O papel do íon ferro nas infecções por Pythium insidiosum

Abstract

The oomycete Pythium insidiosum, classified in the kingdom Straminipila, is the agent of pythiosis, a chronic disease that can be hard to treat and life-threatening. The predisposing factors in mammals are unknown, but the iron overload is suspected to act directly or indirectly in the development of the infection. This aim of this study was: to evaluate the iron metabolism in rabbits with experimental pythiosis; to verify the antifungal activity and the mechanism of action in vitro of the iron chelator deferasirox against P. insidiosum isolates and to compared the treatment and the mechanism of action of the drug with immunotherapy in vivo; to verify the antifungal activity of micafungin, alone and in combination with deferasirox, in vitro and in vivo; to develop an experimental model of pythiosis using the fruit-fly Drosophila melanogaster with iron overload. The drug susceptibility tests included 17 P. insidiosum clinical isolates, and were performed according to the CLSI M38-A2 guidelines. The mechanism of action of deferasirox was evaluated by the XTT and DiBAC assays. For the in vivo tests, five P. insidiosum infected rabbits were included per group, as follows: infected treated with placebo, treated with immunotherapy, treated with deferasirox, treated with immunotherapy and deferasirox, treated with micafungin and treated with micafungin and deferasirox. Five uninfected rabbits were used as controls. Hematological and biochemical analyses were performed at days 0 25, 50 and 75 post-infection. The quantification of iron in the hepatocytes was performed after 50 days of treatment (day 75). The mechanism of action of deferasirox was evaluated by the quantification of the catabolic enzyme adenosine deaminase and by the histology of the subcutaneous lesions. The results show that P. insidiosum is poorly susceptible to deferasirox in vitro, with minimum inhibition concentrations (MICs) ranging from 12.5 to 50 μg/ml and minimum fungicidal concentrations between 50 and 100 μg/ml. No activity against P. insidiosum was observed for micafungin in vitro (MICs > 128 μg/ml) and in rabbits with pythiosis. Notwithstanding, synergism was observed in 88% of the isolates when micafungin was combined with deferasirox, and the lesions were decreased in comparison to the other groups (P = 0.06). In general, the rabbits showed microcytic hypochromic anemia with depleted iron stores, which was less prominent in the groups treated with immunotherapy or deferasirox. Despite the iron chelator has failed to thwart the infection, deferasirox showed toxicity against P. insidiosum hyphae in vitro and an immunomodulatory action was observed in the lesions, in a similar pattern to that seen in immunotherapy-treated rabbits. However, the use of deferasirox favored the dissemination of the disease to the lung by unrevealed mechanisms. D. melanogaster flies were resistant to P. insidiosum infection, independently of the iron overload. Conversely, toll-deficient D. melanogaster flies were susceptible to the infection, highlighting the importance of these receptors in pythiosis. In conclusion, rabbits with pythiosis showed iron deficiency anemia and, despite the chelating effect of deferasirox, the administration of the drug should be evaluated with care because of the dissemination of the disease observed in some of the treated animals.