Cargando…
Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis
BACKGROUND: Cystic Fibrosis (CF) is a genetic disorder caused by mutation(s) in the CF-transmembrane conductance regulator (Cftr) gene. The most common mutation, ΔF508, leads to accumulation of defective-CFTR protein in aggresome-bodies. Additionally, Pseudomonas aeruginosa (Pa), a common CF pathoge...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597233/ https://www.ncbi.nlm.nih.gov/pubmed/28902888 http://dx.doi.org/10.1371/journal.pone.0184793 |
_version_ | 1783263676223979520 |
---|---|
author | Brockman, Scott Mackenzie Bodas, Manish Silverberg, David Sharma, Ajit Vij, Neeraj |
author_facet | Brockman, Scott Mackenzie Bodas, Manish Silverberg, David Sharma, Ajit Vij, Neeraj |
author_sort | Brockman, Scott Mackenzie |
collection | PubMed |
description | BACKGROUND: Cystic Fibrosis (CF) is a genetic disorder caused by mutation(s) in the CF-transmembrane conductance regulator (Cftr) gene. The most common mutation, ΔF508, leads to accumulation of defective-CFTR protein in aggresome-bodies. Additionally, Pseudomonas aeruginosa (Pa), a common CF pathogen, exacerbates obstructive CF lung pathology. In the present study, we aimed to develop and test a novel strategy to improve the bioavailability and potentially achieve targeted drug delivery of cysteamine, a potent autophagy-inducing drug with anti-bacterial properties, by developing a dendrimer (PAMAM-DEN)-based cysteamine analogue. RESULTS: We first evaluated the effect of dendrimer-based cysteamine analogue (PAMAM-DEN(CYS)) on the intrinsic autophagy response in IB3-1 cells and observed a significant reduction in Ub-RFP and LC3-GFP co-localization (aggresome-bodies) by PAMAM-DEN(CYS) treatment as compared to plain dendrimer (PAMAM-DEN) control. Next, we observed that PAMAM-DEN(CYS) treatment shows a modest rescue of ΔF508-CFTR as the C-form. Moreover, immunofluorescence microscopy of HEK-293 cells transfected with ΔF508-CFTR-GFP showed that PAMAM-DEN(CYS) is able to rescue the misfolded-ΔF508-CFTR from aggresome-bodies by inducing its trafficking to the plasma membrane. We further verified these results by flow cytometry and observed significant (p<0.05; PAMAM-DEN vs. PAMAM-DEN(CYS)) rescue of membrane-ΔF508-CFTR with PAMAM-DEN(CYS) treatment using non-permeabilized IB3-1 cells immunostained for CFTR. Finally, we assessed the autophagy-mediated bacterial clearance potential of PAMAM-DEN(CYS) by treating IB3-1 cells infected with PA01-GFP, and observed a significant (p<0.01; PAMAM-DEN vs. PAMAM-DEN(CYS)) decrease in intracellular bacterial counts by immunofluorescence microscopy and flow cytometry. Also, PAMAM-DEN(CYS) treatment significantly inhibits the growth of PA01-GFP bacteria and demonstrates potent mucolytic properties. CONCLUSIONS: We demonstrate here the efficacy of dendrimer-based autophagy-induction in preventing sequestration of ΔF508-CFTR to aggresome-bodies while promoting its trafficking to the plasma membrane. Moreover, PAMAM-DEN(CYS) decreases Pa infection and growth, while showing mucolytic properties, suggesting its potential in rescuing Pa-induced ΔF508-CF lung disease that warrants further investigation in CF murine model. |
format | Online Article Text |
id | pubmed-5597233 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-55972332017-09-15 Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis Brockman, Scott Mackenzie Bodas, Manish Silverberg, David Sharma, Ajit Vij, Neeraj PLoS One Research Article BACKGROUND: Cystic Fibrosis (CF) is a genetic disorder caused by mutation(s) in the CF-transmembrane conductance regulator (Cftr) gene. The most common mutation, ΔF508, leads to accumulation of defective-CFTR protein in aggresome-bodies. Additionally, Pseudomonas aeruginosa (Pa), a common CF pathogen, exacerbates obstructive CF lung pathology. In the present study, we aimed to develop and test a novel strategy to improve the bioavailability and potentially achieve targeted drug delivery of cysteamine, a potent autophagy-inducing drug with anti-bacterial properties, by developing a dendrimer (PAMAM-DEN)-based cysteamine analogue. RESULTS: We first evaluated the effect of dendrimer-based cysteamine analogue (PAMAM-DEN(CYS)) on the intrinsic autophagy response in IB3-1 cells and observed a significant reduction in Ub-RFP and LC3-GFP co-localization (aggresome-bodies) by PAMAM-DEN(CYS) treatment as compared to plain dendrimer (PAMAM-DEN) control. Next, we observed that PAMAM-DEN(CYS) treatment shows a modest rescue of ΔF508-CFTR as the C-form. Moreover, immunofluorescence microscopy of HEK-293 cells transfected with ΔF508-CFTR-GFP showed that PAMAM-DEN(CYS) is able to rescue the misfolded-ΔF508-CFTR from aggresome-bodies by inducing its trafficking to the plasma membrane. We further verified these results by flow cytometry and observed significant (p<0.05; PAMAM-DEN vs. PAMAM-DEN(CYS)) rescue of membrane-ΔF508-CFTR with PAMAM-DEN(CYS) treatment using non-permeabilized IB3-1 cells immunostained for CFTR. Finally, we assessed the autophagy-mediated bacterial clearance potential of PAMAM-DEN(CYS) by treating IB3-1 cells infected with PA01-GFP, and observed a significant (p<0.01; PAMAM-DEN vs. PAMAM-DEN(CYS)) decrease in intracellular bacterial counts by immunofluorescence microscopy and flow cytometry. Also, PAMAM-DEN(CYS) treatment significantly inhibits the growth of PA01-GFP bacteria and demonstrates potent mucolytic properties. CONCLUSIONS: We demonstrate here the efficacy of dendrimer-based autophagy-induction in preventing sequestration of ΔF508-CFTR to aggresome-bodies while promoting its trafficking to the plasma membrane. Moreover, PAMAM-DEN(CYS) decreases Pa infection and growth, while showing mucolytic properties, suggesting its potential in rescuing Pa-induced ΔF508-CF lung disease that warrants further investigation in CF murine model. Public Library of Science 2017-09-13 /pmc/articles/PMC5597233/ /pubmed/28902888 http://dx.doi.org/10.1371/journal.pone.0184793 Text en © 2017 Brockman et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Brockman, Scott Mackenzie Bodas, Manish Silverberg, David Sharma, Ajit Vij, Neeraj Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis |
title | Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis |
title_full | Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis |
title_fullStr | Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis |
title_full_unstemmed | Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis |
title_short | Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis |
title_sort | dendrimer-based selective autophagy-induction rescues δf508-cftr and inhibits pseudomonas aeruginosa infection in cystic fibrosis |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597233/ https://www.ncbi.nlm.nih.gov/pubmed/28902888 http://dx.doi.org/10.1371/journal.pone.0184793 |
work_keys_str_mv | AT brockmanscottmackenzie dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis AT bodasmanish dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis AT silverbergdavid dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis AT sharmaajit dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis AT vijneeraj dendrimerbasedselectiveautophagyinductionrescuesdf508cftrandinhibitspseudomonasaeruginosainfectionincysticfibrosis |