Dataset Information

IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface.

ABSTRACT:

Background

Autotransporters 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).

Results

Expression 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.

Conclusions

The 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.

SUBMITTER: Lum M

PROVIDER: S-EPMC3298707 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Publications

IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface.

Lum Mabel M Morona Renato R

Microbial cell factories 20120207

<h4>Background</h4>Autotransporters 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 (α domain) and translocation domain (β 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 β-lactamase (Bla) and maltose binding protein (MalE or MBP) fus ...[more]

PMID: 22309506

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Project description:The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded beta-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen.

Dataset Information

IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface.

Background

Results

Conclusions

Publications

IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface.

Similar Datasets

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