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

Lum M, Morona R - Microb. Cell Fact. (2012)

Bottom Line: UT5600 expressing IcsA-Bla was more resistant compared to UT5600 expressing IcsAβ-Bla.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.Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native α domain.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.

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 (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.

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Related in: MedlinePlus

Summary of IcsA and IcsAβ fusion proteins. Black boxes represent the IcsA signal peptide (ss), light grey boxes represent the 80 kDa IcsAα (α) and white boxes represent the 37 kDa IcsAβ (β). TEM-1 beta lactamase (Bla) and maltose binding protein (MalE/MBP) are represented by dark grey boxes. Predicted molecular weights are shown in parentheses.
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Figure 2: Summary of IcsA and IcsAβ fusion proteins. Black boxes represent the IcsA signal peptide (ss), light grey boxes represent the 80 kDa IcsAα (α) and white boxes represent the 37 kDa IcsAβ (β). TEM-1 beta lactamase (Bla) and maltose binding protein (MalE/MBP) are represented by dark grey boxes. Predicted molecular weights are shown in parentheses.

Mentions: IcsA and IcsAβ proteins fused to the 29 kDa Bla and 44 kDa MalE were generated to investigate the contribution of the IcsA passenger domain to the overall stability of the chimeric proteins (Figure 2). Both Bla and MalE are relatively globular and have been used in various autotransporter studies [15,31]. The bla and malE coding regions were subcloned from pKMRM1 and pMAL-c2, respectively, with forward and reverse primers incorporating a NotI restriction site resulting in pMLRM28 (pMLRM1::bla), pMLRM20 (pMLRM2::bla), pMLRM39 (pMLRM1::malE) and pMLRM37 (pMLRM2::malE). Figure 1 outlines the construction of these plasmids. The plasmids were transformed into an ΔompT E. coli K-12 strain, UT5600, to prevent fusion protein cleavage by OmpT, an OM protease [7,9].


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

Lum M, Morona R - Microb. Cell Fact. (2012)

Summary of IcsA and IcsAβ fusion proteins. Black boxes represent the IcsA signal peptide (ss), light grey boxes represent the 80 kDa IcsAα (α) and white boxes represent the 37 kDa IcsAβ (β). TEM-1 beta lactamase (Bla) and maltose binding protein (MalE/MBP) are represented by dark grey boxes. Predicted molecular weights are shown in parentheses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298707&req=5

Figure 2: Summary of IcsA and IcsAβ fusion proteins. Black boxes represent the IcsA signal peptide (ss), light grey boxes represent the 80 kDa IcsAα (α) and white boxes represent the 37 kDa IcsAβ (β). TEM-1 beta lactamase (Bla) and maltose binding protein (MalE/MBP) are represented by dark grey boxes. Predicted molecular weights are shown in parentheses.
Mentions: IcsA and IcsAβ proteins fused to the 29 kDa Bla and 44 kDa MalE were generated to investigate the contribution of the IcsA passenger domain to the overall stability of the chimeric proteins (Figure 2). Both Bla and MalE are relatively globular and have been used in various autotransporter studies [15,31]. The bla and malE coding regions were subcloned from pKMRM1 and pMAL-c2, respectively, with forward and reverse primers incorporating a NotI restriction site resulting in pMLRM28 (pMLRM1::bla), pMLRM20 (pMLRM2::bla), pMLRM39 (pMLRM1::malE) and pMLRM37 (pMLRM2::malE). Figure 1 outlines the construction of these plasmids. The plasmids were transformed into an ΔompT E. coli K-12 strain, UT5600, to prevent fusion protein cleavage by OmpT, an OM protease [7,9].

Bottom Line: UT5600 expressing IcsA-Bla was more resistant compared to UT5600 expressing IcsAβ-Bla.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.Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native α domain.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.

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 (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.

Show MeSH
Related in: MedlinePlus