Author:
K Rosam1*, M Keniya2, L Zenz1, C Müller3, P Schuchter1, L Bachstein1, BC Monk2, M Lackner1
Author address:
1Institute of Hygiene and medical Microbiology, Medical University, Innsbruck, Austria
2Sir John Walsh Research Institute and Department of oral Sciences, University of Otago, Dunedin, New Zealand
3Department of Pharmacy, Ludwig-Maximilians University, Munich, Germany
Full conference title:
10th Advances Against Aspergillosis and Mucormycosis
Date: 2 February 2022
Abstract:
Purpose:
The COVID-19 pandemic has caused a dramatic increase in the global incidence of mucormycosis. Particularly in India and Brazil cohorts of SARS-COV2 survivors experienced mucormycosis epidemics. Mucor circinelloides is one of the causative agents of this lethal fungal infection. Mucormycetes are intrinsically resistant to echinocandins and short-tailed azoles, leaving amphotericin B (AMB), posaconazole (PSC), and isavuconazole (IVU) as the only systemic treatment options.The mechanism responsible for mucormycete resistance to short-tailed azoles is not fully understood. We have tested the hypothesis that the amino acid (AA) substitutions Y129F and V293A in the ligand-binding pocket (LBP) of the azole target sterol-14α-demethylase (SDM) confer intrinsic resistance to short-tailed azoles.
Methods:
Mucor circinelloides SDM-F1 and SDM-F5 and their cognate NADPH-cytochrome-P450-reductase (CPR) were expressed in a Saccharomyces cerevisiae host at the PDR5 and PDR15 loci, respectively. The hypersensitive host is deleted in seven ABC transporters, harbours a pdr1-3 gain of function mutation, and has a galactose-inducible/glucose-repressible native ScERG11. The effect of the two postulated AAs on drug binding were studied in SDM-F1 by introducing Y129F and V293A substitutions, individually and in combination, in SDM-F5 with the revertants F129Y, A293V, and F129Y A293V.
All recombinant strains were confirmed by PCR and DNA sequence analysis. Susceptibility testing used EUCAST guidelines for AMB, voriconazole (VRC), fluconazole (FLC), IVU, PSC, and itraconazole (ITC). Growth kinetics plus SDS-PAGE and Western blots were used to characterize recombinant protein expression. The accumulation of ergosterol and its precursors as well as toxic intermediates were analysed by gas chromatography mass spectrometry under azole treatment.
Results:
Wild-type McSDM-F1 and McSDM-F5, with and without CPR, were functionally expressed in the S. cerevisiae host. Highest MICs against short-tailed azoles were found for McSDM-F5-CPR (VRC: 4 mg/L and FLC: 128 mg/L), followed by McSDM-F5 (VRC: 0.6 mg/L and FLC: 42.7 mg/L), and McSDM-F1-CPR (VRC: 0.3 mg/L and FLC: 14.5 mg/L). For McSDM-F5-CPR, IVU showed high MIC values like VRC (4 mg/L). The susceptibilities to long-tailed azoles and AmB were unchanged. Susceptible phenotypes were obtained with the McSDM-F5 revertants and single AA substitutions in the McSDM-F1 paralog.
Genetic and phenotypic tests confirmed the expected genomic sites of the recombinant genes and strain fitness. Recombinant protein expression relative to the ScERG11 recombinant control was comparable for McSDM-F1 and McSDM-F1-CPR (98% and 90%) and higher for McSDM-F5 (162%) and McSDM-F5-CPR (177%). Only SDM-F1 Y129F V293A was not expressed. VRC treatment caused accumulation of a toxic sterol (14-methylergosta-8,24(28)-diene-3beta,6alpha-diol in variants expressing single expressing McSDM-F1 or McSDM-F5 (both 14%). The reduction in ergosterol content was more extensive in McSDM-F1 constructs (McSDM-F1: 17%, McSDM-F1-CPR 6%) than in McSDM-F5 constructs (McSDM-F5: 5% McSDM-F5-CPR: 3%).
Conclusion:
Wild type M. circinelloides SDM and a range of its variants have been functionally expressed in S. cerevisiae. Susceptibility profiling, protein expression studies and sterol analysis demonstrate that the intrinsic AA substitutions F129 and A291 in the McSDM-F5 paralog confer intrinsic resistance to short-tailed azole drugs.
Abstract Number: 46
Conference Year: 2022
Link to conference website: https://aaam2022.org/
URL Conference abstract: