Resumo

Differential expression of genes involved in copper metabolism in Cryptococcus gattii Santos, PR ¹; Kmetzsch, L ^1,2; Staats, CC ^1,2; Schrank, A^1,2; Vainstein, MH^1,2 1 Programa de Pós-graduação em Biologia Celular e Molecular (UFRGS), Porto Alegre, Brasil 2 Centro de Biotecnologia (CBiot-UFRGS), Porto Alegre, Brasil patriciaribsant@yahoo.com.br Keywords: C. gattii, transcriptome, copper, RT-PCR The yeast Cryptococcus gattii has been considered an emerging fungal pathogen of humans and animals. Cryptococcosis caused by C. gattii and the congeneric pathogen Cryptococcus neoformans usually results in pneumonia and meningoencephalitis. Although Canada has the highest number of cases of cryptococcosis caused by C. gattii, spread of cases of cryptococcosis by C. gattii in the States of Washington and Oregon in the USA was recently identified. The ability to adapt to changes in the environment is critical for pathogen survival and for the ability to cause disease. In this context, an important factor is the copper availability in the host during infection. Recent reports have shown that copper may play a critical role in C. neoformans virulence. Our aim was to evaluate the C. gattii transcriptional profile under copper limitation conditions and to confirm the differential expression of selected genes by qRT-PCR. Samples containing yeast cells were submitted to 4 hours in different copper conditions (0, 1 or 10 µM CuSO₄). Cells were recovered and storage for posterior RNA extraction and cDNA synthesis. Quantitative RT-PCR was performed on a Real-time PCR StepOne PCR System (Applied Biosystems). All experiments were performed using three independent cultures, and each cDNA sample was analyzed in triplicate. Melting curve analysis was performed at the end of the reaction to confirm the presence of a single PCR product. Data were normalized to actin cDNAs amplified in each set of PCR experiments. Relative expression was determined by the 2 ^-ΔCT method. Statistical analyses were conducted via a two-tailed Student’s t-test. The C. gattii transcriptional profile revealed 7,704 transcripts that were identified in copper deprivation samples, without excluding isoforms of the transcripts. We selected 10 genes to validate the differential expression in different copper conditions, based on FPKM (fragments per kilobase per million fragments mapped) observed in RNAseq analysis. Seven of these genes are involved with copper transport or need copper as a co-factor and three encoded hypothetical proteins. Financial Support: CNPq, CAPES.