Bacterial and Host Determinants of MAL Activation upon EPEC Infection: The Roles of Tir, ABRA, and FLRT3

Abstract

Infection of host cells by pathogenic microbes triggers signal transduction pathways leading to a multitude of host cell responses including actin cytoskeletal re-arrangements and transcriptional programs. The diarrheagenic pathogens Enteropathogenic E. coli (EPEC) and the related Enterohemorrhagic E. coli (EHEC) subvert the host-cell actin cytoskeleton to form attaching and effacing lesions on the surface of intestinal epithelial cells by injecting effector proteins via a type III secretion system. Here we use a MAL translocation assay to establish the effect of bacterial pathogens on host cell signaling to transcription factor activation. MAL is a cofactor of Serum response factor (SRF), a transcription factor with important roles in the regulation of the actin cytoskeleton. We show that EPEC induces nuclear accumulation of MAL-GFP. The translocated intimin receptor is essential for this process and phosphorylation of Tyrosine residues 454 and 474 is important. Using an expression screen we identify FLRT3, C22orf28 and TESK1 as novel activators of SRF. Importantly we demonstrate that ABRA (actin-binding Rho-activating protein, also known as STARS) is necessary for EPEC-induced nuclear accumulation of MAL and the novel SRF activator FLRT3, is a component of this pathway. We further demonstrate that ABRA is important for structural maintenance of EPEC pedestals. Our results uncover novel components in pathogen-activated cytoskeleton signalling to MAL activation.

Author Summary

Many significant immune diseases are caused by bacterial pathogens that deliver effector proteins into their host. The pathogen uses these proteins to subvert the hosts' normal cytosolic defense in a way that services the pathogen. It is therefore important to understand the normal processes of a cell and how they are affected by bacterial infection. We have established the effect of bacteria on host cell signalling to the transcription factor serum response factor. Serum response factor is a widely expressed transcription factor that controls the expression of many important genes. We show that Enteropathogenic E. coli infection can activate serum response factor and that the effector protein Tir is essential for this activation. Furthermore, we identify new genes that are important in this infection-induced activation and show that they are important in maintaining structures necessary for Enteropathogenic E. coli infection.