Abstract
BackgroundThe recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level.Methodology/Principal FindingsWe examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate Mr ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high Mr fraction. Hundreds of proteins were enriched in a Mr range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster.SignificanceNearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.
Highlights
Enterohemorrhagic E. coli (EHEC) are a group of bacteria containing many serotypes that are responsible for outbreaks of bloody diarrhea often leading to serious systemic complications such as the hemolytic uremic syndrome (HUS) and neurological abnormalities which in severe cases can be fatal [1]
Quantitative proteomic analysis was followed by data mining, including the correlation of enrichment of proteins in a distinct cell lysate fraction or a distinct size exclusion chromatography (SEC) fraction (Mr ranges of .280 kDa vs. 280-80 kDa vs. 80-10 kDa) with its biochemical properties such as sequence-based and native Mr value, participation as a subunit in higher order protein assemblies, subcellular localization and attachment to or integration in phospholipid membranes
The combined solubilized fraction was separated by SEC yielding fractions in the Mr ranges .280 kDa, 280-80 kDa and 80-10 kDa, with estimated Mrs based on retention times for five globular protein standards
Summary
Enterohemorrhagic E. coli (EHEC) are a group of bacteria containing many serotypes that are responsible for outbreaks of bloody diarrhea often leading to serious systemic complications such as the hemolytic uremic syndrome (HUS) and neurological abnormalities which in severe cases can be fatal [1]. These complications are attributed to Shiga toxins (Stx), shared with Shigella dysenteriae and acquired via horizontal gene transfer through phages [2]. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level
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