The Influence of Genetic Stability onAspergillus fumigatusVirulence and Azole Resistance

Abstract

Genetic stability is extremely important for the survival of every living organism, and a very complex set of genes has evolved to cope with DNA repair upon DNA damage. Here, we investigated the Aspergillus fumigatus AtmA (Ataxia-telangiectasia mutated, ATM) and AtrA kinases, and how they impact virulence and the evolution of azole resistance. We demonstrated that A. fumigatus atmA and atrA null mutants are haploid and have a discrete chromosomal polymorphism. The ΔatmA and ΔatrA strains are sensitive to several DNA-damaging agents, but surprisingly both strains were more resistant than the wild-type strain to paraquat, menadione, and hydrogen peroxide. The atmA and atrA genes showed synthetic lethality emphasizing the cooperation between both enzymes and their consequent redundancy. The lack of atmA and atrA does not cause any significant virulence reduction in A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonela. Wild-type, ΔatmA, and ΔatrA populations that were previously transferred 10 times in minimal medium (MM) in the absence of voriconazole have not shown any significant changes in drug resistance acquisition. In contrast, ΔatmA and ΔatrA populations that similarly evolved in the presence of a subinhibitory concentration of voriconazole showed an ∼5–10-fold increase when compared to the original minimal inhibitory concentration (MIC) values. There are discrete alterations in the voriconazole target Cyp51A/Erg11A or cyp51/erg11 and/or Cdr1B efflux transporter overexpression that do not seem to be the main mechanisms to explain voriconazole resistance in these evolved populations. Taken together, these results suggest that genetic instability caused by ΔatmA and ΔatrA mutations can confer an adaptive advantage, mainly in the intensity of voriconazole resistance acquisition.

The Aspergillus fumigatus Mismatch Repair MSH2 Homolog Is Important for Virulence and Azole Resistance

“…As previously described (19), the karyotype of the A. fumigatus wild type is composed of five chromosome-sized bands distributed as follows: three megabase bands of 5.7, 4.9, and 3.9 Mb; two middle-sized bands of 2.2 and 1.8 Mb (Fig.…”

“…We have assigned the in silico chromosomes (Chr1 to Chr8) of A. fumigatus Af293 to the chromosomal bands separated by pulsed-field gel electrophoresis (PFGE) (19). In the wild type, each chromosomal band is associated with only one chromosome, with the exception of the 3.9-Mb band, which harbors four in silico chromosomes (Chr3, Chr4, Chr5, and Chr6).…”

“…mellonella obtained by breeding adult moths (31). The larvae infection protocol was done according to the method reported previously by dos Reis et al (19).…”

“…We previously investigated the A. fumigatus AtmA (Ataxia-telangiectasia mutated) and AtrA kinases and how they impact virulence and the evolution of azole resistance (19). We have observed that genetic instability caused by Δ atmA and Δ atrA mutations can confer an adaptive advantage mainly in the intensity of voriconazole resistance acquisition but not in virulence.…”

Ploidy Determination in the Pathogenic Fungus Sporothrix spp.

“…Conidia were recovered through successive gaze filtration. Aspergillus nidulans A4 and R21/R153 (dos Reis et al., 2018), haploid and diploid strains, respectively, were maintained in YPD solid medium at 30°C. For subsequent analysis, conidia were collected and washed with phosphate buffered saline (PBS) (1×) (8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH 2 PO 4 per liter of sterilized water).…”