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Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells
Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa. However, the precise characterization of the intracellular trafficking and fate of P. gingivalis in GECs remains incomplete. Therefore,...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Taylor & Francis
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955440/ https://www.ncbi.nlm.nih.gov/pubmed/29616874 http://dx.doi.org/10.1080/21505594.2018.1454171 |
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author | Lee, Kyulim Roberts, JoAnn S. Choi, Chul Hee Atanasova, Kalina R. Yilmaz, Özlem |
author_facet | Lee, Kyulim Roberts, JoAnn S. Choi, Chul Hee Atanasova, Kalina R. Yilmaz, Özlem |
author_sort | Lee, Kyulim |
collection | PubMed |
description | Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa. However, the precise characterization of the intracellular trafficking and fate of P. gingivalis in GECs remains incomplete. Therefore, we employed high-resolution three-dimensional-transmission-electron-microscopy to determine the subcellular location of P. gingivalis in human primary GECs upon invasion. Serial sections of infected-GECs and their tomographic reconstruction depicted ER-rich-double-membrane autophagosomal-vacuoles harboring P. gingivalis. Western-blotting and fluorescence confocal microscopy showed that P. gingivalis significantly induces LC3-lipidation in a time-dependent-manner and co-localizes with LC3, ER-lumen-protein Bip, or ER-tracker, which are major components of the phagophore membrane. Furthermore, GECs that were infected with FMN-green-fluorescent transformant-strain (PgFbFP) and selectively permeabilized by digitonin showed rapidly increasing large numbers of double-membrane-vacuolar-P. gingivalis over 24 hours of infection with a low-ratio of cytosolically free-bacteria. Moreover, inhibition of autophagy using 3-methyladenine or ATG5 siRNA significantly reduced the viability of intracellular P. gingivalis in GECs as determined by an antibiotic-protection-assay. Lysosomal marker, LAMP-1, showed a low-degree colocalization with P. gingivalis (∼20%). PgFbFP was used to investigate the fate of vacuolar- versus cytosolic-P. gingivalis by their association with ubiquitin-binding-adaptor-proteins, NDP52 and p62. Only cytosolic-P. gingivalis had a significant association with both markers, which suggests cytosolically-free bacteria are likely destined to the lysosomal-degradation pathway whereas the vacuolar-P. gingivalis survives. Therefore, the results reveal a novel mechanism for P. gingivalis survival in GECs by harnessing host autophagy machinery to establish a successful replicative niche and persistence in the oral mucosa. |
format | Online Article Text |
id | pubmed-5955440 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-59554402018-05-21 Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells Lee, Kyulim Roberts, JoAnn S. Choi, Chul Hee Atanasova, Kalina R. Yilmaz, Özlem Virulence Research Paper Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa. However, the precise characterization of the intracellular trafficking and fate of P. gingivalis in GECs remains incomplete. Therefore, we employed high-resolution three-dimensional-transmission-electron-microscopy to determine the subcellular location of P. gingivalis in human primary GECs upon invasion. Serial sections of infected-GECs and their tomographic reconstruction depicted ER-rich-double-membrane autophagosomal-vacuoles harboring P. gingivalis. Western-blotting and fluorescence confocal microscopy showed that P. gingivalis significantly induces LC3-lipidation in a time-dependent-manner and co-localizes with LC3, ER-lumen-protein Bip, or ER-tracker, which are major components of the phagophore membrane. Furthermore, GECs that were infected with FMN-green-fluorescent transformant-strain (PgFbFP) and selectively permeabilized by digitonin showed rapidly increasing large numbers of double-membrane-vacuolar-P. gingivalis over 24 hours of infection with a low-ratio of cytosolically free-bacteria. Moreover, inhibition of autophagy using 3-methyladenine or ATG5 siRNA significantly reduced the viability of intracellular P. gingivalis in GECs as determined by an antibiotic-protection-assay. Lysosomal marker, LAMP-1, showed a low-degree colocalization with P. gingivalis (∼20%). PgFbFP was used to investigate the fate of vacuolar- versus cytosolic-P. gingivalis by their association with ubiquitin-binding-adaptor-proteins, NDP52 and p62. Only cytosolic-P. gingivalis had a significant association with both markers, which suggests cytosolically-free bacteria are likely destined to the lysosomal-degradation pathway whereas the vacuolar-P. gingivalis survives. Therefore, the results reveal a novel mechanism for P. gingivalis survival in GECs by harnessing host autophagy machinery to establish a successful replicative niche and persistence in the oral mucosa. Taylor & Francis 2018-04-04 /pmc/articles/PMC5955440/ /pubmed/29616874 http://dx.doi.org/10.1080/21505594.2018.1454171 Text en © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 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 work is properly cited. |
spellingShingle | Research Paper Lee, Kyulim Roberts, JoAnn S. Choi, Chul Hee Atanasova, Kalina R. Yilmaz, Özlem Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells |
title | Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells |
title_full | Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells |
title_fullStr | Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells |
title_full_unstemmed | Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells |
title_short | Porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells |
title_sort | porphyromonas gingivalis traffics into endoplasmic reticulum-rich-autophagosomes for successful survival in human gingival epithelial cells |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955440/ https://www.ncbi.nlm.nih.gov/pubmed/29616874 http://dx.doi.org/10.1080/21505594.2018.1454171 |
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