Protein Kinase C Mediates Enterohemorrhagic Escherichia coli O157:H7-Induced Attaching and Effacing Lesions

Abstract

Enterohemorrhagic Escherichia coli serotype O157:H7 causes outbreaks of diarrhea, hemorrhagic colitis, and the hemolyticuremic syndrome. E. coli O157:H7 intimately attaches to epithelial cells, effaces microvilli, and recruits F-actin into pedestals to form attaching and effacing lesions. Lipid rafts serve as signal transduction platforms that mediate microbe-host interactions. The aims of this study were to determine if protein kinase C (PKC) is recruited to lipid rafts in response to E. coli O157:H7 infection and what role it plays in attaching and effacing lesion formation. HEp-2 and intestine 407 tissue culture epithelial cells were challenged with E. coli O157:H7, and cell protein extracts were then separated by buoyant density ultracentrifugation to isolate lipid rafts. Immunoblotting for PKC was performed, and localization in lipid rafts was confirmed with an anti-caveolin-1 antibody. Isoform-specific PKC small interfering RNA (siRNA) was used to determine the role of PKC in E. coli O157:H7-induced attaching and effacing lesions. In contrast to uninfected cells, PKC was recruited to lipid rafts in response to E. coli O157:H7. Metabolically active bacteria and cells with intact lipid rafts were necessary for the recruitment of PKC. PKC recruitment was independent of the intimin gene, type III secretion system, and the production of Shiga toxins. Inhibition studies, using myristoylated PKC pseudosubstrate, revealed that atypical PKC isoforms were activated in response to the pathogen. Pretreating cells with isoform-specific PKC siRNA showed that PKC plays a role in E. coli O157:H7-induced attaching and effacing lesions. We concluded that lipid rafts mediate atypical PKC signal transduction responses to E. coli O157:H7. These findings contribute further to the understanding of the complex array of microbe-eukaryotic cell interactions that occur in response to infection.

Protein kinase C δ signaling is required for dietary prebiotic-induced strengthening of intestinal epithelial barrier function

“…Cani and colleagues previously found that prebiotics increased the expression of both ZO-1 and occludin in mice via a microbe-dependent mechanism21, but the possibility of direct non-microbial mechanism has also been suggested23. We previously identified that atypical PKC isoforms are involved in the signaling responses to EHEC24, we extend these findings by now showing PKC signaling can also be triggered by exposure to dietary prebiotics to alter epithelial barrier and TJ function. This observation was validated through PKC loss-of-function experiments, which abolished the protective effects of prebiotics on epithelial barrier integrity.…”

“…Caco-2Bbe1 cells, a differentiated Caco-2 subclone which attains faster confluency, were purchased from American Type Culture Collection and grown using cell culture conditions as previously described824. Cells were harvested with trypsin-EDTA and seeded onto 6.5-mm Transwell filter supports (~1 × 10 5 cells/insert, Corning, Mississauga, Canada) and maintained for 3–4 d until TER reached >800 Ω cm −2 measured using chopstick electrodes (Millipore, Etobicoke, Canada)939.…”

“…Cell monolayers were lysed, as previously described24, and lysates separated using SDS-PAGE followed by transfer onto nitrocellulose membranes (BioTrace NT, Ann Arbor, MI). Descriptions of primary antibodies used can be found in the supplemental information.…”

Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells

“…coli is capable of determining the deamidation of Rho GTPases through the production of CNF-1 (Cytotoxic Necrotizing Factor-1) [ 65 ]. Previous studies suggested that the ability of E. coli to raise intracellular calcium levels and generate diacylglycerol (DAG) led to the proposal that EPEC activates calcium- dependent protein kinases, including protein kinase C (PKC), in host epithelia [ 66 ]. It has been also reported that activation of PKC results in up-regulation of iPLA 2 ß expression that leads to activation of RhoA/Rho kinase/CPI-17 signalling [ 67 , 68 ].…”

Screening for Host Proteins Interacting with Escherichia Coli O157:H7 EspF using Bimolecular Fluorescence Complementation

“…Meanwhile, ANXA6 can negatively control the EGFR/Ras pathway through binding to p120GAP and PKCα [76]. Both EPEC and EHEC infections can modulate the host cytoskeleton by future science group www.futuremedicine.com activating PKCα and recruiting PKC to form adhesion pedestals via functionally intact lipid rafts [77][78][79], but the precise factor that mediates this event is unknown. PKC activation is also controlled by the PI3K/Akt signaling pathway, on which the EPEC depends to escape phagocytosis by host cell macrophages [78,79].…”