Dataset Information

Subdomain 2 of the Autotransporter Pet Is the Ligand Site for Recognizing the Pet Receptor on the Epithelial Cell Surface.

ABSTRACT: Most autotransporter passenger domains, regardless of their diversity in function, fold or are predicted to fold as right-handed ?-helices carrying various loops that are presumed to confer functionality. Our goal here was to identify the subdomain (loop) or amino acid sequence of the Pet passenger domain involved in the receptor binding site on the host cell for Pet endocytosis. Here, we show that d1 and d2 subdomains, as well as the amino acid sequence linking the subdomain d2 and the adjacent ?-helix (PDWET), are not required for Pet secretion through the autotransporter system and that none of our deletion mutants altered the predicted long right-handed ?-helical structure. Interestingly, Pet lacking the d2 domain (Pet?d2) was unable to bind on the epithelial cell surface, in contrast to Pet lacking d1 (Pet?d1) subdomain or PDWET sequences. Moreover, the purified d1 subdomain, the biggest subdomain (29.8 kDa) containing the serine protease domain, was also unable to bind the cell surface. Thus, d2 sequence (54 residues without the PDWET sequence) was required for Pet binding to eukaryotic cells. In addition, this d2 sequence was also needed for Pet internalization but not for inducing cell damage. In contrast, Pet?d1, which was able to bind and internalize inside the cell, was unable to cause cell damage. Furthermore, unlike Pet, Pet?d2 was unable to bind cytokeratin 8, a Pet receptor. These data indicate that the surface d2 subdomain is essential for the ligand-receptor (Pet-Ck8) interaction for Pet uptake and to start the epithelial cell damage by this toxin.

SUBMITTER: Chavez-Duenas L

PROVIDER: S-EPMC4936364 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Subdomain 2 of the Autotransporter Pet Is the Ligand Site for Recognizing the Pet Receptor on the Epithelial Cell Surface.

Chavez-Dueñas Lucia L Serapio-Palacios Antonio A Nava-Acosta Raul R Navarro-Garcia Fernando F

Infection and immunity 20160623 7

Most autotransporter passenger domains, regardless of their diversity in function, fold or are predicted to fold as right-handed β-helices carrying various loops that are presumed to confer functionality. Our goal here was to identify the subdomain (loop) or amino acid sequence of the Pet passenger domain involved in the receptor binding site on the host cell for Pet endocytosis. Here, we show that d1 and d2 subdomains, as well as the amino acid sequence linking the subdomain d2 and the adjacent ...[more]

PMID: 27113356

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Project description:UnlabelledThe 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.ImportanceReceptor-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.

Project description:BackgroundAutotransporters are attractive cell surface display vehicles as they lack complex adaptor proteins necessary for protein export. Recent reports have suggested that the native effector domain (? domain) and translocation domain (? domain) interact with each other to drive translocation of the effector domain to the outer membrane. In this report we compared the expression, surface localisation and folding of TEM-1 ?-lactamase (Bla) and maltose binding protein (MalE or MBP) fused to either full length Shigella flexneri IcsA (IcsA) autotransporter or to the ? domain alone (IcsA?) to determine the contribution of the native IcsA ? domain in presenting the fusion proteins on the surface of E. coli K-12 UT5600 (?ompT).ResultsExpression of IcsA-Bla was greater than IcsA?-Bla. High levels of IcsA-MalE were detected but IcsA?-MalE was not expressed. All fusion proteins other than IcsA?-MalE were localised to the outer membrane and were detected on the surface of UT5600 via immunofluorescence microscopy. All bacteria expressing IcsA-MalE were labelled with both ?-IcsA and ?-MBP. UT5600 expressing IcsA?-MalE was not labelled with ?-MBP. A third of UT5600 expressing IcsA-Bla were detectable with ?-Bla but only 5% of UT5600 (IcsA?-Bla) were labelled with ?-Bla. The correct folding of the Bla moiety when fused to IcsA and IcsA? was also retained as UT5600 expressing either fusion protein exhibited a decreased zone of inhibition in the presence of ampicillin. UT5600 expressing IcsA-Bla was more resistant compared to UT5600 expressing IcsA?-Bla.ConclusionsThe export mechanism of autotransporters is not well understood but accumulating evidence suggest a critical role for the native effector or ? domain in facilitating its own export via interactions with the translocation or ? domain. This is the first report directly comparing expression of heterologous proteins fused to the full length IcsA autotransporter and fusion to the ? domain alone. Protein expression and surface presentation of the fusion proteins were dramatically improved when fused to IcsA rather than IcsA?. Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native ? domain. This work also provides further evidence for a key interaction between the autotransporter ? and ? domains.

Project description:Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca) autotransporter (AT) family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1-3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis). Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i) to infect mammalian cells should the flea bite a host, ii) to remain infectious when extracellular and iii) to infect the flea midgut when ingested with a blood meal. Each Sca protein may be important for survival of R. typhi and the lack of host restricted expression may indicate a strategy of preparedness for infection of a new host.

Dataset Information

Subdomain 2 of the Autotransporter Pet Is the Ligand Site for Recognizing the Pet Receptor on the Epithelial Cell Surface.

Publications

Subdomain 2 of the Autotransporter Pet Is the Ligand Site for Recognizing the Pet Receptor on the Epithelial Cell Surface.

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