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. 2013 May 31;164(1-2):164-70.
doi: 10.1016/j.vetmic.2013.01.028. Epub 2013 Feb 4.

The enterohemorrhagic Escherichia coli effector protein NleF binds mammalian Tmp21

Rachel L Olsen  1 , Frank Echtenkamp, Dilyara Cheranova, Wanyin Deng, B Brett Finlay, Philip R Hardwidge
Affiliations

The enterohemorrhagic Escherichia coli effector protein NleF binds mammalian Tmp21

Rachel L Olsen et al. Vet Microbiol. .

Abstract

The human pathogens enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC), as well as the mouse pathogen Citrobacter rodentium encode type III secretion system (T3SS) effector proteins to promote their survival in the infected host. The mechanisms of action and the host targets of T3SS effectors are under active investigation because of their importance to bacterial virulence. The non-locus of enterocyte effacement (LEE)-encoded protein F, NleF, contributes to E. coli and C. rodentium colonization of piglets and mice, respectively. Here we sought to characterize the host binding partners of NleF. Using a yeast two-hybrid screen, we identified Tmp21, a type-I integral membrane protein and COPI-vesicle receptor involved in trans-Golgi network function, as an NleF-binding partner. We confirmed this interaction using immunoprecipitation and bimolecular fluorescence complementation (BiFC). We expressed a temperature-sensitive vesicular stomatitis virus glycoprotein (tsVSVG) to monitor protein trafficking and determined that NleF slows the intracellular trafficking of tsVSVG from the endoplasmic reticulum to the Golgi.

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Figures

Fig. 1
Fig. 1
NleF binds Tmp21. (A) β-Galactosidase assays. NleF was expressed in yeast in the presence or absence of human Tmp21 and β-galactosidase activity was quantified. Asterisks indicate significantly different β-galactosidase activity as compared with untransformed yeast (p < 0.05, t-test). (B) Coimmunoprecipitation. His-Tmp21 was co-expressed with NleF-FLAG in E. coli BL21(DE3). NleF was immunoprecipitated and its binding to His-Tmp21 binding was assessed using immunoblotting. (C) Immunofluorescence microscopy. HeLa cells were infected with C. rodentium DBS100/pnleF-FLAG and stained with an α-NleF antibody (red). (D) NleF and Tmp21 co-localize. HeLa cells were infected with C. rodentium/pnleF-FLAG, transfected with Tmp21-HA and stained with α-NleF (green) and α-HA (red) antibodies.
Fig. 2
Fig. 2
BiFC. (A) NleF and Tmp-21 expression. NleF and Tmp21 were cloned into eYFP-VN and eYFP-VC vectors and protein expression was evaluated by immunoblotting. (B) BiFC quantification. Relative fluorescence intensity after co-transfecting indicated NleF- and Tmp21-eYFP plasmid combinations (n = 3). Asterisks indicate significantly different fluorescence intensity as compared with untransfected samples (p < 0.05, ANOVA). (C) NleF truncations binding to Tmp21. NleF truncations were cloned into eYFP-VC vectors and protein expression was evaluated by immunoblotting. Binding to Tmp21-eYFP-VN was measured using BiFC and is scored as positive (+) or negative (−) in the respective columns. (D) Tmp21 truncations binding to NleF. Tmp21 truncations were cloned into eYFP-VC vectors and protein expression was evaluated by immunoblotting. Binding to NleF-eYFP-VN was measured using BiFC and is scored as positive (+) or negative in the respective columns.
Fig. 3
Fig. 3
NleF alters VSVG-GFP trafficking. (A) VSVG-GFP localization schematic. At 40 °C, VSVG-GFP becomes reversibly misfolded and retained in the ER. Incubation at 19 °C allows VSVG-GFP refolding and transport to the TGN. Further incubation at 32 °C allows VSVG-GFP trafficking to the PM. (B) VSVG-GFP colocalization with calnexin. HeLa cells were transfected with VSVG-GFP and then incubated at 37 °C or shifted to 19 °C, 32 °C, or 40 °C. Cells were stained with an α-calnexin antibody. (C) VSVG-GFP colocalization with golgin-97. Experiment performed as in (B). Cells were stained with an α-golgin-97 antibody. (D) Impact of NleF on VSVG-GFP colocalization with calnexin. HeLa cells were cotransfected with both VSVG-GFP and NleF-HA and then incubated at 37 °C or shifted to 19 °C, 32 °C, or 40 °C. Cells were stained with α-calnexin (red) and α-HA (blue) antibodies. (E) Impact of NleF on VSVG-GFP colocalization with golgin-97. Experiment performed as in (D). Cells were stained with α-golgin-97 (red) and α-HA (blue) antibodies.
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