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Candida spp. are opportunistic yeasts capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. We screened the Pandemic Response Box containing 400 diverse drug-like molecules active against bacteria, viruses or fungi, for inhibitors of Candida albicans and Candida auris biofilm formation. Initial hits were identified based on the demonstration of >70% inhibitory activity. Dose-response assays were used to confirm the antifungal activity of initial hits and establish their potency. The spectrum of antifungal activity of the leading compounds was determined against a panel of medically important fungi, and the in vivo activity of the leading repositionable agent was evaluated in murine models of C. albicans and C. auris systemic candidiasis. The primary screening identified 20 hit compounds, and their antifungal activity and potency against C. albicans and C. auris were validated using dose-response measurements. From these experiments, the rapalog everolimus, emerged as the leading repositionable candidate. Everolimus displayed potent antifungal activity against different Candida spp., but more moderate levels of activity against filamentous fungi. Treatment with everolimus increased survival of mice infected with C. albicans, but not those with C. auris. The screening of the Pandemic Response Box resulted in the identification of several drugs with novel antifungal activity, with everolimus emerging as the main repositionable candidate. Further in vitro and in vivo studies are needed to confirm its potential therapeutic use., (© 2023 Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Repositioning old drugs can significantly decrease the time and effort that it takes to develop novel antifungal therapeutics, which represents a pressing and unmet clinical need due to the devastating nature of fungal infections. We have previously described the activity of auranofin, a gold thiol compound used to treat rheumatoid arthritis, against Candida albicans biofilms. Here we evaluate its antifungal spectrum of action and describe its activity against a variety of medically important fungi.

Understanding the pathogenesis of infectious disease requires the examination and successful integration of parameters related to both microbial virulence and host responses. As a practical and powerful method to control microbial gene expression, including in vivo, the tetracycline-regulatable system has recently gained the favor of many investigative groups. However, some immunomodulatory effects of the tetracyclines, including doxycycline, could potentially limit its use to evaluate host responses during infection. Here we have used a well-established murine model of disseminated candidiasis, which is highly dependent on both the virulence displayed by the fungal cells and on the host immune status, to validate the use of this system. We demonstrate that the pathogenesis of the wild type C. albicans CAF2-1 strain, which does not contain any tet-regulatable element, is not affected by the presence of doxycycline. Moreover levels of key cytokines, chemokines and many other biomarkers, as determined by multi-analyte profiling, remain essentially unaltered by the presence of the antibiotic during infection. Our results indicate that the levels of doxycycline needed to control the tetracycline regulatable promoter gene expression system have no detectable effect on global host responses during candidiasis. Because tet-regulatable systems are now being increasingly used in a variety of pathogenic microorganisms, these observations have wide implications in the field of infectious diseases.

In Candida albicans, calcineurin is essential for virulence and survival during membrane perturbation by azoles. Crz1 is a proposed downstream target of calcineurin based on studies of Saccharomyces cerevisiae. However, the in vitro phenotypes of C. albicans crz1/crz1 and calcineurin mutants differ and Crz1 is not required for virulence.

Calcineurin is a calcium-activated protein phosphatase that is the target of the immunosuppressants cyclosporin A and FK506. In T cells, calcineurin controls nuclear import of the NF-AT transcription factor and gene activation. In plants and fungi, calcineurin functions in stress responses (e.g., temperature, cations, and pH) and is necessary for the virulence of the fungal pathogen Cryptococcus neoformans. Here we show that calcineurin is also required for the virulence of another major fungus that is pathogenic to humans, Candida albicans. C. albicans calcineurin mutants had significantly reduced virulence in a murine model of systemic infection. In contrast to its role in C. neoformans, calcineurin was not required for C. albicans survival at 37 degrees C. Moreover, C. albicans calcineurin mutant strains exhibited no defects in known Candida virulence traits associated with host invasion, including filamentous growth, germ tube formation, and adherence to and injury of mammalian cells. C. albicans calcineurin mutant strains failed to colonize and grow in the kidneys of infected animals and were unable to survive when exposed to serum in vitro. Our studies illustrate that calcineurin has evolved to control aspects of the virulence of two divergent fungal pathogens via distinct mechanisms that can be targeted to achieve broad-spectrum antifungal action.

Protective host defense mechanisms against vaginal Candida albicans infections are poorly understood. Although cell-mediated immunity (CMI) is the predominant host defense mechanism against most mucosal Candida infections, the role of CMI against vaginal candidiasis is uncertain, both in humans and in an experimental mouse model. The role of humoral immunity is equally unclear. While clinical observations suggest a minimal role for antibodies against vaginal candidiasis, an experimental rat model has provided evidence for a protective role for Candida-specific immunoglobulin A (IgA) antibodies. Additionally, Candida vaccination-induced IgM and IgG3 antibodies are protective in a mouse model of vaginitis. In the present study, the role of infection-induced humoral immunity in protection against experimental vaginal candidiasis was evaluated through the quantification of Candida-specific IgA, IgG, and IgM antibodies in serum and vaginal lavage fluids of mice with primary and secondary (partially protected) infection. In naïve mice, total, but not Candida-specific, antibodies were detected in serum and lavage fluids, consistent with lack of yeast colonization in mice. In infected mice, Candida-specific IgA and IgG antibodies were induced in serum with anamnestic responses to secondary infection. In lavage fluid, while Candida-specific antibodies were detectable, concentrations were extremely low with no anamnestic responses in mice with secondary infection. The incorporation of alternative protocols-including infections in a different strain of mice, prolongation of primary infection prior to secondary challenge, use of different enzyme-linked immunosorbent assay capture antigens, and concentration of lavage fluid-did not enhance local Candida-specific antibody production or detection. Additionally, antibodies were not removed from lavage fluids by being bound to Candida during infection. Together, these data suggest that antibodies are not readily present in vaginal secretions of infected mice and thus have a limited natural protective role against infection.