Cargando…
Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation
Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2016
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729943/ https://www.ncbi.nlm.nih.gov/pubmed/26858708 http://dx.doi.org/10.3389/fmicb.2016.00035 |
_version_ | 1782412326230556672 |
---|---|
author | Aguilera, Paulina Marcoleta, Andrés Lobos-Ruiz, Pablo Arranz, Rocío Valpuesta, José M. Monasterio, Octavio Lagos, Rosalba |
author_facet | Aguilera, Paulina Marcoleta, Andrés Lobos-Ruiz, Pablo Arranz, Rocío Valpuesta, José M. Monasterio, Octavio Lagos, Rosalba |
author_sort | Aguilera, Paulina |
collection | PubMed |
description | Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts. |
format | Online Article Text |
id | pubmed-4729943 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-47299432016-02-08 Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation Aguilera, Paulina Marcoleta, Andrés Lobos-Ruiz, Pablo Arranz, Rocío Valpuesta, José M. Monasterio, Octavio Lagos, Rosalba Front Microbiol Microbiology Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts. Frontiers Media S.A. 2016-01-28 /pmc/articles/PMC4729943/ /pubmed/26858708 http://dx.doi.org/10.3389/fmicb.2016.00035 Text en Copyright © 2016 Aguilera, Marcoleta, Lobos-Ruiz, Arranz, Valpuesta, Monasterio and Lagos. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Aguilera, Paulina Marcoleta, Andrés Lobos-Ruiz, Pablo Arranz, Rocío Valpuesta, José M. Monasterio, Octavio Lagos, Rosalba Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation |
title | Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation |
title_full | Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation |
title_fullStr | Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation |
title_full_unstemmed | Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation |
title_short | Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation |
title_sort | identification of key amino acid residues modulating intracellular and in vitro microcin e492 amyloid formation |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729943/ https://www.ncbi.nlm.nih.gov/pubmed/26858708 http://dx.doi.org/10.3389/fmicb.2016.00035 |
work_keys_str_mv | AT aguilerapaulina identificationofkeyaminoacidresiduesmodulatingintracellularandinvitromicrocine492amyloidformation AT marcoletaandres identificationofkeyaminoacidresiduesmodulatingintracellularandinvitromicrocine492amyloidformation AT lobosruizpablo identificationofkeyaminoacidresiduesmodulatingintracellularandinvitromicrocine492amyloidformation AT arranzrocio identificationofkeyaminoacidresiduesmodulatingintracellularandinvitromicrocine492amyloidformation AT valpuestajosem identificationofkeyaminoacidresiduesmodulatingintracellularandinvitromicrocine492amyloidformation AT monasteriooctavio identificationofkeyaminoacidresiduesmodulatingintracellularandinvitromicrocine492amyloidformation AT lagosrosalba identificationofkeyaminoacidresiduesmodulatingintracellularandinvitromicrocine492amyloidformation |