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Front Microbiol
2018 Jan 01;9:2286. doi: 10.3389/fmicb.2018.02286.
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Extracellular Vesicles From Sporothrix brasiliensis Are an Important Virulence Factor That Induce an Increase in Fungal Burden in Experimental Sporotrichosis.
Ikeda MAK
,
de Almeida JRF
,
Jannuzzi GP
,
Cronemberger-Andrade A
,
Torrecilhas ACT
,
Moretti NS
,
da Cunha JPC
,
de Almeida SR
,
Ferreira KS
.
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Sporotrichosis is a mycosis that affects the skin, lymphatic system and other organs in humans and animals. The disease has a worldwide distribution, with endemic areas in Brazil, and is caused by a complex of species, including Sporothrix brasiliensis. Some fungi release extracellular vesicles (EVs) that can interact with the host cell and modulate the host immune response. The aim of this study was to analyze the participation of S. brasiliensis EVs in the modulation of dendritic cells (DCs) and in the control of infection in vivo. Our results showed that in vitro, the EVs isolated from S. brasiliensis induced an increase in the phagocytic index and fungal burden in DCs. In addition, we observed a significant increase in IL-12p40 and TNF-α cytokine production. Then, the EVs were inoculated into BALB/c mice before subcutaneous infection with yeast, and the lesion was analyzed after 21, 35, and 42 days. An increase in fungal burden and lesion diameter were observed after 21 days in mice inoculated with a high concentration of EVs. However, after 35 days, we observed a regression of the lesion, which persisted until 42 days after infection. Interestingly, we observed an increase in fungal burden in these mice. In addition, we observed the presence of immunogenic components and proteins that could be related with virulence in EVs. These results suggest that EVs can play an important role in virulence and modulation of the host immune system during experimental S. brasiliensis infection.
FIGURE 1. Extracellular vesicle (EV) release from S. brasiliensis yeasts. Yeasts were obtained from a culture of yeasts for 6 days in BHI broth and processed for TEM analysis. At a magnification of 29,000x (A) and 80,000x (B,C), EVs (arrows) were observed at the surface of the cell wall (CW) (A,B) or secreted (C) from yeasts.
FIGURE 2. EV quantification. EVs were isolated by differential centrifugation of supernatant from a culture of yeasts for 6 days in BHI broth and analyzed by NTA. The graph shows the average concentration per size of EVs in samples (black), with a standard error of the mean (red). We obtained samples with a mean concentration of 1.19 × 1011 particles/mL with a mean size between 50 and 150 nm. Representatives from the average taken from the experiments are shown in three 30-s videos.
FIGURE 3. Presence of immunoreactive components in S. brasiliensis EVs. Analysis of EVs (1) in comparison with exoantigen (2) from yeasts. (A) SDS-PAGE gel stained with silver nitrate shows bands of 100 and 70 kDa. (B) Western blot with serum from infected mice with S. brasiliensis reveals a signal at approximately 100 kDa. The analysis was performed in three independent experiments.
FIGURE 5. Progression of the subcutaneous infection in a murine model (A). Graph represents the evolution of the average diameter of skin lesions for 42 days resulting from subcutaneous fungal infection. A representative of 3 independent experiments, containing 5 animals each group at each timepoint. (B) Figures represent the external appearance of skin lesions formed at the fungus inoculation site, with the presence of ulcerative nodular formation at 21 days, nodular healing at 35 days, and complete regression at 42 days. (C) Internal appearance of a skin lesion at 35 days. A subcutaneous (left) encapsulated formation is observed, which when cut contains a purulent exudate (right).
FIGURE 6. EVs increase the fungal load of a skin lesion in a murine model. From a skin lesion collected on the 21st and 35th day, CFU was analyzed. Animals that previously received EVs had a higher fungal burden than animals that did not receive EVs. A representative of 3 independent experiments, containing 5 animals per group, at each timepoint. One-way analysis ANOVA, with multiple comparisons by the Tukey test, was considered significant if p < 0.05. ∗∗p < 0.001, ∗∗∗p < 0.0001 compared with S. brasiliensis (positive control).
FIGURE 7. Histological analysis of a skin lesion after 21 days of infection. GMS stain, 40× magnification. We can observe the higher number of yeast structures stained in lesions of animals that received EVs before infection.
FIGURE 9. Proteomic analysis of EVs released from S. brasiliensis and S. schenckii
(A). Venn diagram representing the number of proteins found in both EVs. (B) Graphs representing the percentage of proteins characterized and not characterized in each EVs. (C) Representation of the predicted associated cellular component by GO analysis in each EVs. (D) Distribution of proteins according to the predicted biological process by GO analysis in each EVs.
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