Insights into the Pathogenesis of Enteropathogenic E. coli Using an Improved Intestinal Enterocyte Model

Abstract

Enteropathogenic E. coli (EPEC) is a human pathogen that targets the small intestine, causing severe and often fatal diarrhoea in infants. A defining feature of EPEC disease is the loss (effacement) of absorptive microvilli (MV) from the surface of small intestinal enterocytes. Much of our understanding of EPEC pathogenesis is derived from studies using cell lines such as Caco-2 – the most extensively used small intestinal model. However, previous work has revealed fundamental differences between Caco-2 cells and in vivo differentiated enterocytes in relation to MV effacement. This, and the high heterogeneity and low transfection efficiency of the Caco-2 cell line prompted the isolation of several sub-clones (NCL-1–12) to identify a more tractable and improved in vivo-like cell model. Along with established Caco-2 clones (TC-7, BBE1), sub-clones were assessed for growth rate, apical surface morphology, epithelial barrier function and transfection efficiency. TC-7 cells provided the best all-round clone and exhibited highest levels of ectopic gene expression following cell polarisation. Novel alterations in EGFP-labelled mitochondria, that were not previously documented in non-polarised cell types, highlighted the potential of the TC-7 model for defining dynamic enterocyte-specific changes during infection. Crucially, the TC-7 cell line also mimicked ex vivo derived enterocytes with regard to MV effacement, enabling a better dissection of the process. Effacement activity caused by the EPEC protein Map in the Caco-2 but not ex vivo model, was linked to a defect in suppressing its Cdc42-dependent functionality. MV effacement activity of the EPEC protein EspF in the TC-7 model was dependent on its N-WASP binding motif, which is also shown to play an essential role in epithelial barrier dysfunction. Together, this study highlights the many advantages of using TC-7 cells as a small intestinal model to study host-pathogen interactions.

Dynamics of brush border remodeling induced by enteropathogenicE. coli

“…Consistent with previous work describing MLPs, 7,11-14 we observed numerous examples of elongated protrusions on the surface of EPEC-infected Caco-2 BBE cells at both early and late time points post-confluency (Fig. 2A and B).…”

“…10 Microvillar-like processes (MLPs), which are the result of microvillar elongation, have long been observed as part of the normal A/E process during EPEC infection. 7,11-14 Ultimately, microvilli surrounding the microcolony are destroyed during infection, leaving an area around the bacteria devoid of brush border and giving rise to a cellular morphology referred to as the “A/E lesion.”…”

Genetic variants inCDC42andNXPH1as susceptibility factors for constipation and diarrhoea predominant irritable bowel syndrome

“…Intestinal stem cells lacking CDC42 undergo defective cell division, abnormal morphogenesis, elevated apoptosis and failed Paneth cell differentiation,41 42 indicating that genetic variants in CDC42 may alter epithelial barrier function. Moreover, the loss of absorptive microvilli from the surface of small intestinal enterocytes (Caco-2 cells) in response to Escherichia coli or its toxins was linked to CDC42 functionality 43. Additionally, CDC42 regulates epithelial membrane transport and secretion 41 44.…”

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

“…Interestingly, the fully differentiated NCL2 clone recently isolated from the parental Caco-2 cell line shows the homogeneous presence of cells apically secreting a glycocalyx-like or mucin-like material (93).…”

“…tEPEC-induced effacement of microvilli in fully differentiated Caco-2 cells requires the cooperative action of T3SS effectors Map (mitochondrion-associated protein), EspF, and Tir as well as intimin (606). Microvillus effacement activity of the tEPEC protein EspF in the Caco-2/TC7 cells was dependent on its N-WASP binding motif (93). EspB binds to myosin and blocks its interaction with actin in fully differentiated Caco-2 cells (607).…”