Fig 1.
Soil organisms sharing the putative habitat of P. brasiliensis.
A) Schematic representation of the soil amoeba isolation methodology. Soil samples from armadillo burrows positive for P. brasiliensis DNA were collected and used for the isolation of soil amoebae. The samples were plated in non-nutrient agar plates containing a bacterial lawn as a food source and observed in an inverted microscope B) Bright field microscopy of ciliate trophozoites (black arrowheads) present in a soil sample. Scale bar = 20 μm, C) Bright field microscopy of a nematode present in the soil sample. Scale bar = 50 μm, D) and E) DIC microscopy of trophozoites of A. spelaea. Scale bar = 10 μm. F and G) DIC microscopy of trophozoites of V. vermiformis. Scale bar = 10 μm. H and I) DIC microscopy of trophozoites of Acanthamoeba spp. Scale bar = 10 μm.
Fig 2.
Interaction between P. brasiliensis Pb18 with amoebae isolated from soil of armadillo burrows positive for P. brasiliensis.
The amoeba isolates were co-incubated with Pb18 previously dyed with pHrodo or FITC at an MOI of two at 25°C for 24 hours in PYG medium. A) Percentage of amoeba cells interacting with P. brasiliensis Pb18. After the interaction, non-internalized Pb18 cells were dyed using Uvitex 2B. B) Viability of the different amoeba isolates after 24 hours of interaction with Pb18. A and B depict the results of at least three independent experiments. At least 100 cells per replicate of each sample were counted for each assay. The bars represent 95% confidence intervals. C-F) a suspension of A. spelaea cells was placed next to a colony of P. brasiliensis cells in non-nutrient agar. The cells were co-incubated at 25°C and examined daily in an inverted microscope. C) Macroscopic view of the fungal colony in a 35 mm plate. D) Microscopic view of the fungal cell lawn after seven days of interaction. E) Microscopic view of amoeba trophozoites growing in the periphery of the fungal lawn. F) Microscopic view of amoeba trophozoites interacting with a fungal cell. Scale bars = 50 μm. Black arrowheads indicate trophozoites. White arrowheads indicate fungal cells.
Fig 3.
TEM and SEM analysis of the interaction of P. brasiliensis Pb18 cells and different soil amoebae.
The isolates were co-incubated with Pb18 at an MOI of two at 25°C for 4 or 24 hours in PAS and fixed for microscopy. A-B) TEM of the interaction of P. brasiliensis with A. spelaea (4 h, 24 h). Scale bars = 500 nm. C-D) SEM of the interaction of P. brasiliensis with A. spelaea. Scale bars = 10 μm. E-F) TEM of the interaction of P. brasiliensis with Acanthamoeba spp (4 h, 24 h). Scale bars = 2 μm. G-H) SEM of the Interaction of P. brasiliensis with Acanthamoeba spp. Scale bars = 10 μm. I-J) TEM of the interaction of P. brasiliensis with V. vermiformis (4 h, 24 h). Scale bars = 2 μm. K-L) SEM of the interaction of P. brasiliensis with V. vermiformis. Scale bars = 10 μm. White arrows indicate fungal cells, or their remains and black arrowheads indicate amoeba cells.
Fig 4.
P. brasiliensis Pb18 interaction with an axenic A. castellanii strain.
A) P. brasiliensis and A. castellanii were co-incubated at an MOI of one for one hour at 28°C, and then stained with Giemsa and observed by light microscopy. B) Enlargement of the area depicted in the square region of panel A. C) TEM of the interaction of A. castellanii and Pb18 cells. Incubation was at an MOI of one for six hours at 28°C and then fixed. The black arrow indicates an internalized P. brasiliensis (Scale bar = 2 μm). D) Confocal microscopy. A. castellanii was dyed with DiD-DS (red), while P. brasiliensis cells were labeled first with CMFDA (green), and after the interaction with Uvitex 2B (blue). The arrows show fungal cells inside an amoeba. (Scale bar = 10 μm). E and F) Survival of P. brasiliensis after interaction with A. castellanii. Incubation was at an MOI of two at 28°C for six (E) or 24 hours (F), using the fungus alone as a control. After the interaction amoeba cells were lysed and fungal cells were plated for CFU counting. The figure depicts the results of three independent experiments. The error bars represent the standard error of the mean.
Fig 5.
Interaction of Paracoccidioides spp strains with A. castellanii at six hours.
Amoebae and three different strains of Paracoccidiodes spp (Pb18 –P. brasiliensis, Pb01 –P. lutzii, PbT16B1 –P. brasiliensis isolated from an armadillo spleen) were co-incubated at an MOI of two for six hours at 28°C. A) Percentage of A. castellanii cells interacting with Paracoccidioides spp. The interaction was assessed by counting at least 100 phagocytes cells per replicate of each sample after Giemsa staining of the samples. The bars represent 95% confidence intervals. B) Viability of A. castellanii upon interaction with Paracoccidioides spp. The viability was assessed by counting at least 100 cells per replicate of each sample after staining with trypan blue. The bars represent means plus 95% confidence intervals. C) Survival of fungal cells from different strains of Paracoccidioides spp following interaction with amoebae. The error bars represent standard error of the mean. Figures depict the combined results of at least three independent experiments. *All the strains showed a significant difference in the % of dead amoebae at six hours relative to the control amoebae growing alone.
Fig 6.
Effects of sequential passaging of Pb18 with amoebae, assessed in several models of infection.
Pb18 and Pb18 Pb-Ac cells were co-incubated with A. castellanii at an MOI of two at 28°C for six hours. A) Percentage of A. castellanii cells interacting with Pb18 and Pb18-Ac. B) Viability of A. castellanii after six hours of interaction with Pb18 and Pb18-Ac. C) Survival of Pb18 and Pb18-Ac upon interaction with A. castellanii. D) Survival of Pb18 and Pb18 Pb-Ac upon interaction with J774 macrophages. E) Survival curve of G. mellonella infected with Pb18 and Pb18-Ac. The curve is representative of two biological replicates. P<0.0001 for the comparison of the survival curve of larvae infected with the two different strains (log-rank test). F) Survival curve of BALB/c mice infected with Pb18 or Pb18 Pb-Ac. Each group had 15 mice. p = 0.0003 for the comparison of the survival curve of mice infected with the two strains (log-rank test). A-D depict the combined results of at least two independent experiments. The bars represent means plus 95% confidence intervals in A and B and standard error mean in C and D.
Fig 7.
Changes in P. brasiliensis Pb18 gene expression after cycles of interaction with amoeba.
A–G) Modulation of Pb18 gene expression after previous passages with amoebae. Transcript accumulation was determined by the comparative threshold method using the ΔCt value obtained after normalization with the constitutively expressed gene L34. Data are reported as individual 2-ΔΔCt values of three independent experiments for each group and the bar represents their respective means. FC = fold change in mRNA accumulation, obtained as the ratio Pb18-Ac/Pb18. A) AGS1: α-glucan synthase, B) HADH: Hydroxyacyl-CoA Dehydrogenase, C) MS1: malate synthase, D) SOD1: superoxide dismutase 1, E) HSP60: Heat shock protein 60, F) HSP70: Heat shock protein 70, G) HSP90: Heat shock protein 90. H) Cell surface staining of α glucan in the surface of Pb18 cells that were submitted (Pb18-Ac) or not (Pb18) to serial passages of interaction with A. castellanii. Cells from the two cell types (Pb18 and Pb18-Ac) were labeled with the antibody to α-glucan MOPC 104E and then with secondary IgM-Alexa fluor 488 antibody and analyzed by flow cytometry.