E. coli O157:H7 Evades CD8+ T Cell Immunity Through T3SS-Dependent Suppression and Limited MHCI Presentation of translocated proteins

Abstract Introduction Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes severe disease in humans, but colonizes cattle asymptomatically. Our previous research showed a Th1-biased mucosal immune response in colonized cattle, with antigen-specific CD8+ T cell proliferation. EHEC O157:H7 colonizes the bovine gastro-intestinal tract, a process that requires a type 3 secretion system (T3SS) and injection of a cocktail of ‘effector’ proteins into epithelial cells, resulting in tight bacterial/epithelial attachment. Mathematical modeling indicates EHEC O157:H7 effector proteins have reduced MHCI ligand density, potentially an adaptation to evade CD8+ T cell immunity when injected into host cells. Methods In this study, immunopeptidomics was applied to study the presentation of E. coli peptides on MHCI molecules using an in vitro bovine epithelial cell colonization model peptide elution from bovine epithelial cells colonized with E. coli O157:H7 Results immunopeptidomic analysis of bovine epithelial cells colonized with E. coli O157:H7 experimentally confirmed a scarcity of MHCI-presented peptides from secreted bacterial effector proteins. Unexpectedly, two MHCI ligands were identified from Intimin, a bacterial outer membrane protein crucial for cattle colonization, with one ligand overlapping a known CD4+ T cell epitope. Using a bovine epithelial cell line transfected with the BoLA-1*023:01 MHCI allele, we showed that E. coli O157:H7 suppresses antigen-specific CD8+ T-cell activation. Conclusion the study results highlights the importance of engaging both CD4+ and CD8+ T cell responses for rational vaccine design, an area not traditionally emphasized for extracellular pathogens such as E. coli O157:H7. By combining immunopeptidomics and bespoke in silico prediction algorithms, we provide a foundation for next-generation vaccines aimed at more effectively clearing persistent E. coli O157:H7 infections in cattle and reducing the risk of zoonotic transmission.