EspF is crucial for Citrobacter rodentium-induced tight junction disruption and lethality in immunocompromised animals

Abstract

Attaching/Effacing (A/E) bacteria include human pathogens enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E . coli (EHEC), and their murine equivalent Citrobacter rodentium (CR), of which EPEC and EHEC are important causative agents of foodborne diseases worldwide. While A/E pathogen infections cause mild symptoms in the immunocompetent hosts, an increasing number of studies show that they produce more severe morbidity and mortality in immunocompromised and/or immunodeficient hosts. However, the pathogenic mechanisms and crucial host-pathogen interactions during A/E pathogen infections under immunocompromised conditions remain elusive. We performed a functional screening by infecting interleukin-22 (IL-22) knockout ( Il22 -/- ) mice with a library of randomly mutated CR strains. Our screen reveals that interruption of the espF gene, which encodes the Type III Secretion System effector EspF ( E . coli secreted protein F) conserved among A/E pathogens, completely abolishes the high mortality rates in CR-infected Il22 -/- mice. Chromosomal deletion of espF in CR recapitulates the avirulent phenotype without impacting colonization and proliferation of CR, and EspF complement in ΔespF strain fully restores the virulence in mice. Moreover, the expression levels of the espF gene are elevated during CR infection and CR induces disruption of the tight junction (TJ) strands in colonic epithelium in an EspF-dependent manner. Distinct from EspF, chromosomal deletion of other known TJ-damaging effector genes espG and map failed to impede CR virulence in Il22 -/- mice. Hence our findings unveil a critical pathophysiological function for EspF during CR infection in the immunocompromised host and provide new insights into the complex pathogenic mechanisms of A/E pathogens.

Type III secretion system effectors form robust and flexible intracellular virulence networks

“…In C. rodentium, 23 additional effector genes are located outside of the LEE, mainly in genomic insertions and cryptic prophages (28). Following translocation, Tir is integrated into the IECs' plasma membrane where it serves as a receptor for intimin, enabling intimate bacterial attachment and formation of actin-rich pedestal-like structures (29), while Map and EspF target the mitochondria and affect the integrity of the tight junctions (TJs) (30,31).…”

“…This process enables intimate bacterial attachment and the formation of actin-rich pedestal-like structures (30). Map and EspF target the host cell mitochondria and affect the integrity of the tight junctions (TJs) between cells (31,32). Single mutants lacking intimin or Tir (33); NleB, an arginine glycosyltransferase that modifies the death domain-containing proteins such as FADD (Fas-associated death domain protein) or RIPK1 (receptor-interacting protein kinase 1) (16); NleA (also known as EspI), which binds Sec24 and affects endosomal trafficking (34); or EspZ, which serves as the T3SS gate keeper protecting cells from effector overdose cytotoxicity (26,35), are highly attenuated in vivo (16,36,37).…”

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

“…Furthermore, a murine model of infection by EHEC also indicated a rapid disruption of the TJ fence function (Roxas et al, 2010 ). Although the mechanism underlying TJ disruption induced by EHEC/EPEC remains unclear, extensive studies have suggested that EspF may play an important role (Mcnamara et al, 2001 ; Tapia et al, 2017a ; Singh et al, 2018 ; Xia et al, 2019 ).…”

Citrobacter rodentium Infection Induces Persistent Molecular Changes and Interferon Gamma-Dependent Major Histocompatibility Complex Class II Expression in the Colonic Epithelium

“…Infection with C. rodentium is dependent on the injection of effector proteins via a type III secretion system (T3SS), which manipulates multiple cell signaling pathways ( 12 ). For example, Tir mediates intimate bacterial attachment and activates NF-κB signaling ( 13 ), while NleC and NleE inhibit NF-κB signaling ( 14 – 16 ), and EspF and EspI/NleA alter tight junction integrity ( 17 , 18 ).…”