Amino Acid Residues within EnterohemorrhagicEscherichia coliO157:H7 Tir Involved in Phosphorylation, α-Actinin Recruitment, and Nck-Independent Pedestal Formation

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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.

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