Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway

ABSTRACT Trimeric autotransporter adhesins (TAAs) are a family of bacterial outer membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular (“passenger”) domain and a short C-terminal segment that contributes four β strands to a single 1...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Rakesh Sikdar, Harris D. Bernstein
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Bam complex membrane proteins outer membrane protein folding protein secretion trimeric autotransporters Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/043d320418494428bead6e4249e9f957
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:043d320418494428bead6e4249e9f957
record_format	dspace
spelling	oai:doaj.org-article:043d320418494428bead6e4249e9f9572021-11-15T15:59:40ZSequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway10.1128/mBio.01973-192150-7511https://doaj.org/article/043d320418494428bead6e4249e9f9572019-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01973-19https://doaj.org/toc/2150-7511ABSTRACT Trimeric autotransporter adhesins (TAAs) are a family of bacterial outer membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular (“passenger”) domain and a short C-terminal segment that contributes four β strands to a single 12-stranded β barrel. The mechanism by which the passenger domains are translocated across the OM and the energetics of the translocation reaction are poorly understood. To address these issues, we examined the secretion of modified versions of the passenger domain of UpaG, a TAA produced by Escherichia coli CFT073. Using the SpyTag-SpyCatcher system to probe passenger domain localization, we found that both intrinsically disordered polypeptides fused to the UpaG passenger domain and artificially disulfide-bonded polypeptides were secreted effectively but relatively slowly. Surprisingly, we also found that in some cases, the three nonnative passenger domain segments associated with a single trimer were secreted sequentially. Photo-cross-linking experiments indicated that incompletely assembled UpaG derivatives remained bound to the barrel assembly machinery (Bam) complex until all three passenger domains were fully secreted. Taken together, our results strongly suggest that the secretion of polypeptides through the TAA pathway is coordinated with the assembly of the β barrel domain and that the folding of passenger domains in the extracellular space maximizes the rate of secretion. Furthermore, our work provides evidence for an unprecedented sequential mode of protein translocation, at least under specific experimental conditions. IMPORTANCE Trimeric autotransporter adhesins (TAAs) are specialized bacterial outer membrane proteins consisting of three identical subunits. TAAs contain large extracellular domains that trimerize and promote virulence, but the mechanism by which they are secreted is poorly understood. We found that the extracellular domains of a native TAA were secreted rapidly but that disordered and artificially folded polypeptides fused to native passenger domains were secreted in a slow, sequential fashion. Our results strongly suggest that the efficient secretion of native extracellular domains is driven by their trimerization following export but that alternative energy sources can be harnessed to secrete nonnative polypeptides. Furthermore, we obtained evidence that TAA extracellular domains are secreted before the assembly of the linked membrane spanning domain is completed.Rakesh SikdarHarris D. BernsteinAmerican Society for MicrobiologyarticleBam complexmembrane proteinsouter membraneprotein foldingprotein secretiontrimeric autotransportersMicrobiologyQR1-502ENmBio, Vol 10, Iss 5 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Bam complex membrane proteins outer membrane protein folding protein secretion trimeric autotransporters Microbiology QR1-502
spellingShingle	Bam complex membrane proteins outer membrane protein folding protein secretion trimeric autotransporters Microbiology QR1-502 Rakesh Sikdar Harris D. Bernstein Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway
description	ABSTRACT Trimeric autotransporter adhesins (TAAs) are a family of bacterial outer membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular (“passenger”) domain and a short C-terminal segment that contributes four β strands to a single 12-stranded β barrel. The mechanism by which the passenger domains are translocated across the OM and the energetics of the translocation reaction are poorly understood. To address these issues, we examined the secretion of modified versions of the passenger domain of UpaG, a TAA produced by Escherichia coli CFT073. Using the SpyTag-SpyCatcher system to probe passenger domain localization, we found that both intrinsically disordered polypeptides fused to the UpaG passenger domain and artificially disulfide-bonded polypeptides were secreted effectively but relatively slowly. Surprisingly, we also found that in some cases, the three nonnative passenger domain segments associated with a single trimer were secreted sequentially. Photo-cross-linking experiments indicated that incompletely assembled UpaG derivatives remained bound to the barrel assembly machinery (Bam) complex until all three passenger domains were fully secreted. Taken together, our results strongly suggest that the secretion of polypeptides through the TAA pathway is coordinated with the assembly of the β barrel domain and that the folding of passenger domains in the extracellular space maximizes the rate of secretion. Furthermore, our work provides evidence for an unprecedented sequential mode of protein translocation, at least under specific experimental conditions. IMPORTANCE Trimeric autotransporter adhesins (TAAs) are specialized bacterial outer membrane proteins consisting of three identical subunits. TAAs contain large extracellular domains that trimerize and promote virulence, but the mechanism by which they are secreted is poorly understood. We found that the extracellular domains of a native TAA were secreted rapidly but that disordered and artificially folded polypeptides fused to native passenger domains were secreted in a slow, sequential fashion. Our results strongly suggest that the efficient secretion of native extracellular domains is driven by their trimerization following export but that alternative energy sources can be harnessed to secrete nonnative polypeptides. Furthermore, we obtained evidence that TAA extracellular domains are secreted before the assembly of the linked membrane spanning domain is completed.
format	article
author	Rakesh Sikdar Harris D. Bernstein
author_facet	Rakesh Sikdar Harris D. Bernstein
author_sort	Rakesh Sikdar
title	Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway
title_short	Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway
title_full	Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway
title_fullStr	Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway
title_full_unstemmed	Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway
title_sort	sequential translocation of polypeptides across the bacterial outer membrane through the trimeric autotransporter pathway
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/043d320418494428bead6e4249e9f957
work_keys_str_mv	AT rakeshsikdar sequentialtranslocationofpolypeptidesacrossthebacterialoutermembranethroughthetrimericautotransporterpathway AT harrisdbernstein sequentialtranslocationofpolypeptidesacrossthebacterialoutermembranethroughthetrimericautotransporterpathway
_version_	1718427008610861056

Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway

Ejemplares similares