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Title: Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages

Abstract

Here, diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus’ EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonized with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host’s immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms formore » a fine-tuned response by fungal cells.« less

Authors:
 [1];  [2];  [3]; ORCiD logo [4];  [5]; ORCiD logo [3];  [2]
  1. Albert Einstein College of Medicine, Bronx, NY (United States); Univ. Federal de Minas Gerais, Minas Gerais (Brazil)
  2. Albert Einstein College of Medicine, Bronx, NY (United States)
  3. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  4. National Univ. of Singapore (Singapore)
  5. Univ. Federal do Rio de Janeiro (UFRJ), Rio de Janeiro (Brazil)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1441220
Report Number(s):
PNNL-SA-124828
Journal ID: ISSN 2045-2322; PII: 25665
Grant/Contract Number:  
AC05-76RL01830
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Baltazar, Ludmila Matos, Zamith-Miranda, Daniel, Burnet, Meagan C., Choi, Hyungwon, Nimrichter, Leonardo, Nakayasu, Ernesto S., and Nosanchuk, Joshua D. Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages. United States: N. p., 2018. Web. doi:10.1038/s41598-018-25665-5.
Baltazar, Ludmila Matos, Zamith-Miranda, Daniel, Burnet, Meagan C., Choi, Hyungwon, Nimrichter, Leonardo, Nakayasu, Ernesto S., & Nosanchuk, Joshua D. Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages. United States. doi:10.1038/s41598-018-25665-5.
Baltazar, Ludmila Matos, Zamith-Miranda, Daniel, Burnet, Meagan C., Choi, Hyungwon, Nimrichter, Leonardo, Nakayasu, Ernesto S., and Nosanchuk, Joshua D. Wed . "Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages". United States. doi:10.1038/s41598-018-25665-5. https://www.osti.gov/servlets/purl/1441220.
@article{osti_1441220,
title = {Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages},
author = {Baltazar, Ludmila Matos and Zamith-Miranda, Daniel and Burnet, Meagan C. and Choi, Hyungwon and Nimrichter, Leonardo and Nakayasu, Ernesto S. and Nosanchuk, Joshua D.},
abstractNote = {Here, diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus’ EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonized with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host’s immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.},
doi = {10.1038/s41598-018-25665-5},
journal = {Scientific Reports},
number = 1,
volume = 8,
place = {United States},
year = {2018},
month = {5}
}

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Figures / Tables:

Figure 1 Figure 1: Protein and sterol quantification in EV from H. capsulatum. Yeast cells were incubated with or without 6 and 20 μg/mL of 6B7 and 7B6 mAb. The EV protein content was determined by BCA assay (A). EV sterol quantification was performed using Amplex reagent kit (B). Graphs represent meansmore » and standard deviation from at least two independent EV isolations and all the analyses were performed in duplicate. *p ≤ 0.05, compared to the untreated control group. #p ≤ 0.05, compared to the groups 6B7 and 7B6 at 6 μg/mL. ϕp ≤0.05, compared to 6B7 mAb treatment at 20 μg/mL.« less

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