Enterohemorrhagic Escherichia coli Effector Protein EspF Interacts With Host Protein ANXA6 and Triggers Myosin Light Chain Kinase (MLCK)-Dependent Tight Junction Dysregulation.

Ying Hua,Bao Zhang,Muqing Fu,Jiali Wu,Wei Zhao,Jinyue Liu,Xiaoxia Li,Chengsong Wan

doi:10.3389/fcell.2020.613061

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen that can cause bloody diarrhea and hemolytic uremic syndrome (HUS) in humans. EspF is one of the best-characterized effector proteins secreted from the type three secretion system to hijack host cell functions. However, the crucial pathogen-host interactions and the basis for the intestinal barrier disruption during infections remain elusive. Our previous study screened and verified the interaction between host protein ANXA6 and EspF protein. Here, by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (CO-IP), we verified that EspF interacts with ANXA6 through its C-terminal domain. Furthermore, we found that both the constitutive expression of EspF or ANXA6 and the co-expression of EspF-ANXA6 could decrease the levels of tight junction (TJ) proteins ZO-1 and occludin, and disrupt the distribution of ZO-1. Moreover, we showed that EspF-ANXA6 activated myosin light chain kinase (MLCK), induced the phosphorylation of myosin light chain (MLC) and PKCα, and down-regulated the expression level of Calmodulin protein. Collectively, this study revealed a novel interaction between the host protein (ANXA6) and EspF. The binding of EspF to ANXA6 may perturb TJs in an MLCK-MLC-dependent manner, and thus may be involved in EHEC pathogenic function.

Highlights

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen; it has been linked to a broad spectrum of diseases ranging from bloody diarrhea to hemorrhagic colitis and even life-threatening hemolytic uremic syndrome (HUS) (Tarr et al, 2005)
We investigated the function of the EspFANXA6 complex by co-expressing CE and YA in HEK293 cells
As shown by the arrow, fluorescence resonance energy transfer (FRET) is mainly displayed in places close to the inner cell membrane (Figure 2B), indicating that these two proteins mostly interact at the inner cell membrane; this result is consistent with a study that described the Annexin A6 (ANXA6) protein as an annexin protein (Cornely et al, 2011)

Summary

Introduction

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen; it has been linked to a broad spectrum of diseases ranging from bloody diarrhea to hemorrhagic colitis and even life-threatening hemolytic uremic syndrome (HUS) (Tarr et al, 2005). EHEC-associated enteritis and diarrhea are closely related to the subversion of intestinal mucosal epithelial cells barrier integrity and the disruption of epithelium permeability (Roxas et al, 2010). TJs are complex structures located in the most apical region of the lateral membrane and modulate two primary functions in epithelia (van Itallie and Anderson, 2014). They form a physical barrier that restricts paracellular transportation of molecules such as ions, solutes, water, and immune cells (Pawlowska and Sobieszczanska, 2017). It is not surprising that TJs are one of the main targets of bacterial proteins and key players in host-pathogen interaction

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