Susceptibility to Cryptococcus neoformans Infection with Bruton's Tyrosine Kinase Inhibition.

Patients receiving the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib have an increased likelihood of fungal infections. The objectives of this study were to determine if Cryptococcus neoformans infection severity was isolate dependent with BTK inhibition and whether blocking BTK impacted infection severity in a mouse model. We compared four clinical isolates from patients on ibrutinib to virulent (H99) and avirulent (A1-35-8) reference strains. BTK knockout (KO) and wild-type (WT) C57 mice and WT CD1 mice were infected by intranasal (i.n.), oropharyngeal aspiration (OPA), and intravenous (i.v.) routes. Infection severity was assessed by survival and fungal burden (CFU per gram of tissue). Ibrutinib (25 mg/kg) or vehicle was administered daily through intraperitoneal injections. In the BTK KO model, no isolate-dependent effect on fungal burden was observed, and infection severity was not significantly different from that of the WT with i.n., OPA, and i.v. routes. Ibrutinib treatment did not impact infection severity. However, when the four clinical isolates were compared to H99, two of these isolates were less virulent, with significantly longer survival and reduced rates of brain infection. In conclusion, C. neoformans infection severity in the BTK KO model does not appear to be isolate dependent. BTK KO and ibrutinib treatment did not result in significantly different infection severities. However, based on repeated clinical observations of increased susceptibility to fungal infections with BTK inhibitor therapy, further work is needed to optimize a mouse model with BTK inhibition to better understand the role that this pathway plays in susceptibility to C. neoformans infection.

Pharmacodynamics of ATI-2307 in a rabbit model of cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is a devastating fungal disease with high morbidity and mortality. The current regimen that is standard-of-care involves a combination of three different drugs administered for up to one year. There is a critical need for new therapies due to both toxicity and inadequate fungicidal activity of the currently available antifungal drugs. ATI-2307 is a novel aryl amidine that disrupts the mitochondrial membrane potential and inhibits the respiratory chain complexes of fungi-it thus represents a new mechanism for direct antifungal action. Furthermore, ATI-2307 selectively targets fungal mitochondria via a fungal-specific transporter that is not present in mammalian cells. It has very potent in vitro anticryptococcal activity. In this study, the efficacy of ATI-2307 was tested in a rabbit model of CM. ATI-2307 demonstrated significant fungicidal activity at dosages between 1 and 2 mg/kg/d, and these results were superior to fluconazole and similar to amphotericin B treatment. When ATI-2307 was combined with fluconazole, the antifungal effect was greater than either therapy alone. While ATI-2307 has potent anticryptococcal activity in the subarachnoid space, its ability to reduce yeasts in the brain parenchyma was relatively less over the same study period. This new drug, with its unique mechanism of fungicidal action and ability to positively interact with an azole, has demonstrated sufficient anticryptococcal potential in this experimental setting to be further evaluated in clinical studies.

A ketogenic diet enhances fluconazole efficacy in murine models of systemic fungal infection.

Invasive fungal infections are a significant public health concern, with mortality rates ranging from 20% to 85% despite current treatments. Therefore, we examined whether a ketogenic diet could serve as a successful treatment intervention in murine models of Cryptococcus neoformans and Candida albicans infection in combination with fluconazole-a low-cost, readily available antifungal therapy. The ketogenic diet is a high-fat, low-carbohydrate diet that promotes fatty acid oxidation as an alternative to glycolysis through the production of ketone bodies. In this series of experiments, mice fed a ketogenic diet prior to infection with C. neoformans and treated with fluconazole had a significant decrease in fungal burden in both the brain (mean 2.66 ± 0.289 log10 reduction) and lung (mean 1.72 ± 0.399 log10 reduction) compared to fluconazole treatment on a conventional diet. During C. albicans infection, kidney fungal burden of mice in the keto-fluconazole combination group was significantly decreased compared to fluconazole alone (2.37 ± 0.770 log10-reduction). Along with higher concentrations of fluconazole in the plasma and brain tissue, fluconazole efficacy was maximized at a significantly lower concentration on a keto diet compared to a conventional diet, indicating a dramatic effect on fluconazole pharmacodynamics. Our findings indicate that a ketogenic diet potentiates the effect of fluconazole at multiple body sites during both C. neoformans and C. albicans infection and could have practical and promising treatment implications.IMPORTANCEInvasive fungal infections cause over 2.5 million deaths per year around the world. Treatments for fungal infections are limited, and there is a significant need to develop strategies to enhance antifungal efficacy, combat antifungal resistance, and mitigate treatment side effects. We determined that a high-fat, low-carbohydrate ketogenic diet significantly potentiated the therapeutic effect of fluconazole, which resulted in a substantial decrease in tissue fungal burden of both C. neoformans and C. albicans in experimental animal models. We believe this work is the first of its kind to demonstrate that diet can dramatically influence the treatment of fungal infections. These results highlight a novel strategy of antifungal drug enhancement and emphasize the need for future investigation into dietary effects on antifungal drug activity.

Efficacy of APX2039 in a Rabbit Model of Cryptococcal Meningitis.

Cryptococcal Meningitis (CM) is uniformly fatal if not treated, and treatment options are limited. We previously reported on the activity of APX2096, the prodrug of the novel Gwt1 inhibitor APX2039, in a mouse model of CM. Here, we investigated the efficacy of APX2039 in mouse and rabbit models of CM. In the mouse model, the controls had a mean lung fungal burden of 5.95 log10 CFU/g, whereas those in the fluconazole-, amphotericin B-, and APX2039-treated mice were 3.56, 4.59, and 1.50 log10 CFU/g, respectively. In the brain, the control mean fungal burden was 7.97 log10 CFU/g, while the burdens were 4.64, 7.16, and 1.44 log10 CFU/g for treatment with fluconazole, amphotericin B, and APX2039, respectively. In the rabbit model of CM, the oral administration of APX2039 at 50 mg/kg of body weight twice a day (BID) resulted in a rapid decrease in the cerebrospinal fluid (CSF) fungal burden, and the burden was below the limit of detection by day 10 postinfection. The effective fungicidal activity (EFA) was -0.66 log10 CFU/mL/day, decreasing from an average of 4.75 log10 CFU/mL to 0 CFU/mL, over 8 days of therapy, comparing favorably with good clinical outcomes in humans associated with reductions of the CSF fungal burden of -0.4 log10 CFU/mL/day, and, remarkably, 2-fold the EFA of amphotericin B deoxycholate in this model (-0.33 log10 CFU/mL/day). A total drug exposure of the area under the concentration-time curve from 0 to 24 h (AUC0-24) of 25 to 50 mg · h/L of APX2039 resulted in near-maximal antifungal activity. These data support the further preclinical and clinical evaluation of APX2039 as a new oral fungicidal monotherapy for the treatment of CM. IMPORTANCE Cryptococcal meningitis (CM) is a fungal disease with significant global morbidity and mortality. The gepix Gwt1 inhibitors are a new class of antifungal drugs. Here, we demonstrated the efficacy of APX2039, the second member of the gepix class, in rabbit and mouse models of cryptococcal meningitis. We also analyzed the drug levels in the blood and cerebrospinal fluid in the highly predictive rabbit model and built a mathematical model to describe the behavior of the drug with respect to the elimination of the fungal pathogen. We demonstrated that the oral administration of APX2039 resulted in a rapid decrease in the CSF fungal burden, with an effective fungicidal activity of -0.66 log10 CFU/mL/day, comparing favorably with good clinical outcomes in humans associated with reductions of -0.4 log10 CFU/mL/day. The drug APX2039 had good penetration of the central nervous system and is an excellent candidate for future clinical testing in humans for the treatment of CM.

Genomic Variation across a Clinical Cryptococcus Population Linked to Disease Outcome.

Cryptococcus neoformans is the causative agent of cryptococcosis, a disease with poor patient outcomes that accounts for approximately 180,000 deaths each year. Patient outcomes may be impacted by the underlying genetics of the infecting isolate; however, our current understanding of how genetic diversity contributes to clinical outcomes is limited. Here, we leverage clinical, in vitro growth and genomic data for 284 C. neoformans isolates to identify clinically relevant pathogen variants within a population of clinical isolates from patients with human immunodeficiency virus (HIV)-associated cryptococcosis in Malawi. Through a genome-wide association study (GWAS) approach, we identify variants associated with the fungal burden and the growth rate. We also find both small and large-scale variation, including aneuploidy, associated with alternate growth phenotypes, which may impact the course of infection. Genes impacted by these variants are involved in transcriptional regulation, signal transduction, glycosylation, sugar transport, and glycolysis. We show that growth within the central nervous system (CNS) is reliant upon glycolysis in an animal model and likely impacts patient mortality, as the CNS yeast burden likely modulates patient outcome. Additionally, we find that genes with roles in sugar transport are enriched in regions under selection in specific lineages of this clinical population. Further, we demonstrate that genomic variants in two genes identified by GWAS impact virulence in animal models. Our approach identifies links between the genetic variation in C. neoformans and clinically relevant phenotypes and animal model pathogenesis, thereby shedding light on specific survival mechanisms within the CNS and identifying the pathways involved in yeast persistence. IMPORTANCE Infection outcomes for cryptococcosis, most commonly caused by C. neoformans, are influenced by host immune responses as well as by host and pathogen genetics. Infecting yeast isolates are genetically diverse; however, we lack a deep understanding of how this diversity impacts patient outcomes. To better understand both clinical isolate diversity and how diversity contributes to infection outcomes, we utilize a large collection of clinical C. neoformans samples that were isolated from patients enrolled in a clinical trial across 3 hospitals in Malawi. By combining whole-genome sequence data, clinical data, and in vitro growth data, we utilize genome-wide association approaches to examine the genetic basis of virulence. Genes with significant associations display virulence attributes in both murine and rabbit models, demonstrating that our approach can identify potential links between genetic variants and patho-biologically significant phenotypes.

Exposure to the antifouling chemical medetomidine slows development, reduces body mass, and delays metamorphosis in wood frog (Lithobates sylvaticus) tadpoles.

Antifouling chemicals have a long history of causing toxicity to aquatic organisms. We measured growth and developmental timing in wood frog tadpoles exposed to the antifouling chemical medetomidine (10 nM-10 µM) starting at two different developmental stages in static renewal experiments. For tadpoles hatched from egg masses and exposed for 3 weeks to 100 nM and 1 µM, head width/total body length ratio was significantly shorter compared to control. For field-collected tadpoles at Gosner stage 24-25 and exposed for 2 weeks, 1 and 10 µM medetomidine significantly slowed development as measured by Gosner stage. Medetomidine (1 and 10 µM) significantly increased the time to metamorphosis by over 16 days on average, and at 100 nM and 1 µM, it significantly decreased mass at metamorphosis. We discuss the possible effects of antifouling chemicals containing medetomidine on globally threatened groups such as amphibians.

Exposure to SSRI-type antidepressants increases righting time in the marine snail Ilyanassa obsoleta.

Exposure to human antidepressants has been shown to disrupt locomotion and other foot-mediated mechanisms in aquatic snails. We tested the effect of three selective serotonin reuptake inhibitor (SSRI)- and one selective serotonin-norepinephrine reuptake inhibitor (SNRI)-type antidepressants on the righting response in the marine snail, Ilyanassa obsoleta. All four antidepressants (fluoxetine, sertraline, paroxetine, venlafaxine) significantly increased righting time compared with controls with an exposure time as short as 1 h. Dose responses were nonmonotonic with effects seen mainly at the lowest exposure concentrations and shortest duration. The lowest concentration to show an effect was 3.45 µg/L fluoxetine with a 2-h exposure period and is about 3.71 times higher than environmental concentrations. Our results highlight rapid disruption of another foot-mediated behavior in aquatic snails by SSRI-type antidepressants. We discuss these and other reported nonmonotonic dose responses caused by antidepressants in terms of the various possible physiological mechanisms of action in nontarget aquatic species.