title: Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations.
abstract: Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.
authors.affiliation: Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy.
authors.forename: Luana
authors.initials: L
authors.lastname: Maggiore
authors.affiliation: Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Lu
authors.initials: L
authors.lastname: Yu
authors.affiliation: Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Omasits
authors.affiliation: Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy.
authors.forename: Omar
authors.initials: O
authors.lastname: Rossi
authors.affiliation: Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Gordon
authors.initials: G
authors.lastname: Dougan
authors.affiliation: Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Thomson
authors.affiliation: Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy. Electronic address: allan.j.saul@GSK.com.
authors.forename: Allan
authors.initials: A
authors.lastname: Saul
authors.affiliation: Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Jyoti S
authors.initials: JS
authors.lastname: Choudhary
authors.affiliation: Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy.
authors.forename: Christiane
authors.initials: C
authors.lastname: Gerke
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics; D000954:Antigens, Surface / Q000032:analysis* / Q000235:genetics; D001428:Bacterial Vaccines; D002462:Cell Membrane / Q000276:immunology / Q000648:ultrastructure; D004405:Dysentery, Bacillary / Q000517:prevention & control; D008565:Membrane Proteins / Q000276:immunology; D033903:Periplasmic Proteins / Q000276:immunology; D040901:Proteomics; D012763:Shigella flexneri / Q000276:immunology* / Q000648:ultrastructure; D012764:Shigella sonnei / Q000276:immunology* / Q000648:ultrastructure
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001428:Bacterial Vaccines; D008565:Membrane Proteins; D033903:Periplasmic Proteins
keywords: GMMA; Proteomics; Shigella flexneri 2a; Shigella sonnei; Vaccine; iBAQ