Clorgyline Analogs Synergize with Azoles against Drug Efflux in Candida auris.

Concern about the global emergence of multidrug-resistant fungal pathogens led us to explore the use of combination therapy to combat azole resistance in Candida auris. Clorgyline had previously been shown to be a multi-target inhibitor of Cdr1 and Mdr1 efflux pumps of Candida albicans and Candida glabrata. A screen for antifungal sensitizers among synthetic analogs of Clorgyline detected interactions with the C. auris efflux pump azole substrates Posaconazole and Voriconazole. Of six Clorgyline analogs, M19 and M25 were identified as potential sensitizers of azole resistance. M19 and M25 were found to act synergistically with azoles against resistant C. auris clade I isolates and recombinant Saccharomyces cerevisiae strains overexpressing C. auris efflux pumps. Nile Red assays with the recombinant strains showed M19 and M25 inhibited the activity of Cdr1 and Mdr1 efflux pumps that are known to play key roles in azole resistance in C. auris clades I, III, and IV. While Clorgyline, M19 and M25 uncoupled the Oligomycin-sensitive ATPase activity of Cdr1 from C. albicans and C. auris, their mode of action is yet to be fully elucidated. The experimental combinations described herein provides a starting point to combat azole resistance dominated by overexpression of CauCdr1 in C. auris clades I and IV and CauMdr1 in C. auris clade III.

Functional Expression of Recombinant Candida auris Proteins in Saccharomyces cerevisiae Enables Azole Susceptibility Evaluation and Drug Discovery.

Candida auris infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in C. auris are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes CDR1 and MDR1. We report the establishment of a novel platform for molecular analysis and drug screening based on acquired azole-resistance mechanisms found in C. auris. Constitutive functional overexpression of wild-type C. auris Erg11, Erg11 with amino acid substitutions Y132F or K143R and the recombinant efflux pumps Cdr1 and Mdr1 has been achieved in Saccharomyces cerevisiae. Phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. Strains overexpressing the Cdr1 protein were pan-azole resistant. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Type II binding spectra showed tight azole binding to the affinity-purified recombinant CauErg11 protein. The Nile Red assay confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 exhibited ATPase activity that was inhibited by Oligomycin. The S. cerevisiae overexpression platform enables evaluation of the interaction of existing and novel azole drugs with their primary target CauErg11 and their susceptibility to drug efflux.

Microbicidal activity of N-chlorotaurine can be enhanced in the presence of lung epithelial cells.

BACKGROUND: N-chlorotaurine (NCT) is an endogenous active chlorine compound that can be used as an antiseptic and anti-infective in different body regions. Recently, tolerability of inhaled NCT has been demonstrated in humans so that it is of interest for future treatment of cystic fibrosis. In the present study, we tested the bactericidal and fungicidal activity of NCT in different lung cell culture models. METHODS: Bacteria (Staphylococcus aureus, Pseudomonas aeruginosa) and fungi (Candida albicans, Exophiala dermatitidis) were co-incubated with lung epithelial cell cultures, and after 4 h NCT was added. After different incubation times, aliquots were removed and quantitative cultures were performed. RESULTS: NCT at the therapeutically applied concentration of 1% (55 mM) completely killed the test pathogens within 15 - 30 min at 20 °C and at 37 °C. Killing by 0.3% NCT lasted up to 4 h dependent on the pathogen at 20 °C and up to 1 h at 37 °C. 0.1% NCT was the threshold concentration for killing since this amount of oxidation capacity was consumed by reactions with the organic compounds of the medium within 3 h (20 °C) and 0.5 h (37 °C). CONCLUSIONS: NCT in therapeutic concentration demonstrated its microbicidal activity in the presence of lung epithelial cells. Remarkably, particularly the fungicidal activity was higher under these conditions than in phosphate buffer. This can be explained by formation of the stronger microbicidal monochloramine in equilibrium by transchlorination. The results suggest the suitability of NCT as inhalation medication in the lung.