouse models.

Candida auris, an emerging fungal pathogen, predominately colonizes human skin leading to serious invasive infections in humans. Though it is assumed that skin colonization can lead to invasive infection, dissemination potential of C. auris from skin to internal organs is still unknown. In this study, immunocompetent and immunocompromised mouse models of intradermal skin infection were used to compare the dissemination potential of C. auris to internal organs. Our results suggest that C. auris persists in the skin tissue of both immunocompetent and immunocompromised infected mice even at 30 days post-infection. Furthermore, C. auris can readily disseminate from skin tissue to internal organs such as the spleen and kidney as early as 24 h post-infection and was detected until 30 days post-infection. Taken together, our findings for the first time indicate that murine skin intradermally infected with C. auris can readily disseminate to internal organs and cause invasive infections. IMPORTANCE: Candida auris is a multi-drug-resistant emerging fungal pathogen colonizes the human skin and causes life-threatening infections. However, whether C. auris can disseminate from the skin to internal organs is unclear. Understanding the dissemination potential of C. auris in both immunocompetent and immunocompromised hosts is necessary to monitor susceptible individuals and to develop novel approaches to prevent and treat this emerging fungal pathogen. Using mouse models of intradermal C. auris skin infection, our findings report a novel observation that mice skin intradermally infected with C. auris can readily disseminate to internal organs leading to systemic disease. These findings help explain the colonization, persistence, and dissemination potential of C. auris in immunocompetent and immunocompromised hosts and reveal that skin infection is a potential source of invasive infection.

Yeast and filamentous Candida auris stimulate distinct immune responses in the skin.

Candida auris, an emerging multidrug-resistant fungal pathogen, predominately colonizes the human skin long term leading to subsequent life-threatening invasive infections. Fungal morphology is believed to play a critical role in modulating mucocutaneous antifungal immunity. In this study, we used an intradermal mouse model of C. auris infection to examine fungal colonization and the associated innate and adaptive immune response to yeast and filamentous C. auris strains. Our results indicate that mice infected with a filamentous C. auris had significantly decreased fungal load compared to mice infected with the yeast form. Mice infected with yeast and filamentous forms of C. auris stimulated distinct innate immune responses. Phagocytic cells (CD11b+Ly6G+ neutrophils, CD11b+Ly6Chi inflammatory monocytes, and CD11b+MHCII+CD64+ macrophages) were differentially recruited to mouse skin tissue infected with yeast and filamentous C. auris. The percentage and absolute number of interleukin 17 (IL-17) producing innate lymphoid cells, TCRγδ+, and CD4+ T cells in the skin tissue of mice infected with filamentous C. auris were significantly increased compared to the wild-type of yeast strain. Furthermore, complementation of filamentous mutant strain of C. auris (Δelm1 + ELM1) strain exhibited wild-type yeast morphology in vivo and induced comparable level of skin immune responses similar to mice infected with yeast strain. Collectively, our findings indicate that yeast and filamentous C. auris induce distinct local immune responses in the skin. The decreased fungal load observed in mouse skin infected with filamentous C. auris is associated with a potent IL-17 immune response induced by this morphotype.IMPORTANCECandida 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 systemic infections. Understanding the factors that regulate C. auris skin colonization and host immune response is critical to develop novel preventive and therapeutic approaches against this emerging pathogen. We identified that yeast and filamentous forms of C. auris induce distinct skin immune responses in the skin. These findings may help explain the differential colonization and persistence of C. auris morphotypes in skin tissue. Understanding the skin immune responses induced by yeast and filamentous C. auris is important to develop novel vaccine strategies to combat this emerging fungal pathogen.

The Candida auris Hog1 MAP kinase is essential for the colonization of murine skin and intradermal persistence.

Candida auris , a multidrug-resistant human fungal pathogen, was first identified in 2009 in Japan. Since then, systemic C. auris infections have now been reported in more than 50 countries, with mortality rates of 30-60%. A major contributing factor to its high inter- and intrahospital clonal transmission is that C. auris, unlike most Candida species, displays unique skin tropism and can stay on human skin for a prolonged period. However, the molecular mechanisms responsible for C. auris skin colonization, intradermal persistence, and systemic virulence are poorly understood. Here, we report that C. auris Hog1 mitogen-activated protein kinase (MAPK) is essential for efficient skin colonization, intradermal persistence, as well as systemic virulence. RNA-seq analysis of wildtype parental and hog1 Δ mutant strains revealed marked down-regulation of genes involved in processes such as cell adhesion, cell-wall rearrangement, and pathogenesis in hog1 Δ mutant compared to the wildtype parent. Consistent with these data, we found a prominent role for Hog1 in maintaining cell-wall architecture, as the hog1 Δ mutant demonstrated a significant increase in cell-surface ß-glucan exposure and a concomitant reduction in chitin content. Additionally, we observed that Hog1 was required for biofilm formation in vitro and fungal survival when challenged with primary murine macrophages and neutrophils ex vivo . Collectively, these findings have important implications for understanding the C. auris skin adherence mechanisms and penetration of skin epithelial layers preceding bloodstream infections. Importance: Candida auris is a World Health Organization (WHO) fungal priority pathogen and an urgent public health threat recognized by the Centers for Disease Control and Prevention (CDC). C. auris has a unique ability to colonize human skin. It also persists on abiotic surfaces in healthcare environments for an extended period of time. These attributes facilitate the inter- and intrahospital clonal transmission of C. auris . Therefore, understanding C. auris skin colonization mechanisms are critical for infection control, especially in hospitals and nursing homes. However, despite its profound clinical relevance, the molecular and genetic basis of C. auris skin colonization mechanisms are poorly understood. Herein, we present data on the identification of the Hog1 MAP kinase as a key regulator of C. auris skin colonization. These findings lay foundation for further characterization of unique mechanisms that promote fungal persistence on human skin.

Intestinal colonization with Candida auris and mucosal immune response in mice treated with cefoperazone oral antibiotic.

Candida auris, an emerging multi-drug resistant fungal pathogen, causes invasive infections in humans. The factors regulating the colonization of C. auris in host niches are not well understood. In this study, we examined the effect of antibiotic-induced gut dysbiosis on C. auris intestinal colonization, dissemination, microbiome composition and the mucosal immune response. Our results indicate that mice treated with cefoperazone alone had a significant increase in C. auris intestinal colonization compared to untreated control groups. A significant increase in the dissemination of C. auris from the intestine to internal organs was observed in antibiotic-treated immunosuppressed mice. Intestinal colonization of C. auris alters the microbiome composition of antibiotic-treated mice. Relative abundance of firmicutes members mainly Clostridiales and Paenibacillus were considerably increased in the cefoperazone-treated mice infected with C. auris compared to cefoperazone-treated uninfected mice. Next, we examined the mucosal immune response of C. auris infected mice and compared the results with Candida albicans infection. The number of CD11b+ CX3CR1+ macrophages was significantly decreased in the intestine of C. auris infected mice when compared to C. albicans infection. On the other hand, both C. auris and C. albicans infected mice had a comparable increase of the number of Th17 and Th22 cells in the intestine. A significant increase in Candida-specific IgA was observed in the serum of C. auris but not in the C. albicans infected mice. Taken together, treatment with broad-spectrum antibiotic increased the colonization and dissemination of C. auris from the intestine. Furthermore, findings from this study for the first time revealed the microbiome composition, innate and adaptive cellular immune response to intestinal infection with C. auris.

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.

Role of Microbiota in the Skin Colonization of Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen that can cause life-threatening infections in humans. Unlike other Candida species that colonize the gut, C. auris efficiently colonizes the skin and contaminates the patient's environment, resulting in rapid nosocomial transmission and outbreaks of systemic infections. As the largest organ of the body, the skin harbors beneficial microbiota that play a critical role to protect from invading pathogens. However, the role of skin microbiota in the colonization and pathogenesis of C. auris remains to be explored. With this perspective, we review and discuss recent insights into skin microbiota and their potential interactions with the immune system in the context of C. auris skin colonization. Understanding microbiota, C. auris, and host interactions in the skin is important to develop microbiome-based therapeutic approaches to prevent and treat this emerging fungal pathogen in humans.

Discovery of spirooxindole-pyrrolidine heterocyclic hybrids with potent antifungal activity against fungal pathogens.

Fungal pathogens mainly Candida and Cryptococcus species causes serious life-threating infections to humans, especially in individuals who are immunocompromised. Increasing frequency of antifungal drug resistance along with paucity of FDA-approved drugs suggest a dire need for new antifungal drugs. Our screening of newly synthesized spirooxindole heterocyclic hybrid compounds revealed that the novel small molecule, DPA-3, has potent antifungal activity without inducing mammalian cell cytotoxicity. Furthermore, DPA-3 significantly reduced hyphal and biofilm formation of Candida albicans ATCC 10231 strain, out-competing two FDA approved antifungal drugs. The results of our study conclude that DPA-3 is a compelling candidate for further development as an antifungal drug.

Bile Acid Regulates Mononuclear Phagocytes and T Helper 17 Cells to Control Candida albicans in the Intestine.

Invasive Candida albicans (CA) infections often arise from the intestine and cause life-threatening infections in immunocompromised individuals. The role of gut commensal microbiota, metabolites, and host factors in the regulation of CA colonization in the intestine is poorly understood. Previous findings from our lab indicate that taurocholic acid (TCA), a major bile acid present in the intestine, promotes CA colonization and dissemination. Here, we report that oral administration of TCA to CA-infected mice significantly decreased the number of mononuclear phagocytes and CD4+ IL17A+ T helper 17 cells that play a critical role in controlling CA in the intestine. Collectively, our results indicate that TCA modulates mucosal innate and adaptive immune responses to promote CA colonization in the intestine.

Evaluation of Candida spp. and Other Fungi in Feces from Dogs with Naturally Occurring Diabetes Mellitus.

Diabetes mellitus is a common endocrinopathy in dogs and in most cases is analogous to type 1 diabetes mellitus (T1DM) in humans. Candida spp. is a common commensal fungi with higher prevalence and magnitude of growth in humans with T1DM. There is currently no published information about the fungal microbiome in diabetic dogs. Therefore, the objectives of this study were to (i) determine whether diabetic dogs were more likely to have Candida spp. or other types of fungi from feces compared to non-diabetic controls, and (ii) identify variables associated with fungi colonization. Fourteen diabetic dogs and 14 age, sex, and breed matched non-diabetic healthy control dogs were included in this prospective case-control study. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for fungal identification. Diabetic dogs had greater quantitative fungal growth compared to controls (p = 0.004). Moreover, female dogs were more likely to have fungi colonization than males (p = 0.02). All instances of Candida spp. and Aspergillus spp. colonization were exclusively identified in diabetic dogs. Serum fructosamine concentration was higher in diabetic dogs with fecal colonization of Candida spp. compared to diabetic dogs without growth (p = 0.03). Our results indicate that the fungal microbiome in feces is altered in diabetic dogs, which seem to favor an increased prevalence of Candida spp. and higher quantitative fungal growth. Moreover, female sex and glycemic control could affect the intestinal mycobiome.