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.

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.

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 albicans FRE8 encodes a member of the NADPH oxidase family that produces a burst of ROS during fungal morphogenesis.

Until recently, NADPH oxidase (NOX) enzymes were thought to be a property of multicellularity, where the reactive oxygen species (ROS) produced by NOX acts in signaling processes or in attacking invading microbes through oxidative damage. We demonstrate here that the unicellular yeast and opportunistic fungal pathogen Candida albicans is capable of a ROS burst using a member of the NOX enzyme family, which we identify as Fre8. C. albicans can exist in either a unicellular yeast-like budding form or as filamentous multicellular hyphae or pseudohyphae, and the ROS burst of Fre8 begins as cells transition to the hyphal state. Fre8 is induced during hyphal morphogenesis and specifically produces ROS at the growing tip of the polarized cell. The superoxide dismutase Sod5 is co-induced with Fre8 and our findings are consistent with a model in which extracellular Sod5 acts as partner for Fre8, converting Fre8-derived superoxide to the diffusible H2O2 molecule. Mutants of fre8Δ/Δ exhibit a morphogenesis defect in vitro and are specifically impaired in development or maintenance of elongated hyphae, a defect that is rescued by exogenous sources of H2O2. A fre8Δ/Δ deficiency in hyphal development was similarly observed in vivo during C. albicans invasion of the kidney in a mouse model for disseminated candidiasis. Moreover C. albicans fre8Δ/Δ mutants showed defects in a rat catheter model for biofilms. Together these studies demonstrate that like multicellular organisms, C. albicans expresses NOX to produce ROS and this ROS helps drive fungal morphogenesis in the animal host.

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.

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

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.

Temporal patterns of chronic wasting disease prion excretion in three cervid species.

Chronic wasting disease (CWD) is the only naturally occurring transmissible spongiform encephalopathy affecting free-ranging wildlife populations. Transmission of CWD occurs by direct contact or through contaminated environments; however, little is known about the temporal patterns of CWD prion excretion and shedding in wild cervids. We tested the urine and faeces of three species of captive cervids (elk, mule and white-tailed deer) at 6, 12, 18 and 24 months after oral inoculation to evaluate the temporal, species- and genotype-specific factors affecting the excretion of CWD prions. Although none of the animals exhibited clinical signs of CWD during the study, we determined that all three cervid species were excreting CWD prions by 6 months post-inoculation. Faecal samples were consistently positive for CWD prions for all three cervid species (88 %), and were more likely to be positive than urine samples (28 %). Cervids with genotypes encoding for the prion protein (PRNP) that were considered to be more susceptible to CWD were more likely to excrete CWD prions (94 %) than cervids with genotypes considered to be less susceptible (64 %). All cervids with CWD prions in their urine also had positive faeces (n=5), but the converse was not true. Our study is the first to demonstrate CWD prion excretion in urine by asymptomatic elk and mule deer. Our results indicate that the excretion of CWD prions in faeces and, to a lesser extent, urine may provide an important avenue for depositing prions in the environment.

Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation.

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.

Transcriptomic responses to prion disease in rats.

BACKGROUND: Prions diseases are fatal neurodegenerative diseases of mammals. While the molecular responses to prion infection have been extensively characterized in the laboratory mouse, little is known in other rodents. To explore these responses and make comparisons, we generated a prion disease in the laboratory rat by successive passage beginning with mouse RML prions. RESULTS: We describe the accumulation of rat prions, associated pathology and the transcriptional impact throughout the disease course. Comparative transcriptional profiling between laboratory mice and rats suggests that similar molecular and cellular processes are unfolding in response to prion infection. At the level of individual transcripts, however, variability exists between mice and rats and many genes deregulated by prion infection in mice are not affected in rats. CONCLUSION: Our findings detail the molecular responses to prion disease in the rat and highlight the usefulness of comparative approaches to understanding neurodegeneration and prion diseases in particular.

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.

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.

Emerging prion disease drives host selection in a wildlife population.

Infectious diseases are increasingly recognized as an important force driving population dynamics, conservation biology, and natural selection in wildlife populations. Infectious agents have been implicated in the decline of small or endangered populations and may act to constrain population size, distribution, growth rates, or migration patterns. Further, diseases may provide selective pressures that shape the genetic diversity of populations or species. Thus, understanding disease dynamics and selective pressures from pathogens is crucial to understanding population processes, managing wildlife diseases, and conserving biological diversity. There is ample evidence that variation in the prion protein gene (PRNP) impacts host susceptibility to prion diseases. Still, little is known about how genetic differences might influence natural selection within wildlife populations. Here we link genetic variation with differential susceptibility of white-tailed deer to chronic wasting disease (CWD), with implications for fitness and disease-driven genetic selection. We developed a single nucleotide polymorphism (SNP) assay to efficiently genotype deer at the locus of interest (in the 96th codon of the PRNP gene). Then, using a Bayesian modeling approach, we found that the more susceptible genotype had over four times greater risk of CWD infection; and, once infected, deer with the resistant genotype survived 49% longer (8.25 more months). We used these epidemiological parameters in a multi-stage population matrix model to evaluate relative fitness based on genotype-specific population growth rates. The differences in disease infection and mortality rates allowed genetically resistant deer to achieve higher population growth and obtain a long-term fitness advantage, which translated into a selection coefficient of over 1% favoring the CWD-resistant genotype. This selective pressure suggests that the resistant allele could become dominant in the population within an evolutionarily short time frame. Our work provides a rare example of a quantifiable disease-driven selection process in a wildlife population, demonstrating the potential for infectious diseases to alter host populations. This will have direct bearing on the epidemiology, dynamics, and future trends in CWD transmission and spread. Understanding genotype-specific epidemiology will improve predictive models and inform management strategies for CWD-affected cervid populations.

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.

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.

Chronic wasting disease (CWD) susceptibility of several North American rodents that are sympatric with cervid CWD epidemics.

Chronic wasting disease (CWD) is a highly contagious always fatal neurodegenerative disease that is currently known to naturally infect only species of the deer family, Cervidae. CWD epidemics are occurring in free-ranging cervids at several locations in North America, and other wildlife species are certainly being exposed to infectious material. To assess the potential for transmission, we intracerebrally inoculated four species of epidemic-sympatric rodents with CWD. Transmission was efficient in all species; the onset of disease was faster in the two vole species than the two Peromyscus spp. The results for inocula prepared from CWD-positive deer with or without CWD-resistant genotypes were similar. Survival times were substantially shortened upon second passage, demonstrating adaptation. Unlike all other known prion protein sequences for cricetid rodents that possess asparagine at position 170, our red-backed voles expressed serine and refute previous suggestions that a serine in this position substantially reduces susceptibility to CWD. Given the scavenging habits of these rodent species, the apparent persistence of CWD prions in the environment, and the inevitable exposure of these rodents to CWD prions, our intracerebral challenge results indicate that further investigation of the possibility of natural transmission is warranted.

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.