APEX-based differential protein abundance analysis of Shigella dysenteriae comparing in vitro and in vivo proteomes

摘要

Shigella dysenteriae type 1 (SD1) is the most virulent of the four Shigella species and is a causative agent of shigellosis. While the critical role of Shiga toxin as a cytotoxin and the organism's ability to invade host epithelial cells have been elucidated, the overall pathogenesis is still not completely understood. In this study, protein abundance changes in SD1 occurring in response to its exposure to the host (piglet) gut environment were analyzed quantitatively using APEX, a label-free quantitation method for absolute protein expression measurements based on spectral counts from mass spectrometric (MS) data and computational predictions of proteotypic peptides. The predicted number of unique proteotypic peptides for each Shigella protein required for APEX calculations was derived from a species-specific SD1 training dataset, which was generated from the 100 most abundant Shigella proteins utilizing an integrated in-house APEX tool. Differential protein abundance analysis comparing the in vitro and in vivo proteome profiles identified more than 150 proteins altered quantitatively at 99percent statistical confidence, including enzymes involved in nucleotide, protein and energy metabolism. Dramatic increases in protein abundance were also observed for several virulence-associated proteins, thus strongly indicative of the induction of genes encoding virulence-associated proteins upon contact between pathogen and host cells. The identification of differentially expressed virulence-associated proteins from the Shigella in vivo proteome will facilitate future studies of pathogenicity and vaccine development against SD1.