Abstract
Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, β1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.
Highlights
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are diarrheagenic extracellular pathogens affecting humans worldwide [1]
EPEC reaches the small intestine, where an injection device termed the type III secretion system is utilized to inject a set of effector proteins from the bacteria into the host cell
We show that early upon infection, two such effector proteins, EspF and Map, hijack host endosomes at bacterial adherence sites to facilitate endocytosis and recycling of plasma membrane proteins at these sites
Summary
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are diarrheagenic extracellular pathogens affecting humans worldwide [1]. EPEC (strain O127:H6 E2348/69) translocates via its T3SS at least 21 ‘effector’ proteins into its host These effectors are encoded by genes in the locus of enterocyte effacement (LEE) pathogenicity island, and by outside the LEE (non-LEE) genomic sites [7,8]. Tir is incorporated into the host cell plasma membrane and its exposed extracellular domain binds the bacterial outer membrane protein, intimin. Tir-intimin interactions allow intimate attachment of the bacterium to its host plasma membrane and the formation of the F-actin rich pedestal [3]. Limited information exists about the capacity of such effectors to manipulate membrane trafficking pathways of Endosomal remodeling by extracellular diarrheagenic bacterial pathogens their host and, in particular, endosomal trafficking. Exploring the capacity of these effectors to hijack and manipulate endocytic trafficking pathways in a way that benefits the pathogen is important for better understanding the mechanisms by which EPEC colonizes its host
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