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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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Detection of IcsA and IcsAβ fusion proteins in the OM. UT5600 strains were grown to mid-log phase, induced with 0.2% (w/v) arabinose and were fractionated into soluble fraction (S), whole membrane (WM), inner membrane (IM) and outer membrane (OM). Equivalent amounts of protein extracts from each fraction were loaded and analysed by Western immunoblotting. Immunobloting of fractions from (A) MLRM28 (pMLRM1::bla) and MLRM20 (pMLRM2::bla) with anti-Bla; (B) MLRM28 (pMLRM1::bla) with anti-IcsA; (C) MLRM39 (pMLRM1::malE) with anti-MBP and (D) anti-IcsA. The apparent molecular sizes of the proteins are indicated.
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Figure 4: Detection of IcsA and IcsAβ fusion proteins in the OM. UT5600 strains were grown to mid-log phase, induced with 0.2% (w/v) arabinose and were fractionated into soluble fraction (S), whole membrane (WM), inner membrane (IM) and outer membrane (OM). Equivalent amounts of protein extracts from each fraction were loaded and analysed by Western immunoblotting. Immunobloting of fractions from (A) MLRM28 (pMLRM1::bla) and MLRM20 (pMLRM2::bla) with anti-Bla; (B) MLRM28 (pMLRM1::bla) with anti-IcsA; (C) MLRM39 (pMLRM1::malE) with anti-MBP and (D) anti-IcsA. The apparent molecular sizes of the proteins are indicated.

Mentions: The 150 kDa band corresponding to IcsA-Bla was detected in the whole membrane (WM) and OM when immunoblotted with α-Bla and α-IcsA (Figure 4A and 4B, lanes 2 and 4). IcsAβ-Bla was also detected in the WM and OM (Figure 4A, lanes 6 and 8). A band corresponding to the size of the mature 29 kDa Bla was observed in the soluble fraction (S) and the inner membrane (IM) of UT5600 strains expressing IcsA-Bla (MLRM28) and IcsAβ-Bla (MLRM20) (Figure 4A, lanes 1, 3, 5 and 7). The association of the Bla protein at the IM has not been reported previously and is most likely due to an artefact from fractionation. Degraded proteins (< 120 kDa) were observed for IcsA-Bla (Figure 4A and 4B, lanes 2 and 4) but not IcsAβ-Bla in the WM and OM fractions (Figure 4A, lanes 6 and 8).


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

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

Detection of IcsA and IcsAβ fusion proteins in the OM. UT5600 strains were grown to mid-log phase, induced with 0.2% (w/v) arabinose and were fractionated into soluble fraction (S), whole membrane (WM), inner membrane (IM) and outer membrane (OM). Equivalent amounts of protein extracts from each fraction were loaded and analysed by Western immunoblotting. Immunobloting of fractions from (A) MLRM28 (pMLRM1::bla) and MLRM20 (pMLRM2::bla) with anti-Bla; (B) MLRM28 (pMLRM1::bla) with anti-IcsA; (C) MLRM39 (pMLRM1::malE) with anti-MBP and (D) anti-IcsA. The apparent molecular sizes of the proteins are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Detection of IcsA and IcsAβ fusion proteins in the OM. UT5600 strains were grown to mid-log phase, induced with 0.2% (w/v) arabinose and were fractionated into soluble fraction (S), whole membrane (WM), inner membrane (IM) and outer membrane (OM). Equivalent amounts of protein extracts from each fraction were loaded and analysed by Western immunoblotting. Immunobloting of fractions from (A) MLRM28 (pMLRM1::bla) and MLRM20 (pMLRM2::bla) with anti-Bla; (B) MLRM28 (pMLRM1::bla) with anti-IcsA; (C) MLRM39 (pMLRM1::malE) with anti-MBP and (D) anti-IcsA. The apparent molecular sizes of the proteins are indicated.
Mentions: The 150 kDa band corresponding to IcsA-Bla was detected in the whole membrane (WM) and OM when immunoblotted with α-Bla and α-IcsA (Figure 4A and 4B, lanes 2 and 4). IcsAβ-Bla was also detected in the WM and OM (Figure 4A, lanes 6 and 8). A band corresponding to the size of the mature 29 kDa Bla was observed in the soluble fraction (S) and the inner membrane (IM) of UT5600 strains expressing IcsA-Bla (MLRM28) and IcsAβ-Bla (MLRM20) (Figure 4A, lanes 1, 3, 5 and 7). The association of the Bla protein at the IM has not been reported previously and is most likely due to an artefact from fractionation. Degraded proteins (< 120 kDa) were observed for IcsA-Bla (Figure 4A and 4B, lanes 2 and 4) but not IcsAβ-Bla in the WM and OM fractions (Figure 4A, lanes 6 and 8).

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