Introduction: Enterobacter sakazakii (ES) is a gram-negative bacterium that has been linked to severe outbreaks of necrotizing enterocolitis (NEC) in neonates. Previous studies from our lab demonstrated that ES induces apoptosis in intestinal epithelial cells in a rat model of NEC. We have also shown that ES strains isolated from humans exhibit higher binding affinity to intestinal epithelial cells and lead to greater permeability of intestinal monolayers than ES strains isolated from non-human sources. However, the mechanism by which ES induces tight junction disruption is unknown.

Objective: Because protein kinase C (PKC) has been shown to play a role in intestinal permeability, we sought to determine if PKC mediates the observed increase in intestinal epithelium permeability and alteration in tight junction morphology induced by ES.

Methods: Caco-2 cell monolayers were grown in transwells and treated with either the PKC activator, Phorbol 12-Myristate 13-Acetate (PMA), or the PKC inhibitor, Gö 6983, followed by inoculation with ES and horseradish peroxidase (HRP). The transepithelial electrical resistances (TEER) and HRP leakage were then measured at different time points. In addition, the activity of PKC was measured by non-radioactive PepTag assay in the total cell lysates of Caco-2 cells.

Results: Our data show that exposure to ES increased Caco-2 monolayer permeability (decreased TEER and increased HRP leakage). High-binding ES increased PKC activity by two-fold compared to low-binding ES (20.07 units of kinase/mL compared to 10.4 units of kinase/mL). The PKC activator, PMA, decreased the TEER of the Caco-2 monolayers by 21 – 26% over 4 hours while the PKC inhibitor, Gö 6983, increased the TEER by 9 – 12%. Treatment with the PKC inhibitor, Gö 6983, helped preserve epithelial barrier integrity in Caco-2 monolayers exposed to high-binding ES strains, as evidenced by a decrease in TEER of 18% with Gö 6983 treatment vs. 52% without, and HRP leakage of 230 picograms with Gö 6983 vs. 323 picograms without.

Conclusion: This study suggests that high-binding ES increases Caco-2 cell monolayer permeability by enhancing the activation of PKC, possibly via tight junction disassembly. The isoform of PKC involved in this phenomenon has yet to be determined.