Cryptococcus - PLANTA: buscando novos modelos para compreender os mecanismos dessa interação

Abstract

Cryptococcosis is a systemic mycosis whose main etiological agents are Cryptococcus gattii (Cg) and C. neoformans (Cn). These pathogens live saprophytically in the soil and yeasts of the genus Cryptococcus have been isolated from dozens of plant species. However, it is not known how the Cryptococcus-plant interaction can affect the plant model and, especially, the fungus. In this sense, the present study aimed to develop a plant model to study the interaction between Cryptococcus-plant and to verify the influence of resistance to agrochemicals on this interaction. Therefore, C. gattii L27/01 was inoculated by different methods in three plant models: Arabidopsis thaliana (At), Nicotiana tabacum (Nt) and N. benthamiana (Nb). After inoculation, changes in plant models and in Cg were evaluated. Greater fungal recovery was verified after 7 days of infection in all plant models and different inoculation methods. Yeast has no visible pathological effect in plant models and also does not alter their primary and secondary metabolism (starch, lipids, phenolic compounds and lignin). Infection of Cg to Nb results in an increase in the hydrogen peroxide content and in the activity of enzymes of the antioxidant system, such as catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase. Inoculation of Cg in Nb also increased indolylacetic acid auxin. Therefore, inoculation in Nb by the scarification method was the best strategy for interaction studies. Cg exhibited increased cell body diameter after interaction with At and Nb. There was a decrease in capsular thickness and surface/volume ratio after passage in At and Nb. The fungus showed greater growth and lower ergosterol content after passage in Nb. In the interaction with murine macrophages, the phagocytic index of Cg that passed through Nb was increased in 3 hours and decreased in 24 hours. The production of reactive oxygen species was lower for Cg after passage in Nb. The recovery of fungal load of macrophages was high for Cg after passage in Nb. After establishing the most adequate plant model to evaluate the Cryptococcus-plant interaction, the model was inoculated with different strains of Cg and Cn not adapted and adapted to the agrochemicals pyraclostrobin (PCT) and tebuconazole (TBZ). With regard to the interaction of non-adapted and adapted strains to agrochemicals, the same profile was observed. Cn adapted to PCT (A PCT) was less recovered from Nb, while Cn adapted and subcultured for 10 passages in medium without agrochemical (10p PCT) showed greater recovery. Cn A PCT and 10p PCT showed greater cell body diameter and capsular thickness after passage in Nb. Unadapted Cn (NA) exhibited a decrease in capsular thickness and Cn A PCT and 10p PCT showed a reduction in surface/volume ratio after passage in Nb. Cn A TBZ was less recovered and Cn 10p TBZ was more recovered from Nb when compared to the Cn NA strain. Cn TBZ showed decreased cell body diameter and increased capsular thickness and surface/volume ratio after passage in Nb. Cn 10p TBZ exhibited increased cell body diameter and capsular thickness, as well as decreased surface/volume ratio after passage in Nb. Cn A TBZ exhibited lower susceptibility to amphotericin B (AMB) and Cn 10p TBZ also exhibited lower susceptibility to fluconazole (FCZ). Cn A TBZ and A PCT exhibited lower growth at times 24 and 48 hours after passage in Nb. There was an increase in laccase production after passage in Nb to Cn NA, A TBZ, 10p TBZ, A PCT. Phospholipase activity was reduced after passage on Nb in Cn NA, A TBZ and 10p TBZ. Phospholipase activity increased for Cn A PCT and 10p PCT after passage in Nb. When evaluating the interaction of Cg with the plant model, it was observed that Cg 10p PCT exhibited greater Nb recovery. It was verified an increase in cell diameter for Cg NA after passage in Nb. Capsule thickness and surface/volume ratio were reduced in both strains after passage in Nb. Cg NA and 10p PCT exhibited lower susceptibility to AMB and PCT after passage in Nb. Cg 10p PCT was more susceptible to FCZ after passage on Nb. Ergosterol content was increased to Cg 10p PCT and laccase activity was reduced to Cg NA after passage in Nb. Cn and Cg infection reduced Tenebrio molitor survival. Thus, the present work demonstrates that the interaction that plant models can have on the morphophysiology and virulence of Cryptococcus, opening perspectives for using the plant model in the search for new mechanisms involved in the resistance and virulence of the fungus in the environment.