25º Congresso Brasileiro de Microbiologia

25º Congresso Brasileiro de Microbiologia

ResumoID:794-1

Área: Genética e Biologia Molecular ( Divisão N )

IDENTIFICATION OF GENES INVOLVED IN IRON HOMESTAIS IN THE PATHOGENIC YEAST CRYPTOCOCCUS GATTII

Charley Christian Staats (UFRGS); Karina Bohrer do Amaral (UFRGS); Elisa Simon (UFRGS); Anaméli Lipréri (UFRGS); Lívia Kmetzsch Rosa E Silva (UFRGS); Augusto Schrank (UFRGS); Marilene Henning Vainstein (UFRGS)

Resumo

Species from the Cryptococcus neoformans complex are encapsulated yeasts and the etiological agent of cryptococcosis, a potentially fatal disease that is characterized mainly by meningitis. The disease development is limited by the microorganism viability in the host milieu, which is highly dependent of nutrients availability, such as carbon and nitrogen sources. Microelements (e.g. iron, copper and zinc) are also fundamental for the microorganisms, since they are incorporated in a number of enzymes involved in several biological processes. Iron metabolism is the target of numerous studies and has show to be fundamental to development and virulence in human pathogenic microorganisms. For the opportunistic pathogen C. neoformans, some genes that codes for proteins involved in uptake, storage and in regulation of iron homeostasis were already characterized. However, in the primary pathogen Cryptococcus gattii, no reports of genes involved in micronutrients metabolism were described. In order to elucidate the molecular mechanisms that controls the micronutrients homeostasis in Cryptococcus gattii R265 strain, the responsible for the outbreak in Vancouver Island in 1999, we generated mutant library by Agrobacterium tumefaciens mediated transformation. Three experiments of co-cultivation between C. gattii and A. tumefaciens EHA105 harboring pPZP-HYG yielded 8,000 hygromycin resistant colonies. Three distinct iron metabolism related phenotypes were screened in this mutant library: (i) TTC reduction; (ii) growth defects in alkaline environment; (iii) growth defects in zinc overload. Three mutants displayed a higher reduction capacity over TTC in comparison to the wild-type. Two of these mutants (50-F8 and 75-H4) harbor T-DNA insertions in genes that codes for proteins involved in hypoxia response (Sre1 and Sce1, respectively), while the third mutant contain two copies of the T-DNA, as revealed by Southern analysis. seven mutants were also selected for its defects in alkaline (four mutants) or zinc overload (three mutants) growth defects. The mutated genes of these mutants are under identification by chromosome walking methodologies. Knock-out cassettes will be generated by the Delsgate strategy and null mutants will be constructed. This work will contribute to a better understand of the iron metabolism in C. gattii. Finantial support: CAPES, CNPq, FINEP

Palavras-chave: Cryptococcus gattii, iron homeostasis, mutant library

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