Influence of the agrochemical benomyl on Cryptococcus gattii-plant interaction in vitro and in vivo

Abstract

Cryptococcus gattii, an environmental fungus, is one of the agents of cryptococcosis. The influence of agrochemicals on fungal resistance to antifungals is widely discussed. However, the effects of benomyl (BEN) on fungal interaction with different hosts is still to be understood. Here we studied the influence of adaptation to BEN in the interaction with a plant model, phagocytes and with Tenebrio molitor. First, the strain C. gattii L24/01 non-adapted (NA), adapted (A) to BEN, and adapted with further culture on drug-free media (10p) interact with Nicotiana benthamiana, with a peak in the yeast burden on the 7th day post-inoculation. C. gattii L24/01 A and 10p provided lower fungal burden, but these strains increased cell diameter and capsular thickness after the interaction, together with decreased fungal growth. The strains NA and A showed reduced ergosterol levels, while 10p exhibited increased activity of laccase and urease. L24/01 A recovered from N. benthamiana was less engulfed by murine macrophages, with lower production of reactive oxygen species. This phenotype was accompanied by increased ability of this strain to grow inside macrophages. Otherwise, L24/01 A showed reduced virulence in the T. molitor larvae model. Here, we demonstrate that the exposure to BEN, and interaction with plants interfere in the morphophysiology and virulence of the C. gattii.

Keywords: Cryptococcus gattii; Nicotiana benthamiana; Benomyl; Host-pathogen interaction.

Fig. 1

Impact of benomyl (BEN) adaptation on Cryptococcus gattii recovery from Nicotiana benthamiana. (A) Kinetics of C. gattii L24/01 NA (non-adapted), A (adapted to BEN) or 10p (adapted to BEN and cultivated 10 times in culture medium without BEN) recovery (CFU/g) from N. benthamiana leaves over 28 days post-inoculation. (B) Quantified fungal burden (CFU/g) at 7 days post-inoculation. * statistically significant difference (p < 0.05) using analysis of variance (ANOVA), followed by Tukey’s test

Fig. 2

Effects of benomyl (BEN) on cell diameter and capsular thickness of Cryptococcus gattii after interaction with Nicotiana benthamiana. (A) Cell body diameter (µm), (B) capsular thickness (µm), (C) surface/volume ratio for C. gattii L24/01 NA (non-adapted), A (adapted to BEN) or 10p (adapted to BEN and cultivated 10 times in culture medium without BEN) strains before (SDA) and after inoculation into N. benthamiana and recovery 7 days post-inoculation (Nb). Columns represent the average of fifty repetitions ± standard deviation. * statistically significant difference (p < 0.05) using Mann-Whitney test

Fig. 3

Impact of benomyl (BEN) and interaction with Nicotiana benthamiana on the growth and enzymatic activity of Cryptococcus gattii. (A) Growth curve (CFU/mL), (B) ergosterol content (µg/mL), (C) laccase (absorbance), (D) urease (absorbance) and (E) phospholipase and proteinase activities from C. gattii L24 (non-adapted), A (adapted to BEN) or 10p (adapted to BEN and cultivated 10 times in culture medium without BEN) strains before (SDA) and after interaction with N. benthamiana (Nb). Leaves of N. benthamiana were infected with NA, A or 10p and the fungal burden was recovered 7 days post-inoculation. * statistically significant difference (p < 0.05) using Mann-Whitney test, while grown curve was estimated using area under curve analysis followed by T-test

Fig. 4

Effects of benomyl (BEN) and interaction with Nicotiana benthamiana on the interaction of Cryptococcus gattii with phagocytes. (A) Phagocytic index (%), (B) reactive oxygen species (ROS; AU), (C) peroxynitrite (PRN; AU) production and (D) intracellular proliferation rate (IPR) after 3 and 24 h of interaction of C. gattii L24/01 NA and A with macrophages. The intracellular proliferation rate (IPR) is the ratio of the fungal burden recovered from macrophages between 24 and 3 h. * statistically significant difference (p < 0.05) using Mann-Whitney test. Nb: C. gattii strain after interaction with N. benthamiana. SDA: C. gattii strain before interaction with N. benthamiana

Fig. 5

Impact of benomyl (BEN) and interaction with Nicotiana benthamiana on the virulence of Cryptococcus gattii in Tenebrio molitor. (A) Survival curve of T. molitor inoculated with (A) C. gattii L24/01 NA before and after interaction with N. benthamiana, and with (B) C. gattii L24/01 A before and after interaction with N. benthamiana. * statistically significant difference (p < 0.05) using log-rank test