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.

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.

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.

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.

Tetraspanin CD82 restrains phagocyte migration but supports macrophage activation.

Phagocytes migrate into tissues to combat infection and maintain tissue homeostasis. As dysregulated phagocyte migration and function can lead to inflammation or susceptibility to infection, identifying molecules that control these processes is critical. Here, we show that the tetraspanin CD82 restrains the migration of neutrophils and macrophages into tissues. Cd82 -/- phagocytes exhibited excessive migration during in vivo models of peritoneal inflammation, superfusion of CXCL1, retinopathy of prematurity, and infection with the protozoan parasite L. mexicana. However, with the latter, while Cd82 -/- macrophages infiltrated infection sites at higher proportions, cutaneous L. mexicana lesions were larger and persisted, indicating a failure to control infection. Analyses of in vitro bone-marrow-derived macrophages showed CD82 deficiency altered cellular morphology, and impaired gene expression and metabolism in response to anti-inflammatory activation. Altogether, this work reveals an important role for CD82 in restraining phagocyte infiltration and mediating their differentiation in response to stimulatory cues.

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.

Development of a Novel In Vitro Primary Human Monocyte-Derived Macrophage Model To Study Reactivation of HIV-1 Transcription.

Latent HIV reservoirs persist in people living with HIV despite effective antiretroviral therapy and contribute to rebound viremia upon treatment interruption. Macrophages are an important reservoir cell type, but analysis of agents that modulate latency in macrophages is limited by lack of appropriate in vitro models. We therefore generated an experimental system to investigate this by purifying nonproductively infected human monocyte-derived macrophages (MDM) following in vitro infection with an M-tropic enhanced green fluorescent protein reporter HIV clone and quantified activation of HIV transcription using live-cell fluorescence microscopy. The proportion of HIV-infected MDM was quantified by qPCR detection of HIV DNA, and GFP expression was validated as a marker of productive HIV infection by colabeling of HIV Gag protein. HIV transcription spontaneously reactivated in latently infected MDM at a rate of 0.22% ± 0.04% cells per day (mean ± the standard error of the mean, n = 10 independent donors), producing infectious virions able to infect heterologous T cells in coculture experiments, and both T cells and TZM-bl cells in a cell-free infection system using MDM culture supernatants. Polarization to an M1 phenotype with gamma interferon plus tumor necrosis factor resulted in a 2.3-fold decrease in initial HIV infection of MDM (P < 0.001, n = 8) and a 1.4-fold decrease in spontaneous reactivation (P = 0.025, n = 6), whereas M2 polarization using interleukin-4 prior to infection led to a 1.6-fold decrease in HIV infectivity (P = 0.028, n = 8) but a 2.0-fold increase in the rate of HIV reactivation in latently infected MDM (P = 0.023, n = 6). The latency reversing agents bryostatin and vorinostat, but not panobinostat, significantly induced HIV reactivation in latently infected MDM (P = 0.031 and P = 0.038, respectively, n = 6). IMPORTANCE Agents which modulate latent HIV reservoirs in infected cells are of considerable interest to HIV cure strategies. The present study characterizes a robust, reproducible model enabling quantification of HIV reactivation in primary HIV-infected human MDM which is relatively insensitive to the monocyte donor source and hence suitable for evaluating latency modifiers in MDM. The rate of initial viral infection was greater than the rate of HIV reactivation, suggesting that different mechanisms regulate these processes. HIV reactivation was sensitive to macrophage polarization, suggesting that cellular and tissue environments influence HIV reactivation in different macrophage populations. Importantly, latently infected MDM showed different susceptibilities to certain latency-reversing agents known to be effective in T cells, indicating that dedicated strategies may be required to target latently infected macrophage populations in vivo.

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.

Priority effects dictate community structure and alter virulence of fungal-bacterial biofilms.

Polymicrobial biofilms are a hallmark of chronic wound infection. The forces governing assembly and maturation of these microbial ecosystems are largely unexplored but the consequences on host response and clinical outcome can be significant. In the context of wound healing, formation of a biofilm and a stable microbial community structure is associated with impaired tissue repair resulting in a non-healing chronic wound. These types of wounds can persist for years simmering below the threshold of classically defined clinical infection (which includes heat, pain, redness, and swelling) and cycling through phases of recurrent infection. In the most severe outcome, amputation of lower extremities may occur if spreading infection ensues. Here we take an ecological perspective to study priority effects and competitive exclusion on overall biofilm community structure in a three-membered community comprised of strains of Staphylococcus aureus, Citrobacter freundii, and Candida albicans derived from a chronic wound. We show that both priority effects and inter-bacterial competition for binding to C. albicans biofilms significantly shape community structure on both abiotic and biotic substrates, such as ex vivo human skin wounds. We further show attachment of C. freundii to C. albicans is mediated by mannose-binding lectins. Co-cultures of C. freundii and C. albicans trigger the yeast-to-hyphae transition, resulting in a significant increase in neutrophil death and inflammation compared to either species alone. Collectively, the results presented here facilitate our understanding of fungal-bacterial interactions and their effects on host-microbe interactions, pathogenesis, and ultimately, wound healing.

Neutrophils From Patients With Invasive Candidiasis Are Inhibited by Candida albicans Biofilms.

Invasive candidiasis frequently involves medical device placement. On the surfaces of these devices, Candida can form biofilms and proliferate in adherent layers of fungal cells surrounded by a protective extracellular matrix. Due in part to this extracellular matrix, biofilms resist host defenses and antifungal drugs. Previous work (using neutrophils from healthy donors) found that one mechanism employed to resist host defenses involves the inhibition of neutrophil extracellular traps (NET) formation. NETs contain nuclear DNA, as well as antimicrobial proteins that can ensnare pathogens too large or aggregated to be effectively killed by phagocytosis. Given that these neutrophil structures are anticipated to have activity against the large aggregates of C. albicans biofilms, understanding the role of this inhibition in patients could provide insight into new treatment strategies. However, prior work has not included patients. Here, we examine NET formation by neutrophils collected from patients with invasive candidiasis. When compared to neutrophils from healthy participants, we show that patient neutrophils exhibit a heightened background level of NET release and respond to a positive stimulus by producing 100% more NETs. However, despite these physiologic differences, patient neutrophil responses to C. albicans were similar to healthy neutrophils. For both groups, planktonic cells induce strong NET release and biofilms inhibit NET formation. These results show that a mechanism of immune evasion for fungal biofilms translates to the clinical setting.

Candida auris Forms High-Burden Biofilms in Skin Niche Conditions and on Porcine Skin.

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.

Insight into Neutrophil Extracellular Traps through Systematic Evaluation of Citrullination and Peptidylarginine Deiminases.

In rheumatoid arthritis, an autoimmune inflammatory arthritis, citrullinated proteins are targeted by autoantibodies and thus thought to drive disease. Neutrophil extracellular traps (NETs) are a source of citrullinated proteins and are increased in rheumatoid arthritis and therefore also implicated in disease pathogenesis. However, not all NETs are citrullinated. One theory aiming to clarify the intersection of citrullination, NETs, and rheumatoid arthritis suggests that specific stimuli induce different types of NETs defined by citrullination status. However, most studies do not evaluate uncitrullinated NETs, only citrullinated or total NETs. Further, the requirement for peptidylarginine deiminase (PAD) 2 and 4, two important citrullinating enzymes in neutrophils and rheumatoid arthritis, in the formation of different NETs has not been clearly defined. To determine if specific stimulants induce citrullinated or uncitrullinated NETs and if those structures require PAD2 or PAD4, human and murine neutrophils, including from PAD4-/- and PAD2-/- mice, were stimulated in vitro and NETs imaged and quantified. In humans, phorbol myristate acetate (PMA), ionomycin, monosodium urate (MSU), and Candida albicans induced NETs with MSU and C. albicans inducing primarily citrullinated, PMA primarily uncitrullinated, and ionomycin a mix of NETs. Only ionomycin and C. albicans were strong inducers of NETs in mice with ionomycin-induced NETs mostly citrullinated and C. albicans-induced NETs a mix of citrullinated and uncitrullinated. Interestingly, no stimulus induced exclusively citrullinated or uncitrullinated NETs. Further, PAD4 was required for citrullinated NETs only, whereas PAD2 was not required for either NET in mice. Therefore, specific stimuli induce varying proportions of both citrullinated and uncitrullinated NETs with different requirements for PAD4. These findings highlight the complexity of NET formation and the need to further define the mechanisms by which different NETs form and their implications for autoimmune disease.

Emerging Fungal Pathogen Candida auris Evades Neutrophil Attack.

Candida auris has recently emerged as the first fungal pathogen to cause a global public health threat. The reason this species is causing hospital-associated outbreaks of invasive candidiasis with high mortality is unknown. In this study, we examine the interaction of C. auris with neutrophils, leukocytes critical for control of invasive fungal infections. We show that human neutrophils do not effectively kill C. auris Compared to Candida albicans, neutrophils poorly recruited to C. auris and failed to form neutrophil extracellular traps (NETs), which are structures of DNA, histones, and proteins with antimicrobial activity. In mixed cultures, neutrophils preferentially engaged and killed C. albicans over C. auris Imaging of neutrophils in a zebrafish larval model of invasive candidiasis revealed the recruitment of approximately 50% fewer neutrophils in response to C. auris compared to C. albicans Upon encounter with C. albicans in the zebrafish hindbrain, neutrophils produced clouds of histones, suggesting the formation of NETs. These structures were not observed in C. auris infection. Evasion of neutrophil attack and innate immunity offers an explanation for the virulence of this pathogen.IMPORTANCE The emerging fungal pathogen Candida auris has produced numerous outbreaks of invasive disease in hospitals worldwide. Why this species causes deadly disease is unknown. Our findings reveal a failure of neutrophils to kill C. auris compared to the most commonly encountered Candida species, C. albicans While neutrophils produce neutrophil extracellular traps (NETs) upon encounter with C. albicans, these antimicrobial structures are not formed in response to C. auris Using human neutrophils and a zebrafish model of invasive candidiasis, we show that C. auris poorly recruits neutrophils and evades immune attack. Identification of this impaired innate immune response to C. auris sheds light on the dismal outcomes for patients with invasive disease.

Echinocandin Treatment of Candida albicans Biofilms Enhances Neutrophil Extracellular Trap Formation.

The nosocomial pathogen Candida albicans forms biofilms on medical devices that persist in the face of antifungals and host defenses. Echinocandins, the most effective antibiofilm drugs, have recently been shown to augment the activity of neutrophils against biofilms through an unknown mechanism. Here, we show that treatment of C. albicans biofilms with subinhibitory concentrations of echinocandins promotes the formation of neutrophil extracellular traps (NETs), structures of DNA, histones, and antimicrobial proteins with antifungal activity.

Mineral licks as environmental reservoirs of chronic wasting disease prions.

Chronic wasting disease (CWD) is a fatal neurodegenerative disease of deer, elk, moose, and reindeer (cervids) caused by misfolded prion proteins. The disease has been reported across North America and recently discovered in northern Europe. Transmission of CWD in wild cervid populations can occur through environmental routes, but limited ability to detect prions in environmental samples has prevented the identification of potential transmission "hot spots". We establish widespread CWD prion contamination of mineral licks used by free-ranging cervids in an enzootic area in Wisconsin, USA. We show mineral licks can serve as reservoirs of CWD prions and thus facilitate disease transmission. Furthermore, mineral licks attract livestock and other wildlife that also obtain mineral nutrients via soil and water consumption. Exposure to CWD prions at mineral licks provides potential for cross-species transmission to wildlife, domestic animals, and humans. Managing deer use of mineral licks warrants further consideration to help control outbreaks of CWD.

Gene delivery of medium chain acyl-coenzyme A dehydrogenase induces physiological cardiac hypertrophy and protects against pathological remodelling.

We previously showed that medium chain acyl-coenzyme A dehydrogenase (MCAD, key regulator of fatty acid oxidation) is positively modulated in the heart by the cardioprotective kinase, phosphoinositide 3-kinase (PI3K(p110α)). Disturbances in cardiac metabolism are a feature of heart failure (HF) patients and targeting metabolic defects is considered a potential therapeutic approach. The specific role of MCAD in the adult heart is unknown. To examine the role of MCAD in the heart and to assess the therapeutic potential of increasing MCAD in the failing heart, we developed a gene therapy tool using recombinant adeno-associated viral vectors (rAAV) encoding MCAD. We hypothesised that increasing MCAD expression may recapitulate the cardioprotective properties of PI3K(p110α). rAAV6:MCAD or rAAV6:control was delivered to healthy adult mice and to mice with pre-existing pathological hypertrophy and cardiac dysfunction due to transverse aortic constriction (TAC). In healthy mice, rAAV6:MCAD induced physiological hypertrophy (increase in heart size, normal systolic function and increased capillary density). In response to TAC (~15 weeks), heart weight/tibia length increased by ~60% in control mice and ~45% in rAAV6:MCAD mice compared with sham. This was associated with an increase in cardiomyocyte cross-sectional area in both TAC groups which was similar. However, hypertrophy in TAC rAAV6:MCAD mice was associated with less fibrosis, a trend for increased capillary density and a more favourable molecular profile compared with TAC rAAV6:control mice. In summary, MCAD induced physiological cardiac hypertrophy in healthy adult mice and attenuated features of pathological remodelling in a cardiac disease model.