Enteropathogenic Escherichia coli (EPEC) infects human intestinal cells and causes severe diarrheal disease worldwide. During infection, EPEC utilizes a syringe-like type III secretion system (T3SS) to inject virulence factors directly into the cytoplasm of the infected human cell. Identifying the functions and mechanisms of ≥23 T3SS effector proteins is a substantial challenge. Characterizing human cellular proteome dynamics during infection could substantially contribute to our understanding of how T3SS effectors contribute to EPEC-mediated disease.

This study aims to characterize T3SS-mediated alterations in the human proteome using an N-terminal proteomics approach. Proteases regulate human cellular processes relevant to infection, including inflammation, innate immunity, and cell death. N-terminal proteomics enriches proteolytic events, facilitating the study of protease activity and dysregulation. This study may reveal how these and other global pathways are affected by T3SS effectors and may identify novel substrates of T3SS proteases.

Cultured human epithelial cells were infected for 1.5 hours with wild-type EPEC or a mutant unable to secrete T3SS effectors. The N-terminal proteomes of infected human cells were quantitatively compared using terminal amine isotopic labeling of substrates (TAILS) and tandem mass spectrometry on an Orbitrap Velos mass spectrometer.¹  Data from three independent experiments were analyzed by Mascot and quantified by MaxQuant.

We identified 2112 unique modification-specific N-terminal peptides from 1359 unique proteins at 1% FDR. Preliminary data indicate T3SS-dependent proteolysis of proteins involved in known T3SS-mediated pathways, including innate immunity and cytoskeletal regulation, as well as novel proteolytic events in proteins known to interact with EPEC T3SS effectors. Finally, novel proteolytic events in beta-actin may provide further mechanistic details on how the T3SS mediates host cytoskeletal rearrangements during infection, a hallmark of EPEC infection.