25º Congresso Brasileiro de Microbiologia

25º Congresso Brasileiro de Microbiologia

ResumoID:798-1

Área: Genética e Biologia Molecular ( Divisão N )

A PUTATIVE VACUOLAR CALCIUM ION TRANSPORTER PARTICIPATES IN CALCINEURIN PATHWAY AND VIRULENCE OF CRYPTOCOCCUS NEOFORMANS

Livia Kmetzsch Rosa E Silva (UFRGS); Charley Christian Staats (UFRGS); Ana Lusia Leal (UFRGS); Elisa Simon (UFRGS); Debora Leite Oliveira (UFRJ); Luna Sobrino (UFRJ); Fernanda Lopes Fonseca (UFRJ); Márcio Lourenço Rodrigues (UFRJ); Augusto Schrank (UFRGS); Marilene Henning Vainstein (UFRGS)

Resumo

Cryptococcus neoformans is an encapsulated yeast, that causes a life-threatening meningoencephalitis in immunocompromised individuals. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this microorganism. We previously described that the putative calcium ion transporter Vcx1 gene was upregulated during C. neoformans growth at 37°C. This gene is an ortholog of Saccharomyces cerevisiae Vcx1 gene, which encodes a vacuolar H+/Ca2+ exchanger involved in control of cytosolic calcium concentration. In order to elucidate the function of C. neoformans Vcx1 ortholog, we generated null mutants (∆Vcx1) of this gene. The cassette for gene disruption was constructed using Delsgate methodology. The biolistic transformations were conducted in C. neoformans H99 strain, and the null mutants were obtained by replacing the coding region of Vcx1 gene with a cassette conferring resistance to hygromicin. The constructions were confirmed by PCR and Southern blot analysis. The mutant strain ∆Vcx1 was hypersensitive to the calcineurin inhibitor cyclosporin at 37°C. However, we cannot detect this phenotype at 30°C. These findings indicate a possible role of Vcx1 in the calcium/calcineurin pathway of C. neoformans. We also examined the ability of the wild-type and ∆Vcx1 strains to grow on YPD plates supplemented with increasing concentrations of CaCl₂(100 to 250mM), but no growth inhibition was observed. The virulence of ∆Vcx1 was evaluated by an inhalation model of cryptococcosis in mice. The mutant had strongly attenuated virulence in comparison to the wild-type H99. The complementation of the wild type Vcx1 gene in ∆Vcx1 mutant strain, as well the analysis of capsule and melanin production of the mutant strains are being conducted. These results will contribute to a better understanding of the calcium metabolism during C. neoformans-host interaction.

Palavras-chave: Cryptococcus neoformans, calcium metabolism, virulence

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