Author:
I Gonzalez-Jimenez1*, J Lucio1, A Roldan1, L Alcazar-Fuoli1,2, E Mellado1,2
Author address:
1Mycology Reference Labortory, Instituto de Salud Carlos III, Madrid, Spain
2Spanish Network for Research in Infectious Diseases, Instituto de Salud Carlos III, Madrid, Spain
Full conference title:
10th Advances Against Aspergillosis and Mucormycosis
Date: 2 February 2022
Abstract:
Purpose:
The increasing detection of azole resistant Aspergillus fumigatus isolates is threatening the azole class effectiveness in the aspergilosis management. Fungi exhibit a diversity of mechanisms to create genetic variation that eventually lead to the selection and spread of resistant fungal pathogens. In a previous WGS analysis, a collection of azole susceptible and resistant A. fumigatus genomes from very diverse geographical origins were differentiated in four clusters. The most remarkable finding was that all genomes harboring the azole resistance mechanism TR34/L98H in cyp51A were included within the same cluster. The genetic closeness of the strains harboring TR insertions suggests additional genetic mechanisms operating in them that in turn result in the selection of genotypes that fit better to the environment. Some studies have suggested the genetic instability in A. fumigatus as a possible mechanism of evolving azole resistance. One of the systems in charge of recognition and repairing the mistakes during cell replication is the DNA mismatch repair (MMR) system. Two major protein complexes constitute the MMR pathway: MutS, recognizing the mismatch, and MutL, removing the strand with the mistake. Here, we investigate four A. fumigatus MMR genes: msh2, msh6, pms1 and mlh1 and their relation with azole resistance.
Methods:
We examined the MMR gene variations in 168 A. fumigatus genomes including azole susceptible and resistant strains. The msh6 (Afu4g08300), msh2 (Afu3g09850), pms1 (Afu2g13410) and mlh1 (Afu5g11700) genes were analyzed. The gene msh6 was deleted in an akuBKU80 A. fumigatus strain by protoplast transformation experiments. The msh6 knock out isolates were subjected to different fitness tests: oxidative stress with menadione, cell wall studies with calcofluor and congo red and growth at different temperatures. Mutagenesis experiments were carried out using progressive exposure to progressive concentrations of benomyl, prochloraz and azoxystrobin alone and in combination.
Results:
Three MMR genes (msh2, pms1, mlh1) had low genetic variability. Only a few mutations were detected in some strains showing no relation with their azole susceptibility phenotype. Only the gene msh6 had a mutation (G240A), harbored by 41% of the strains, all of them closely related in the phylogenic tree and with the majority also harboring mutations in the cyp51A gene. The mutagenesis experiments, performed with wild type msh6 and G240A-msh6 strains, resulted in the recovery of isolates with mutations F129L and G143A, in the cytB gene under azoxystrobin selection. All of them occurred in a strain harboring the mutation G240A in Msh6. We were unable to recover any antifungal resistant mutant from the msh6 wild type strain.The Δmsh6
strains were indistinguishable from the akuBKU80 parental strain when they were grown at 37-60°C temperature range or in the osmotic or cell wall stresses testing. Mutagenesis experiments using other drugs (benomyl and prochloraz) and with the Δmsh6 strains are currently in progress.
Conclusion:
Modifications in genes involved in the MMR system could be related to a higher mutation rate and contribute to resistance acquisition. This study suggests a possible link between alterations in Msh6and the development of antifungal resistance in A. fumigatus.
Abstract Number: 53
Conference Year: 2022
Link to conference website: https://aaam2022.org/
URL Conference abstract: