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. 2009 Apr 21;106(16):6754-9.
doi: 10.1073/pnas.0809131106. Epub 2009 Apr 6.

Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation

Affiliations

Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation

Didier Vingadassalom et al. Proc Natl Acad Sci U S A. .

Abstract

Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin "pedestals." One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspF(U) (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY(458)) sequence in the Tir C-terminal cytoplasmic domain. Tir, EspF(U), and N-WASP form a complex, but neither EspF(U) nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspF(U) identified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspF(U), and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal NPY(458) sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
IRTKS and IRSp53 are recruited to actin pedestals but only IRTKS localizes to sites of bacterial attachment independently of EspFU. (A) HeLa cells were infected with EHECΔdam, which generates actin pedestals more efficiently on cultured mammalian cells than does WT EHEC (thereby facilitating evaluation of recruitment; ref. 38), and examined after staining with anti-IRSp53 or anti-IRTKS antibody (green), DAPI to localize attached bacteria (blue), and Alexa568-phalloidin (red). (B) HeLa cells were infected with EHECΔdamΔespFU and examined after staining as in A.
Fig. 2.
Fig. 2.
The IRTKS SH3 domain, EspFU proline-rich domain, and WASP GBD form a tripartite complex in vitro. Interactions between GST-SH3IRTKS (50 μM) and EspFU-5R (10 μM) in complex with FITC-labeled GBD (50 μM) were examined by gel filtration chromatography. The A494 profile, which detects FITC-GBD, is shown.
Fig. 3.
Fig. 3.
The EHEC Tir NPY458 sequence is required for binding of IRTKS to Tir and its recruitment to sites of bacterial attachment. (A) GST-IRTKS derivatives were incubated with TirNPY458 or TirAAA458 and pulled down using glutathione magnetic beads. Proteins present in the original incubation (O), the supernatant (S) or the pull-down (P) were visualized by Coomassie staining after 10% SDS/PAGE. (B) HeLa cells transfected with GFP-IRTKS were infected with KC12Δtir (8) harboring plasmids encoding EHEC Tir carrying the WT NPY458 sequence (Tir NPY) or alanine substitutions of this sequence, as indicated (Left). Monolayers were examined after staining with DAPI to localize bacteria (blue), Alexa568-phalloidin (red), and anti-myc antibody to detect GFP-IRTKS-myc (green).
Fig. 4.
Fig. 4.
Ectopic expression of the IRTKS SH3 or IMD domain inhibits EspFU-dependent pedestal formation. Transfected HeLa cells expressing GFP or GFP fusion proteins were infected with KC12/pEspFU (15). Transfected cells were identified by GFP fluorescence (Merge), and monolayers were stained with DAPI (blue) and Alexa568-phalloidin (red). (The lack of localization of GFP-IMD to sites of bacterial attachment may be related to the unexplained paucity of Tir foci.) The percentage of cells competent for actin pedestal formation after infection is shown (Right). Shown is the mean ± SD of at least 3 experiments; *P < 0.0001; =P < 0.01.
Fig. 5.
Fig. 5.
Genetic depletion of IRTKS inhibits EspFU-dependent pedestal formation. HeLa cells transfected with pairs of control, IRTKS, or IRSp53 siRNAs, or a pool of the pair of IRTKS and IRSp53 siRNAs, were infected with KC12/pEspFU (15). Monolayers were examined after staining with DAPI (blue) and Alexa568-phalloidin (red). The percentage of cells competent for actin pedestal formation after infection is shown (Right). Shown is the mean ± SD of at least 3 experiments; *P < 0.0001.

Comment in

  • Enterohemorrhagic Escherichia coli raises the I-BAR.
    Yi CR, Goldberg MB. Yi CR, et al. Proc Natl Acad Sci U S A. 2009 Apr 21;106(16):6431-2. doi: 10.1073/pnas.0902773106. Epub 2009 Apr 20. Proc Natl Acad Sci U S A. 2009. PMID: 19380713 Free PMC article. No abstract available.

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