Abstract
Nucleolin is a prominent nucleolar protein that is mobilized into the cytoplasm during infection by enteropathogenic Escherichia coli (EPEC). Nucleolin also exists at low levels at the cell surface of eukaryotic cells and here we show that upon infection of an intestinal cell model, EPEC recruits and subsequently sequesters cell-surface EGFP-nucleolin into extracellularly located bacterial microcolonies. The recruitment of nucleolin was evident around bacteria within the centre of the microcolonies that were not directly associated with actin-based pedestals. Incubation of host intestinal cells with different ligands that specifically bind nucleolin impaired the ability of EPEC to disrupt epithelial barrier function but did not inhibit bacterial attachment or other effector-driven processes such as pedestal formation or microvilli effacement. Taken together, this work suggests that EPEC exploits two spatially distinct pools of nucleolin during the infection process.
Highlights
Enteropathogenic Escherichia coli (EPEC) is a non-invasive pathogen that binds to human small intestinal enterocytes and delivers multiple effector proteins into host cells via its type III secretion system (Dean & Kenny, 2009)
Nucleolin has been shown to bind directly to the enterohaemorrhagic E. coli (EHEC) outer-membrane protein intimin, it was not found to colocalize with the bacterial outer membrane during EPEC/ EHEC infection of host cells (Sinclair & O’Brien, 2004) and the role of nucleolin and the nature of nucleolin recruitment during infection remain undetermined
Because of the weak IF signal with intestinal Caco-2 cells, we employed an overexpression system using an enhanced green fluorescent protein (EGFP)-nucleolin fusion expressed in the Caco-2 cell line to follow the behaviour of cell surface nucleolin during EPEC infection
Summary
Enteropathogenic Escherichia coli (EPEC) is a non-invasive pathogen that binds to human small intestinal enterocytes and delivers multiple effector proteins into host cells via its type III secretion system (Dean & Kenny, 2009). These effectors subvert many aspects of host cell physiology, leading to diarrhoeal disease (Dean & Kenny, 2009). As physical contact between the bacterium and the host cell membrane is required for the delivery of the effectors, it follows that many individual bacteria within the attached microcolony will not be engaged in the delivery of effectors
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