Resumo

During the infection process, pathogenic Cryptococcus species are exposed to harsh conditions imposed by defense cells. One of such conditions is the so-called nutritional immunity, which reduces the nutrient availability in the infection site. Among the nutrients that have limited availability during infection process, zinc is widely sequestered by host immune system. In the present work, we aim to characterize the ZIP family of zinc transporters in C. gattii. For this purpose, we performed in silico analysis employing the Saccharomyces cerevisiae sequences of Zrt1p and Zrt2p, high and low zinc affinity transmembrane transporters, respectively. We identified in the C. gattii genome sequence 4 members of the ZIP family of zinc transporters. Our previous results demonstrated that ZIP1 (CNBG_6066), ZIP2 (CNBG_2209) and ZIP3 (CNBG_5153) have elevated transcripts levels on zinc deprivation condition, particularly ZIP1 and ZIP2. To further evaluate the role of ZIP1 and ZIP2 in zinc uptake, we generated mutant strains for ZIP1 (zip1Δ) and ZIP2 (zip2Δ), as well as a double mutant strain (zip1Δ/zip2Δ). We used In-Fusion® HD Ecodry™ Cloning kit (Clontech) for inactivation allele construction and posterior biolistic transformation in C. gattii. PCR and Southern blotting analyses confirmed gene deletion. Preliminary assays revealed that zinc deprivation (DTPA 100µM) induced a severe decreased in growth of zip1Δ and zip1Δ/zip2Δ strains compared to wild-type. In addition, concentrations of up to 2.5 µM of ZnCl₂ did not fully supported growth of the zip1Δ mutant strain. However, such phenotype was not observed in the zip2Δ strain, suggesting that the ZIP1 gene product is the main zinc uptake mechanism in C. gattii. As perspectives, we aim to construct complemented strains for zip1Δ and zip2Δ, challenge the mutant strains for the main virulence factors, like melanin production, capsule formation and virulence assays.