Amino acid residues within enterohemorrhagic Escherichia coli O157:H7 Tir involved in phosphorylation, alpha-actinin recruitment, and Nck-independent pedestal formation

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) adherence to epithelial cells results in the formation of actin pedestals. Pedestal formation requires the bacterial protein Tir, which is inserted into the epithelial cell plasma membrane by the type III secretion system. EPEC and EHEC use different Tir-based mechanisms for pedestal formation, and the EPEC Tir residues required have been well described. In contrast, little is known about the regions of EHEC O157:H7 Tir that are essential for pedestal formation. Additionally, EHEC O157:H7 Tir is serine/threonine phosphorylated, although the residues involved and their role in pedestal formation are not known. In this study, we describe two regions within the carboxy terminus of EHEC O157:H7 Tir that are required for phosphorylation and pedestal formation. Serines 436 and 437 are substrates for protein kinase A phosphorylation, although this is not required to form pedestals. Using a series of internal deletion mutants, we found that amino acids 454 to 463 are required for efficient pedestal formation. Deleting this region resulted in a significant decrease in the recruitment of both filamentous actin and the actin binding protein alpha-actinin. As alpha-actinin binds directly to the EHEC O157:H7 amino terminus, these data suggest that its recruitment is dependent on pedestal formation.

FIG. 1.

The EHEC O157:H7 carboxy terminus is important for Tir phosphorylation. (A) Deletion mutations described in the text. (B) Anti-Tir immunoblot of bacterial pellets derived from EHECΔtir expressing WT Tir or the deletion mutants shown in panel A. (C) Anti-Tir immunoblot of TX-100-soluble fractions from HeLa cells incubated with EHECΔtir expressing WT Tir or the deletion mutants shown in panel A. CVD451 is a type III secretion mutant and does not translocate Tir. Arrowheads indicate phosphorylated Tir. In panels B and C, apparent molecular masses are in kilodaltons.

FIG. 2.

EHEC O157:H7 Tir is a substrate for PKA. (A) In vitro phosphorylation assay. Left panel, anti-Tir immunoblot of WT or mutant Tir incubated with increasing concentrations of PKA. Right panel, WT Tir phosphorylation is blocked by the PKA inhibitor H-89. (B) Tir S4366A/S437A is translocated into the host cell but not phosphorylated. Left panel, anti-Tir immunoblot of TX-100-soluble and insoluble fractions derived from EHEC-infected HeLa cells as described in the text. Right panel, anti-Tir immunoblot of TX-100-soluble fractions from EHEC-infected HeLa cells treated with calf alkaline phosphatase (Alk. Phos). (C) PKA phosphorylation is not required for pedestal formation. EHEC-infected HeLa cells were triple labeled for anti-O157, anti-Tir, and phalloidin (actin).

FIG. 3.

EHEC O157:H7 Tir amino acids 452 to 463 are required for pedestal formation. (A) Immunofluorescence micrograph of HeLa cells infected with EHEC Tir mutants deficient for pedestal formation. Cells are triple labeled with anti-O157, anti-EHEC Tir, and phalloidin. (B) Anti-Tir immunoblot of TX-100-soluble fractions from HeLa cells infected with EHECΔtir expressing WT Tir or the deletion mutants. Apparent molecular mass markers are in kilodaltons.

FIG. 4.

Amino acids 451 to 459 are required for Nck-independent pedestal formation by EPEC Tir. (A) Alignment of EHEC O157:H7 and EPEC C-terminal Tir sequences within the regions of the deletions described within the text. Phosphorylated tyrosines within EPEC Tir are indicated by asterisks, while the conserved region deleted within EPEC Tir is underlined. (B) Immunofluorescence micrograph of HeLa cells infected with either EHECΔtir/EPEC Tir Y474F or EHECΔtir/EPEC Tir Y474F Δ451-459 and labeled for EPEC Tir, actin, and anti-O157. (C) HeLa cells infected with EHECΔtirEPEC Tir or EHECΔtirEPEC TirΔ451-459 and labeled for EPEC Tir, actin, and Nck.

FIG. 5.

The requirements for α-actinin recruitment differ between EHEC and EPEC Tirs. (A) Immunofluorescence micrograph of HeLa cells infected with EHECΔtir expressing either WT EHEC Tir or mutant Tir proteins unable to support pedestal formation. Cells were quadruple labeled with anti-O157, anti-EHEC Tir, anti-α-actinin, and phalloidin. (B) Immunofluorescence micrograph of HeLa cells infected with either EHECΔtir/EPEC Tir Y474F or the non-pedestal-forming mutant EHECΔtir/EPEC Tir Y474FΔ451-459. Cells were quadruple labeled for anti-O157, anti-EPEC Tir, anti-α-actinin, and phalloidin.