In this work we aimed to obtain a C. neoformans mutant strain for the putative ATL1 (Alkyltransferase-like protein) gene in order to characterize its phenotypes and virulence attributes, and to assess the mitotic silencing rate for the URA5 transgene in comparison to the JF289 parental strain.
Bioinformatics analyses for the C. neoformans CNAG_02105 sequence suggested that it might encode for an ATL1, a gene function which has not been described for C. neoformans to date. The double-joint PCR technique was employed for the construction of the deletion cassettes aiming the substitution of the ATL1 ORF by the neomycin resistance marker. Genetic transformation was performed by biolistics and two independent atl1∆ mutant strains were confirmed by PCR analyses. Virulence phenotypes (thermotolerance, capsule formation, melanin production, active urease secretion) and the sexual cycle development were assayed in vitro. Virulence assessment in the Galleria mellonella larvae system was also performed. Subsequently, the growth properties were compared to the H99 and JF289 strains. Cells were grown on YPD medium containing: KCl, NaCl or D-Sorbitol as osmotic stressors; Congo Red or SDS as cell wall stressors; H2O2 or menadione as oxidative stressors and the alkylating agents methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS). Resistance to the genotoxic agent hydroxyurea, to fluconazole, to UV radiation exposure and to culture medium pH variation was also evaluated. Mutant strains URA5 transgene silencing rate analysis by growth on 5-FOA supplemented medium are in progress.
The atl1∆ mutant strains did not present differences in thermotolerance, expansion of the polysaccharide capsule, cell wall melanization, urease production, sexual cycle development and virulence when compared to the control strains. No phenotype alterations were detected for growth under stress conditions either. On the other hand, the mutant strains presented marked hypersensitivity to the EMS alkylating agent, but not to MMS. EMS provokes the guanine O6 position alkylation, thus generating O6-etG, a DNA damage structure that is repaired by the ATL1 protein.
Collectively, the bioinformatics and experimental analyses suggest that the CNAG_02105 DNA sequence encodes an ATL1 protein in C. neoformans.

Obtainment and phenotypic characterization of a mutant strain for the putative ATL gene in the Cryptococcus neoformans JF289 strain