Dataset Information

Functional comparison of serine protease autotransporters of enterobacteriaceae.

ABSTRACT: The plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) belongs to a family of high-molecular-weight serine protease autotransporters of Enterobacteriaceae (SPATEs) which also includes Pic from EAEC and Shigella flexneri, EspC from enteropathogenic E. coli, EspP from enterohemorrhagic E. coli, Sat from uropathogenic E. coli, Tsh from avian pathogenic E. coli, and SepA from S. flexneri. Phylogenetic analysis shows the SPATE proteins to represent a distinct subfamily of autotransporters with amino acid identities ranging from 35 to 55%, providing a powerful resource to direct structure-function studies. In this study, we show that these related proteins are proteases with divergent substrate specificities, suggesting different functions. The cleavage profile of oligopeptides was found to be unique for each SPATE protein. The SPATEs showed proteolytic activity for several substrates, namely mucin, pepsin, human coagulation factor V, and erythroid spectrin. The cleavage of spectrin has been hypothesized as the mechanism through which Pet induces cytopathic effects. However, whereas Pet, Sat, and EspC cleaved spectrin, only Pet and Sat elicited cytopathic effects; the remaining SPATEs did not cause any morphological changes to HEp-2 cell monolayers. EspC and Pet exhibited acid-dissociable binding to HEp-2 cells. However, Pet was more efficient at entering HEp-2 cells, suggesting a basis for the different abilities of these two proteases to damage cells. Our data suggest that, despite the homologies observed among these proteins, the SPATEs have different pathogenetic functions only partly dependent on their substrate specificities.

SUBMITTER: Dutta PR

PROVIDER: S-EPMC133081 | biostudies-literature | 2002 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional comparison of serine protease autotransporters of enterobacteriaceae.

Dutta Pinaki R PR Cappello Renato R Navarro-García Fernando F Nataro James P JP

Infection and immunity 20021201 12

The plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) belongs to a family of high-molecular-weight serine protease autotransporters of Enterobacteriaceae (SPATEs) which also includes Pic from EAEC and Shigella flexneri, EspC from enteropathogenic E. coli, EspP from enterohemorrhagic E. coli, Sat from uropathogenic E. coli, Tsh from avian pathogenic E. coli, and SepA from S. flexneri. Phylogenetic analysis shows the SPATE proteins to represent a distinct subfamily of autotr ...[more]

PMID: 12438392

Similar Datasets

Project description:UNLABELLED: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.

Dataset Information

Functional comparison of serine protease autotransporters of enterobacteriaceae.

Publications

Functional comparison of serine protease autotransporters of enterobacteriaceae.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets