RNA interference of PMR1-like Ca2+-ATPase in Aspergillus fumigatus: phenotypic analysis.
SORIANI, F.M.; MAGNANI, T.; MARTINS, V.P.; TUDELLA, V.G.; and UYEMURA, S.A.
DACTB - Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP fredsori@fcfrp.usp.br
Aspergillus fumigatus is a saprophytic fungus that, in immunocompromised patients, causes a fatal invasive disease. The properly ion homeostasis in pathogenic lower eukaryotes represents a key for surviving during the host infection. The uptake of cytoplasmic calcium can be done by PMR1-like Ca2+-ATPase, which is localized to the medial-Golgi compartment delivering calcium and manganese ions to the secretory pathway.
Recently, PMR1 has been related with the virulence of different pathogens. In this sense, we have silenced a PMR1 gene from A. fumigatus (Afpmr1) by RNA interference and investigated phenotypic alterations of this strain. Inverted repeats of a fragment of the Afpmr1 coding sequence were cloned in the pALB1 plasmid, which uses the E. coli hph and ALB1/PKSP genes as selection markers for transformation and RNAi induction, respectively. The transformant was inoculated in minimal medium supplemented with xylose or maltose. In xylose medium, the phenotype of the Afpmr1/alb1 double silenced strain was the same of the observed for the alb1 silenced. Both strains grew in green color, as the parental strain, indicating that the interference was not induced. However, in maltose supplemented medium, in contrast with the white color of the alb1 strain, the double silenced strain grew in light green color indicating that neither the alb1 nor the Afpmr1 gene was entirely silenced.
With the purpose to verify phenotype alterations of the double silenced strain, it was inoculated in the same medium described earlier supplemented with 100 or 200 mM MnCl2 or 100 mM EGTA. After 10 days at 30ºC incubation, the double silenced strain grew properly in the challenged medium while the control xylose inoculated strains did not grow. These evidences support the idea of the Afpmr1 gene silencing, suggesting that this strain could be used for different phenotypic analysis, like virulence assay, neutrophil oxidative burst and macrophage phagocytosis and killing assay, to determine the role of this gene in pathogenicity of A. fumigatus.
Supported by FAPESP and CNPq.
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