Perturbation of Host Cell Cytoskeleton by Cranberry Proanthocyanidins and Their Effect on Enteric Infections

Cranberry-derived compounds, including a fraction known as proanthocyanidins (PACs) exhibit anti-microbial, anti-infective, and anti-adhesive properties against a number of disease-causing organisms. In this study, the effect of cranberry proanthocyanidins (CPACs) on the infection of epithelial cells by two enteric bacterial pathogens, enteropathogenic Escherichia coli (EPEC) and Salmonella Typhimurium was investigated. Immunofluorescence data showed that actin pedestal formation, required for infection by enteropathogenic Escherichia coli (EPEC), was disrupted in the presence of CPACs. In addition, invasion of HeLa cells by Salmonella Typhimurium was significantly reduced, as verified by gentamicin protection assay and immunofluorescence. CPACs had no effect on bacterial growth, nor any detectable effect on the production of bacterial effector proteins of the type III secretion system. Furthermore, CPACs did not affect the viability of host cells. Interestingly, we found that CPACs had a potent and dose-dependent effect on the host cell cytoskeleton that was evident even in uninfected cells. CPACs inhibited the phagocytosis of inert particles by a macrophage cell line, providing further evidence that actin-mediated host cell functions are disrupted in the presence of cranberry CPACs. Thus, although CPAC treatment inhibited Salmonella invasion and EPEC pedestal formation, our results suggest that this is likely primarily because of the perturbation of the host cell cytoskeleton by CPACs rather than an effect on bacterial virulence itself. These findings have significant implications for the interpretation of experiments on the effects of CPACs on bacteria-host cell interactions

Direct evidence of host-mediated glycosylation of NleA and its dependence on interaction with the COPII complex

ABSTRACTNon-LEE-encoded Effector A (NleA) is a type III secreted effector protein of enterohaemorrhagic and enteropathogenic Escherichia coli as well as the related mouse pathogen Citrobacter rodentium. NleA translocation into host cells is essential for virulence. We previously published several lines of evidence indicating that NleA is modified by host-mediated mucin-type O-linked glycosylation, the first example of a bacterial effector protein modified in this way. In this study, we use lectins to provide direct evidence for the modification of NleA by O-linked glycosylation and determine that the interaction of NleA with the COPII complex is necessary for this modification to occur

R-spondins are expressed by sub-epithelial non-hematopoietic stromal cells.

<p>Colonic epithelial and lamina propria cells from C3Ou mice were isolated, and lamina propria cells were further stained for CD45.2 and sorted into CD45⁺ and CD45^- populations after gating on singlet and live cells. R-spondin (A), <i>EpCAM</i> and <i>Ptprc</i> (B) expression was measured by qRT-PCR and normalized to <i>Gapdh</i> (n = 3). Error bars represent mean ±SEM. # = undetected.</p

R-spondin expression levels are regulated during tissue repair following DSS injury.

<p>C3Ou mice were subjected to 3% DSS administration for 6 days before returning to normal drinking water for 9 days. Body weights (A) were measured every other day during the course of DSS treatment and during repair (n = 4–20 per time point, aggregate of two experiments). Histological changes were examined on untreated controls (B), at day 6 of DSS (C), and on days 3 (D), 6 (E), and 9 (F) following DSS removal at 15X magnification. Scale bars, 200 μm. <i>Rspo1-3</i> levels were measured by qRT-PCR on days 0, 3, and 6 of DSS and on days 3, 6, and 9 post-DSS (G) (n = 4 per time point). Wnt target genes <i>Mmp7</i> and <i>c-Myc</i> were measured by qRT-PCR on days 3 and 6 of DSS and on days 3 and 6 post-DSS and were compared to untreated controls (H) (n = 3 per time point). Error bars represent mean ±SEM.</p