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Stress-induced changes in the lipid microenvironment of β-(1,3)-D-glucan synthase cause clinically important echinocandin resistance in Aspergillus fumigatus

ABSTRACT: Aspergillus species are a leading cause of invasive fungal infections. As resistance to first-line therapy involving triazole antifungal agents is rising, second-line therapy with echinocandins is expanding. Resistance to echinocandins is well-established to result from amino acid substitutions in the echinocandin drug target β-(1,3)-D-glucan synthase encoded by the fks1 gene. Recently, we identified several high MIC clinical isolates of A. fumigatus from patients failing echinocandin therapy that did not contain any mutation in the fks1 gene, indicating that echinocandin resistance in these isolates results from an undefined mechanism. To explore possible new mechanisms of resistance, we used a lab-derived strain, RG101, with a nearly identical susceptibility phenotype, as a model system. This strain does not contain fks1 mutations but showed prominent resistance to the echinocandin class drug caspofungin (CAS), while remaining sensitive to other echinocandins, azoles and polyenes. Glucan synthase isolated from RG101 was fully sensitive to drug. Yet, exposure to CAS during its growth yielded a modified enzyme that was insensitive (4-log orders) in kinetic inhibition assays to CAS, as well as other echinocandins. This induction of cross-resistance by CAS was also observed in clinical isolates. To determine the nature of a presumptive posttranslational modification (PTM) of the enzyme, we analyzed whole enzyme PTMs, including the known hot-spot regions, for methylation, acetylation and phosphorylation. While we did not identify any PTMs linked to resistance, analysis of the lipid microenvironment of CAS-induced resistant enzyme revealed a prominent increase in the abundance of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to sensitive enzyme in in vitro kinetic inhibition assays recapitulated the CAS insensitivity of the cellular-derived enzyme. To further examine induction of drug-induced resistance, we used an in vitro assay to demonstrate that CAS, but not other echinocandin class drugs, prominently induced the production of mitochondrial-derived Reactive Oxygen Species (ROS) in A. fumigatus. RNASeq evaluation of whole cells confirmed a ROS signature in cells treated with CAS. Dampening the formation of ROS by antimycin A or thiourea eliminated the induction of drug resistance by CAS. We conclude that CAS-induced formation of ROS promotes a cellular stress response that alters the composition of plasma membrane lipids surrounding glucan synthase, changing its enzymatic properties to make it insensitive to echinocandins. This stress-induced response constitutes a novel mechanism of echinocandin resistance in Aspergillus, with implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Neosartorya Fumigata (aspergillus Fumigatus)

SUBMITTER: Thomas Krüger

LAB HEAD: Axel A. Brakhage

PROVIDER: PXD012438 | Pride | 2019-09-03

REPOSITORIES: Pride

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Stress-Induced Changes in the Lipid Microenvironment of β-(1,3)-d-Glucan Synthase Cause Clinically Important Echinocandin Resistance in Aspergillus fumigatus.

Satish Shruthi S Jiménez-Ortigosa Cristina C Zhao Yanan Y Lee Min Hee MH Dolgov Enriko E Krüger Thomas T Park Steven S Denning David W DW Kniemeyer Olaf O Brakhage Axel A AA Perlin David S DS

mBio 20190604 3

Aspergillus fumigatus is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) A. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in β-(1,3)-d-glucan synthase encoded by the fks1 gene. Yet these clinical isolates did not cont ...[more]

PMID: 31164462

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			Action	DRS
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	C.mgf	Mgf
	C.msf	Msf
	C.pdResult	Other
	C.pep.xml	Pepxml