NleH1 inhibits RPS3 S209 phosphorylation in vivo
We previously utilized a gnotobiotic piglet infection model to determine that piglets infected with ΔnleH1 mutant died more rapidly than those infected with wild-type E. coli O157:H7. Piglets infected with ΔnleH1 displayed clinical disease consistent with a robust inflammatory response, but with reduced bacterial colonization and little diarrhea9. While seemingly paradoxical, based on our cell culture data (Fig. 6d), we hypothesized that NleH1 blocked RPS3 S209 phosphorylation in vivo, thereby preventing RPS3 nuclear translocation in infected piglets. We isolated piglet colons at necropsy, and subjected them to immunohistochemistry using our phospho-RPS3 antibody. Consistent with in vitro data, piglets infected with wild-type E. coli O157:H7 exhibited diffuse and low intensity phospho-RPS3 staining, whereas in piglets infected with ΔnleH1 mutant, phospho-RPS3 expression was florid and intense (Fig. 6f). These data demonstrate that NleH1 inhibits RPS3 S209 phosphorylation both in vitro and in vivo, which might benefit the bacterium in colonization and transmission.