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Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD
The type two secretion system is a large, trans-envelope apparatus that secretes toxins across the outer membrane of many Gram-negative bacteria. In Aeromonas hydrophila, ExeA interacts with peptidoglycan and forms a heteromultimeric complex with ExeB that is required for assembly of the ExeD secret...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4098917/ https://www.ncbi.nlm.nih.gov/pubmed/25025769 http://dx.doi.org/10.1371/journal.pone.0102038 |
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author | Vanderlinde, Elizabeth M. Zhong, Su Li, Gang Martynowski, Dariusz Grochulski, Pawel Howard, S. Peter |
author_facet | Vanderlinde, Elizabeth M. Zhong, Su Li, Gang Martynowski, Dariusz Grochulski, Pawel Howard, S. Peter |
author_sort | Vanderlinde, Elizabeth M. |
collection | PubMed |
description | The type two secretion system is a large, trans-envelope apparatus that secretes toxins across the outer membrane of many Gram-negative bacteria. In Aeromonas hydrophila, ExeA interacts with peptidoglycan and forms a heteromultimeric complex with ExeB that is required for assembly of the ExeD secretin of the secretion system in the outer membrane. While the peptidoglycan-ExeAB (PG-AB) complex is required for ExeD assembly, the assembly mechanism remains unresolved. We analyzed protein-protein interactions to address the hypothesis that ExeD assembly in the outer membrane requires direct interaction with the PG-AB complex. Yeast and bacterial two hybrid analyses demonstrated an interaction between the periplasmic domains of ExeB and ExeD. Two-codon insertion mutagenesis of exeD disrupted lipase secretion, and immunoblotting of whole cells demonstrated significantly reduced secretin in mutant cells. Mapping of the two-codon insertions and deletion analysis showed that the ExeB-ExeD interaction involves the N0 and N1 subdomains of ExeD. Rotational anisotropy using the purified periplasmic domains of ExeB and ExeD determined that the apparent dissociation constant of the interaction is 1.19±0.16 µM. These results contribute important support for a putative mechanism by which the PG-AB complex facilitates assembly of ExeD through direct interaction between ExeB and ExeD. Furthermore, our results provide novel insight into the assembly function of ExeB that may contribute to elucidating the role of homologous proteins in secretion of toxins from other Gram negative pathogens. |
format | Online Article Text |
id | pubmed-4098917 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-40989172014-07-18 Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD Vanderlinde, Elizabeth M. Zhong, Su Li, Gang Martynowski, Dariusz Grochulski, Pawel Howard, S. Peter PLoS One Research Article The type two secretion system is a large, trans-envelope apparatus that secretes toxins across the outer membrane of many Gram-negative bacteria. In Aeromonas hydrophila, ExeA interacts with peptidoglycan and forms a heteromultimeric complex with ExeB that is required for assembly of the ExeD secretin of the secretion system in the outer membrane. While the peptidoglycan-ExeAB (PG-AB) complex is required for ExeD assembly, the assembly mechanism remains unresolved. We analyzed protein-protein interactions to address the hypothesis that ExeD assembly in the outer membrane requires direct interaction with the PG-AB complex. Yeast and bacterial two hybrid analyses demonstrated an interaction between the periplasmic domains of ExeB and ExeD. Two-codon insertion mutagenesis of exeD disrupted lipase secretion, and immunoblotting of whole cells demonstrated significantly reduced secretin in mutant cells. Mapping of the two-codon insertions and deletion analysis showed that the ExeB-ExeD interaction involves the N0 and N1 subdomains of ExeD. Rotational anisotropy using the purified periplasmic domains of ExeB and ExeD determined that the apparent dissociation constant of the interaction is 1.19±0.16 µM. These results contribute important support for a putative mechanism by which the PG-AB complex facilitates assembly of ExeD through direct interaction between ExeB and ExeD. Furthermore, our results provide novel insight into the assembly function of ExeB that may contribute to elucidating the role of homologous proteins in secretion of toxins from other Gram negative pathogens. Public Library of Science 2014-07-15 /pmc/articles/PMC4098917/ /pubmed/25025769 http://dx.doi.org/10.1371/journal.pone.0102038 Text en © 2014 Vanderlinde 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Vanderlinde, Elizabeth M. Zhong, Su Li, Gang Martynowski, Dariusz Grochulski, Pawel Howard, S. Peter Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD |
title | Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD |
title_full | Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD |
title_fullStr | Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD |
title_full_unstemmed | Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD |
title_short | Assembly of the Type Two Secretion System in Aeromonas hydrophila Involves Direct Interaction between the Periplasmic Domains of the Assembly Factor ExeB and the Secretin ExeD |
title_sort | assembly of the type two secretion system in aeromonas hydrophila involves direct interaction between the periplasmic domains of the assembly factor exeb and the secretin exed |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4098917/ https://www.ncbi.nlm.nih.gov/pubmed/25025769 http://dx.doi.org/10.1371/journal.pone.0102038 |
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