PKC Signaling Regulates Drug Resistance of the Fungal Pathogen Candida albicans via Circuitry Comprised of Mkc1, Calcineurin, and Hsp90

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections

Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease

Invasive fungal infections are a leading cause of mortality among immunocompromised individuals. Treatment is notoriously difficult with the limited armamentarium of antifungal drugs, whose efficacy is compromised by host toxicity, a limited activity spectrum, or the emergence of drug resistance. We previously established that the molecular chaperone Hsp90 enables the emergence and maintenance of fungal drug resistance. For the most prevalent fungal pathogen of humans, Candida albicans, Hsp90 mediates resistance to azoles, which inhibit ergosterol biosynthesis and are the most widely deployed antifungals in the clinic. For the emerging opportunistic pathogen Aspergillus terreus, Hsp90 is required for basal resistance to echinocandins, which inhibit β(1, 3)-glucan synthesis and are the only new class of antifungals to reach the clinic in decades. Here, we explore the therapeutic potential of Hsp90 inhibitors in fungal disease using a tractable host-model system, larvae of the greater wax moth Galleria mellonella, and a murine model of disseminated disease. Combination therapy with Hsp90 inhibitors that are well tolerated in humans and an azole rescued larvae from lethal C. albicans infections. Combination therapy with an Hsp90 inhibitor and an echinocandin rescued larvae from infections with the most lethal mold, Aspergillus fumigatus. In a murine model of disseminated candidiasis, genetic compromise of C. albicans HSP90 expression enhanced the therapeutic efficacy of an azole. Thus, harnessing Hsp90 provides a much-needed strategy for improving the treatment of fungal disease because it enhances the efficacy of existing antifungals, blocks the emergence of drug resistance, and exerts broad-spectrum activity against diverse fungal pathogens

Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits

D-amino acid oxidase (DAO) has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs) obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016). This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001) and replicated samples (corrected p = 0.0003). The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia