IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface.
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 (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) fused to either full length Shigella flexneri IcsA (IcsA) autotransporter or to the β domain alone (IcsAβ) to determine the contribution of the native IcsA α domain in presenting the fusion proteins on the surface of E. coli K-12 UT5600 (ΔompT).


RESULTS
Expression of IcsA-Bla was greater than IcsAβ-Bla. High levels of IcsA-MalE were detected but IcsAβ-MalE was not expressed. All fusion proteins other than IcsAβ-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 α-IcsA and α-MBP. UT5600 expressing IcsAβ-MalE was not labelled with α-MBP. A third of UT5600 expressing IcsA-Bla were detectable with α-Bla but only 5% of UT5600 (IcsAβ-Bla) were labelled with α-Bla. The correct folding of the Bla moiety when fused to IcsA and IcsAβ 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 IcsAβ-Bla.


CONCLUSIONS
The export mechanism of autotransporters is not well understood but accumulating evidence suggest a critical role for the native effector or α domain in facilitating its own export via interactions with the translocation or β domain. This is the first report directly comparing expression of heterologous proteins fused to the full length IcsA autotransporter and fusion to the β domain alone. Protein expression and surface presentation of the fusion proteins were dramatically improved when fused to IcsA rather than IcsAβ. Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native α domain. This work also provides further evidence for a key interaction between the autotransporter α and β domains.
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.
Mabel
M
Lum
Renato
R
Morona
D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors; D058619:Maltose-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D012763:Shigella flexneri / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C058483:virG protein, Shigella flexneri; D000667:Ampicillin; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1