Andrew W. Weflen

The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways.

Bacterial toxins and effector proteins hijack eukaryotic enzymes that are spatially localized and display rapid signaling kinetics. However, the molecular mechanisms by which virulence factors engage highly dynamic substrates in the host cell environment are poorly understood. Here, we demonstrate that the enteropathogenic Escherichia coli (EPEC) type III effector protein EspF nucleates a multiprotein signaling complex composed of eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott-Aldrich syndrome protein (N-WASP). We demonstrate that a specific and high affinity association between EspF and SNX9 induces membrane remodeling in host cells. These membrane-remodeling events are directly coupled to N-WASP/Arp2/3–mediated actin nucleation. In addition to providing a biochemical mechanism of EspF function, we find that EspF dynamically localizes to membrane-trafficking organelles in a spatiotemporal pattern that correlates with SNX9 and N-WASP activity in living cells. Thus, our findings suggest that the EspF-dependent assembly of SNX9 and N-WASP represents a novel form of signaling mimicry used to promote EPEC pathogenesis and gastrointestinal disease.

The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII‐dependent pathways. These results suggest that NleAs striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.

Enteropathogenic E. coli non‐LEE encoded effectors NleH1 and NleH2 attenuate NF‐κB activation

Enteric bacterial pathogens have evolved sophisticated strategies to evade host immune defences. Some pathogens deliver anti‐inflammatory effector molecules into the host cell cytoplasm via a type III secretion system (T3SS). Enteropathogenic Escherichia coli (EPEC) inhibits inflammation by an undefined, T3SS‐dependent mechanism. Two proteins encoded outside of the EPEC locus of enterocyte effacement (LEE) pathogenicity island, non‐LEE‐encoded effector H1 (NleH1) and H2 (NleH2), display sequence similarity to Shigella flexneri OspG, which inhibits activation of the pro‐inflammatory transcription factor NF‐κB. We hypothesized that the anti‐inflammatory effects of EPEC were mediated by NleH1 and NleH2. In this study, we examined the effect of NleH1/H2 on the NF‐κB pathway. We show that NleH1/H2 are secreted via the T3SS and that transfection of cells with plasmids harbouring nleH1 or nleH2 decreased IKK‐β‐induced NF‐κB activity and attenuated TNF‐α‐induced degradation of phospho‐IκBα by preventing ubiquitination. Serum KC levels were higher in mice infected with ΔnleH1H2 than those infected with WT EPEC, indicating that NleH1/H2 dampen pro‐inflammatory cytokine expression. ΔnleH1H2 was cleared more rapidly than WT EPEC while complementation of ΔnleH1H2 with either NleH1 or NleH2 prolonged colonization. Together, these data show that NleH1 and NleH2 function to dampen host inflammation and facilitate EPEC colonization during pathogenesis.

Enteropathogenic E. coli-induced barrier function alteration is not a consequence of host cell apoptosis

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death.

E. coli secreted protein F promotes EPEC invasion of intestinal epithelial cells via an SNX9-dependent mechanism.

Enteropathogenic Escherichia coli (EPEC) infection requires the injection of effector proteins into intestinal epithelial cells (IECs) via type 3 secretion. Type 3‐secreted effectors can interfere with IEC signalling pathways via specific protein–protein interactions. For example, E. coli secreted protein F (EspF) binds sorting nexin 9 (SNX9), an endocytic regulator, resulting in tubulation of the plasma membrane. Our aim was to determine the mechanism of EspF/SNX9‐induced membrane tubulation. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. Although characterized as non‐invasive, EPEC are known to invade IECs in vitro and in vivo. Indeed, we found significant invasion of Caco‐2 cells by EPEC, which, like tubulation, was blocked by pharmacological inhibition of CCPs. Interestingly, however, inhibition of dynamin activity did not prevent tubulation or EPEC invasion, which is in contrast to Salmonella invasion, which requires dynamin activity. Our data also indicate that EPEC invasion is dependent on EspF and its interaction with SNX9. Together, these findings suggest that EspF promotes EPEC invasion of IECs by harnessing the membrane‐deforming activity of SNX9.

Tight Junctions and Enteropathogenic E. coli

Enteropathogenic E. coli (EPEC) are a leading cause of infantile diarrhea in developing countries, resulting in millions of deaths each year. EPEC secrete virulence factors, also called effectors, directly into host intestinal epithelial cells via type three secretion systems. Secreted effectors then affect host signaling pathways to induce several phenotypes, which ultimately lead to disease. Among the over 20 secreted effectors is E. coli secreted protein F (EspF), a 206 amino acid protein believed to be central to EPEC pathogenesis, as it disrupts tight junction structure and function. Although the mechanism by which this occurs is unknown, EspF has recently been found to contain several protein–protein interaction domains that may be involved. We have shown EspF to interact with the endocytic regulators sorting nexin 9 (SNX9) and N‐WASP via non‐exclusive binding sites. These interactions induce actin polymerization in vitro, and interaction with SNX9 alters its endocytic activity, as EspF induces the formation of tubular vesicles in a manner dependent upon its interaction with SNX9. EspF, therefore, appears to hijack endocytic regulation via SNX9 and possibly N‐WASP interaction, to affect an as yet unidentified pathogenic phenotype.