Dataset S2. Proteins identified by Scaffold in outer membrane vesicles produced by E. cloacae in the presence and absence of murine norovirus.
Norovirus interactions induce widespread changes in gene expression in E. cloacae. These changes in gene expression occur alongside changes in the surface architecture of the bacterium and an increased production in outer membrane vesicles. While vesicle production is increased, the size of vesicles are smaller. Shifts in vesicle size are correlated with changes in vesicle content. To determine if murine norovirus interactions altered the content of membrane vesicles produced by E. cloacae, proteomic analysis of vesicles produced in the presence and absence of the virus was performed.
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E. cloacae was grown until stationary phase. The bacterial pellet was washed twice with PBS, and the cell count adjusted to a final concentration of 10^8 cells/ml. Cells were inoculated with either MNV (0.1 MOI), silver nanoparticle (AgNP; equivalent volume to that of virus), or PBS. The mixtures were incubated for 1 h at 37°C with constant mixing. Following this step virus:bacteria cultures were inoculated into 200 ml of LB and grown at 37 °C for 12 h. OMVs were harvested first by clarification of supernatants by ultracentrifugation at 25,000 x g for 20 mins, followed by filtration and centrifugation at 150,000 x g for 2 h to obtain OMV pellets. The OMVs were resuspended in dPBS and ultracentrifuged at 150,000 x g for 2 h at 4°C. The OMV pellets was resuspended in 500-µl dPBS supplemented with protease inhibitor cocktail (Thermo Fisher #A32955) and stored at 4°C for use within two weeks or at -80°C for later applications. OMV proteins were purified and precipitated using TCA protein precipitation assay. The final sample in Bolt™ LDS sample buffer and Bolt™ sample reducing agent was heated at 70°C for 10 mins. Samples were run through 30 % of the length of 4-12% Bis-Tris SDS PAGE gel and stained using GelCode™ Blue. Sample lanes were cut into 1-2 mm cubes and in gel tryptic digestion. The final peptide solution was lyophilized and analyzed by mass spectrophotometry. Liquid chromatography was performed in Thermo EASY nano-LC. This was directly interfaced with Orbitrap Fusion MS, where the scan rate was 350-1800 m/z and data were acquired at 120K resolution. Tandem mass spectra were extracted, charge state deconvoluted and deisotoped by Proteome Discoverer version 126.96.36.199. All MS/MS samples were analyzed using Sequest (v. IseNode in Proteome Discoverer 188.8.131.52) and X! Tandem [The GPM, thegpm.org; version X! Tandem Alanine (2017.2.1.4)]. Sequest and X! Tandem were searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 PPM. Scaffold (version Scaffold_4.11.0) was used to validate MS/MS based peptide and protein identifications. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained a minimum of two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. The quantification between the samples was performed by using weighted spectral count, and the ratio was calculated between the treatment and control samples. Student’s t-test was used to assign statistical significance (P>0.05). In addition, proteins were annotated with GO terms from NCBI(16) (downloaded Nov 13, 2020). The functions of OMV proteins were acquired from Uniprot, InterPro, Pfam, and Kegg. UniProt denoted the localization of most proteins or the functions were predicted by Cello2go and psortb.