Sa1746 Cationic Amino Acid Transporter 2 and L-Arginine Availability Are Required for Talin-1-Dependent Adherence of the Colonic Pathogen Citrobacter Rodentium and Subsequent Pro-Inflammatory Responses

Abstract

Background: L-arginine (L-Arg) uptake in cells is mediated by cationic amino acid transporter (CAT) proteins, and CAT2 is the inducible form. We have found that CAT2-/mice exhibit less colitis in the IBD model induced by infection with Citrobacter rodentium, a rodent pathogen similar to EPEC that acts by forming attaching and effacing lesions on epithelial cells. The translocated intimin receptor is injected into host cells by C. rodentium and forms a complex with talin-1 that mediates bacterial binding and downstream signaling. Our aim was to investigate the role of talin-1 in C. rodentium infection. Methods: Male C57BL/6 wild-type (WT) or CAT2-/mice were infected with C. rodentium for 14 days. Talin-1 and the C. rodentium protein espB were assesed by confocal microscopy and Western blotting. mRNA levels of talin-1 and the proinflammatory CXC chemokines, KC and MIP-2, were determined by qPCR. Young adult mouse colon (YAMC) cells were transduced with control or talin-1 shRNA, and activated with C. rodentium at an MOI of 200. Bacterial adherence was assessed by culture after saponin treatment of cells. Levels of the NF-κB subunit p65 were assessed by immunofluorescence at 30 min, and of talin-1, KC and MIP-2 by qPCR at 4 h. Talin-1 mRNA stability was assessed in YAMC cells using the transcriptional inhibitor actinomycin D followed by serial assessment of mRNA levels. Results: C. rodentium infection in vivo induced a 4-fold increase in talin-1 mRNA and protein expression in whole tissues and a 236-fold increase in mRNA levels in colonic epithelial cells (CECs). Talin-1 and espB protein levels were markedly decreased in colon tissues from infected CAT2-/vs. WT mice; this was confirmed by confocal microscopy and there was also less colocalization of these proteins. Talin-1, KC and MIP-2 mRNA levels were each decreased by >92% in CAT2-/vs. WT mice (p<0.01). L-lysine, a competitive inhibitor of L-Arg uptake, reduced induction of talin-1, KC, and MIP-2 mRNA expression by 60.4 ± 18.3%, 91.7 ± 2.6%, and 68.9 ± 12.2%, respectively, in YAMC cells (p<0.01). In talin-1 knockdown cells, levels of adherent C. rodentium decreased from 4.9 ± 0.9 to 0.8 ± 0.2 bacteria/cell (p<0.05), and there was less nuclear translocation of p65. Similarly, KC and MIP-2 levels were decreased by 90.6 ± 0.3% and 92.5 ± 3.1%, respectively, in knockdown cells (p<0.01). There was a 4.2 ± 0.5-fold increase in talin-1 mRNA levels when cells starved of L-Arg were repleted with L-Arg for 4 h. Talin-1 mRNA was less stable in the absence of L-Arg; the mRNA half-life was 5.2fold (p<0.01) greater in cells repleted with L-Arg. Conclusions: Talin-1 is essential for C. rodentium adherence to colonic epithelial cells and induction of host inflammatory responses. L-Arg availability is needed for talin-1 mRNA stability, and thus mediates the immunopathogenesis of C. rodentium infection.