Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>

ABSTRACT The group of proteins known as serine protease autotransporters of Enterobacteriaceae (SPATE) is a growing family of serine proteases secreted to the external milieu by the type V secretion system. Pet toxin and some other SPATE belong to the class 1 cytotoxic SPATE, which have comparable p...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Raul Nava-Acosta, Fernando Navarro-Garcia
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/7888a2203da84b93bd823c120fd669c6
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:7888a2203da84b93bd823c120fd669c6
record_format	dspace
spelling	oai:doaj.org-article:7888a2203da84b93bd823c120fd669c62021-11-15T15:42:31ZCytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>10.1128/mBio.00838-132150-7511https://doaj.org/article/7888a2203da84b93bd823c120fd669c62013-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00838-13https://doaj.org/toc/2150-7511ABSTRACT The group of proteins known as serine protease autotransporters of Enterobacteriaceae (SPATE) is a growing family of serine proteases secreted to the external milieu by the type V secretion system. Pet toxin and some other SPATE belong to the class 1 cytotoxic SPATE, which have comparable protease strength on fodrin. Pet is internalized and is directed to its intracellular substrate by retrograde transport. However, the epithelial cell receptor for Pet has yet to be identified. We show that Pet has affinity for the epithelial cell surface until the saturation of the binding sites at 100 nM Pet. Affinity column assays and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis identified a cytokeratin (CK8) which directly binds to Pet, and both proteins colocalized on the cell surface. Interestingly, CK8 is not present in kidney cell lines, which are not susceptible to Pet. Inhibition experiments by using anti-CK8 and ck8 small interfering RNA (siRNA) blocked the cytotoxic effect induced by Pet, while exogenous CK8 expression in kidney cells made them susceptible to Pet intoxication. Recombinant CK8 showed a Pet-binding pattern similar to that seen by using fixed cells. Remarkably, Pet colocalized with CK8 and clathrin at early times (receptor-mediated endocytosis), and subsequently, Pet colocalized with CK8 and Rab5b in the early endosomes. These data support the idea that CK8 is an important receptor for Pet on epithelial cells for starting its cytotoxic effects. These data suggest that therapeutics that block Pet-CK8 interaction may improve outcome of diseases caused by Pet-secreting Enterobacteriaceae such as enteroaggregative Escherichia coli. IMPORTANCE Receptor-ligand binding is one mechanism by which cells sense and respond to external cues. Receptors may also be utilized by toxins to mediate their own internalization. Pet toxin is secreted by enteroaggregative Escherichia coli, an organism that causes persistent diarrhea in children, traveler’s diarrhea, and acute and persistent diarrhea in patients with HIV. Pet is a member of the family of serine protease autotransporters of Enterobacteriaceae (SPATE). SPATE in different pathogens are virulence factors, and Pet belongs to the class 1 cytotoxic SPATE, which have comparable protease strength on their biological substrate, fodrin (a cytoskeletal protein important for maintaining cell viability). To cleave fodrin, Pet enters the cells by clathrin-mediated endocytosis. This mechanism includes receptor-mediated endocytosis (a receptor-ligand complex triggers the endocytosis). We show that CK8 is an important receptor for Pet on epithelial cells and that it may be useful for identifying molecules that block the interaction of CK8 with Pet.Raul Nava-AcostaFernando Navarro-GarciaAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 6 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Raul Nava-Acosta Fernando Navarro-Garcia Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>
description	ABSTRACT The group of proteins known as serine protease autotransporters of Enterobacteriaceae (SPATE) is a growing family of serine proteases secreted to the external milieu by the type V secretion system. Pet toxin and some other SPATE belong to the class 1 cytotoxic SPATE, which have comparable protease strength on fodrin. Pet is internalized and is directed to its intracellular substrate by retrograde transport. However, the epithelial cell receptor for Pet has yet to be identified. We show that Pet has affinity for the epithelial cell surface until the saturation of the binding sites at 100 nM Pet. Affinity column assays and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis identified a cytokeratin (CK8) which directly binds to Pet, and both proteins colocalized on the cell surface. Interestingly, CK8 is not present in kidney cell lines, which are not susceptible to Pet. Inhibition experiments by using anti-CK8 and ck8 small interfering RNA (siRNA) blocked the cytotoxic effect induced by Pet, while exogenous CK8 expression in kidney cells made them susceptible to Pet intoxication. Recombinant CK8 showed a Pet-binding pattern similar to that seen by using fixed cells. Remarkably, Pet colocalized with CK8 and clathrin at early times (receptor-mediated endocytosis), and subsequently, Pet colocalized with CK8 and Rab5b in the early endosomes. These data support the idea that CK8 is an important receptor for Pet on epithelial cells for starting its cytotoxic effects. These data suggest that therapeutics that block Pet-CK8 interaction may improve outcome of diseases caused by Pet-secreting Enterobacteriaceae such as enteroaggregative Escherichia coli. IMPORTANCE Receptor-ligand binding is one mechanism by which cells sense and respond to external cues. Receptors may also be utilized by toxins to mediate their own internalization. Pet toxin is secreted by enteroaggregative Escherichia coli, an organism that causes persistent diarrhea in children, traveler’s diarrhea, and acute and persistent diarrhea in patients with HIV. Pet is a member of the family of serine protease autotransporters of Enterobacteriaceae (SPATE). SPATE in different pathogens are virulence factors, and Pet belongs to the class 1 cytotoxic SPATE, which have comparable protease strength on their biological substrate, fodrin (a cytoskeletal protein important for maintaining cell viability). To cleave fodrin, Pet enters the cells by clathrin-mediated endocytosis. This mechanism includes receptor-mediated endocytosis (a receptor-ligand complex triggers the endocytosis). We show that CK8 is an important receptor for Pet on epithelial cells and that it may be useful for identifying molecules that block the interaction of CK8 with Pet.
format	article
author	Raul Nava-Acosta Fernando Navarro-Garcia
author_facet	Raul Nava-Acosta Fernando Navarro-Garcia
author_sort	Raul Nava-Acosta
title	Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>
title_short	Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>
title_full	Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>
title_fullStr	Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>
title_full_unstemmed	Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>
title_sort	cytokeratin 8 is an epithelial cell receptor for pet, a cytotoxic serine protease autotransporter of <italic toggle="yes">enterobacteriaceae</italic>
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/7888a2203da84b93bd823c120fd669c6
work_keys_str_mv	AT raulnavaacosta cytokeratin8isanepithelialcellreceptorforpetacytotoxicserineproteaseautotransporterofitalictoggleyesenterobacteriaceaeitalic AT fernandonavarrogarcia cytokeratin8isanepithelialcellreceptorforpetacytotoxicserineproteaseautotransporterofitalictoggleyesenterobacteriaceaeitalic
_version_	1718427592108802048

Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of <italic toggle="yes">Enterobacteriaceae</italic>

Ejemplares similares