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18796

The Role of RIP1 Kinase In the Activation of Apoptosis In Enterocytes by Cronobacter Species

Friday, October 19, 2012
Room R02-R05 (Morial Convention Center)
Daniel Wei, MD1, Rahul Mittal, PhD2 and Nemani V. Prasadarao, PhD2, (1)Neonatology, LAC+USC Medical Center, Los Angeles, CA, (2)Infectious Disease, CHLA, Los Angeles, CA

Purpose

Cronobacter sakazakii (CS) is an emerging pathogen that has been implicated in outbreaks of necrotizing enterocolitis (NEC) in neonates.  Previous studies have shown that Cronobacter sakazakii binds to intestinal epithelial cells during infection and induces tight-junction disruption and apoptosis.  The aim of the present study was to examine the role of RIP1 kinase in the activation of apoptosis during Cronobacter infection in vitro using intestinal epithelial cells.

Methods

HT29 cells were cultured and infected with various clinical strains of Cronobacter spp.  Activation of RIP1 kinase, Caspase 3, Caspase 8, and FADD was examined by Western blotting.  Immunoprecipitation studies were used to examine the interaction of RIP1 with other proteins involved in the apoptotic process. The cells were also stained with anti-RIP1 and anti-E-cadherin antibodies and their association was examined by immunofluorescence microscopy.  RIP1 was silenced using siRNA and apoptosis was measured by AnnexinV-7AAD staining using flow cytometry after infection.  

Results

Infection of HT29 cells with Cronobacter species results in the activation of RIP1, Caspase 3, Caspase 8, and FADD.  A complex is formed between phospho-PKC-α, RIP1, and Caspase 8 as shown by immunoprecipitation studies.  RIP1 also co-localizes with E-cadherin at the tight-junctions of HT29 cells.  Silencing of RIP1 expression in HT29 cells decreased apoptosis after Cronobacter infection.

Conclusion

The results of this study suggest that Cronobacter spp. activates RIP1 kinase in intestinal epithelial cells and interacts with phospho-PKC-α, Caspase 8 and FADD. The formation of this complex ultimately causes the apoptosis of intestinal epithelial cells. Interestingly, silencing of RIP1 protects HT29 cells from apoptosis after infection.  These in vitro studies suggest a significant role for RIP1 in apoptosis of intestinal epithelial cells upon Cronobacter infection.  Further studies in animal models are warranted to evaluate the role of RIP1 in intestinal insult caused by Cronobacter spp.