Characterizing the Escherichia coli O157:H7 proteome including protein associations with higher order assemblies

Abstract

Background: The 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 findings: We 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 M(r) 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 M(r) fraction. Hundreds of proteins were enriched in a M(r) 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.

Significance: Nearly 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.

Figure 1. Experimental design combining two-dimensional LC-MS/MS with upfront fractionation of proteins derived from insoluble and soluble, SEC-separated cell lysate fractions.

Abbreviations: EHEC, enterohemorrhagic E. coli; SEC, size exclusion chromatography; FASP, filter-aided sample preparation for digestion of complex protein mixtures; SCX, strong cation exchange chromatography; BR, biological replicate; T₁R and T₄R, technical replicate stages. The total number of SCX fractions subjected to LC-MS/MS sequentially was 52 in a given experiment.

Figure 2. Size exclusion chromatography fractionating E. coli O157:H7 cell lysate proteins prior to 2D-LC-MS/MS.

The fractions (F1-s, F2-s and F3-s) were eluted from the G3000-SWXL column in PBS and 0.01% Triton X-100 and pooled as shown in the chromatogram. The A₂₈₀ profiles are shown for an EHEC lysate sample and a mixture of five M_r protein standards, the latter of which pertains to the line with a low void volume peak at 667 kDa. Protein separations occurred in the 500-10 kDa range. On the right, protein bands visualized by Coomassie Blue staining in a 4–12%T SDS-PAGE gel are shown in the same order as the SEC fractions (with F3-s before pooling in two consecutive SEC fractions).

Figure 3. Correlation of native and sequence-based M_r values of proteins with their quantitative elution profiles in SEC fractions associated with three approximate M_r ranges (F1-s_SEC, >280 kDa, F2-s_SEC 280-80 kDa, F3-s_SEC 80-10 kDa).

Each heat map column under the broad blue arrow indicating a M_r range represents 11 individual, replicate APEX_i datasets. Each row corresponds to one of 760 EHEC proteins, quantified with the APEX_i tool, that were altered with statistical significance using the Kruskal-Wallis test (p-value<0.02) comparing protein abundances in the three SEC fractions (F1-s_SEC, F2-s_SEC and F3-s_SEC). The range of colors in the heat map corresponds to APEX_i values from 0 to 5000. Left heap map: proteins are ordered based on calculated, amino acid sequence-based M_r values; right heat map: proteins are ordered based on native M_r values including homooligomers and multi-protein complexes (for calculations, see Table S7). The maximum M_r value was 999 kDa, including larger complexes such as the ribosome. Protein positions are indicated for the monomer/complex as follows: Ftn/Ftn₂₄, Udp/Udp₆ and Hfq/Hfq₆. The correlation of protein elution patterns by SEC with calculated native M_rs is improved.