Different Cellular Origins and Functions of Extracellular Proteins from Escherichia coli O157:H7 and O104:H4 as Determined by Comparative Proteomic Analysis

Abstract

Extracellular proteins play important roles in bacterial interactions with the environmental matrices. In this study, we examined the extracellular proteins from Escherichia coli O157:H7 and O104:H4 by tandem mass spectrometry. We identified 500 and 859 proteins from the growth media of E. coli O157:H7 and O104:H4, respectively, including 371 proteins common to both strains. Among proteins that were considered specific to E. coli O157:H7 or present at higher relative abundances in O157:H7 medium, most (57 of 65) had secretion signal sequences in their encoding genes. Noticeably, the proteins included locus of enterocyte effacement (LEE) virulence factors, proteins required for peptidyl-lipoprotein accumulation, and proteins involved in iron scavenging. In contrast, a much smaller proportion of proteins (37 of 150) that were considered specific to O104:H4 or presented at higher relative abundances in O104:H4 medium had signals targeting them for secretion. These proteins included Shiga toxin 2 subunit B and O104:H4 signature proteins, including AAF/1 major fimbrial subunit and serine protease autotransporters. Most of the abundant proteins from the growth medium of E. coli O104:H4 were annotated as having functions in the cytoplasm. We provide evidence that the extensive presence of cytoplasmic proteins in E. coli O104:H4 growth medium was due to biological processes independent of cell lysis, indicating alternative mechanisms for this potent pathogen releasing cytoplasmic contents into the growth milieu, which could play a role in interaction with the environmental matrices, such as pathogenesis and biofilm formation.

Importance: In this study, we compared the extracellular proteins from two of the most prominent foodborne pathogenic E. coli organisms that have caused severe outbreaks in the United States and in Europe. E. coli O157:H7 is a well-studied Shiga toxigenic foodborne pathogen of the enterohemorrhagic pathotype that has caused numerous outbreaks associated with various contaminated foods worldwide. E. coli O104:H4 is a newly emerged Shiga toxigenic foodborne pathogen of the enteroaggregative pathotype that gained notoriety for causing one of the most deadly foodborne outbreaks in Europe in 2011. Comparison of proteins in the growth medium revealed significant differences in the compositions of the extracellular proteins for these two pathogens. These differences may provide valuable information regarding the cellular responses of these pathogens to their environment, including cell survival and pathogenesis.

FIG 1

LIVE/DEAD BacLight staining of E. coli O104:H4 (A) and O157:H7 (B) at cell harvest. Membrane-permeative green fluorescent dye SYTO9 stains all cells green, while impermeative red fluorescent dye propiodium only stains the nucleic acids of cells with compromised membrane integrity (dead cells). The calculated cell viability (O104:H4, 99.4%; O157:H7, 97.4%) was based on counting totals of 7,952 and 1,735 cells, respectively. Scale bar: 25 μm.