title: Tracking the Dynamic Relationship between Cellular Systems and Extracellular Subproteomes in Pseudomonas aeruginosa Biofilms.
abstract: The transition of the opportunistic pathogen Pseudomonas aeruginosa from free-living bacteria into surface-associated biofilm communities represents a viable target for the prevention and treatment of chronic infectious disease. We have established a proteomics platform that identified 2443 and 1142 high-confidence proteins in P. aeruginosa whole cells and outer-membrane vesicles (OMVs), respectively, at three time points during biofilm development (ProteomeXchange identifier PXD002605). The analysis of cellular systems, specifically the phenazine biosynthetic pathway, demonstrates that whole-cell protein abundance correlates to end product (i.e., pyocyanin) concentrations in biofilm but not in planktonic cultures. Furthermore, increased cellular protein abundance in this pathway results in quantifiable pyocyanin in early biofilm OMVs and OMVs from both growth modes isolated at later time points. Overall, our data indicate that the OMVs being released from the surface of the biofilm whole cells have unique proteomes in comparison to their planktonic counterparts. The relative abundance of OMV proteins from various subcellular sources showed considerable differences between the two growth modes over time, supporting the existence and preferential activation of multiple OMV biogenesis mechanisms under different conditions. The consistent detection of cytoplasmic proteins in all of the OMV subproteomes challenges the notion that OMVs are composed of outer membrane and periplasmic proteins alone. Direct comparisons of outer-membrane protein abundance levels between OMVs and whole cells shows ratios that vary greatly from 1:1 and supports previous studies that advocate the specific inclusion, or "packaging", of proteins into OMVs. The quantitative analysis of packaged protein groups suggests biogenesis mechanisms that involve untethered, rather than absent, peptidoglycan-binding proteins. Collectively, individual protein and biological system analyses of biofilm OMVs show that drug-binding cytoplasmic proteins and porins are potentially shuttled from the whole cell into the OMVs and may contribute to the antibiotic resistance of P. aeruginosa whole cells within biofilms.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada , N1G 2W1.
authors.forename: Amber J
authors.initials: AJ
authors.lastname: Park
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada , N1G 2W1.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Murphy
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada , N1G 2W1.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Surette
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada , N1G 2W1.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bandoro
authors.affiliation: SPARC BioCentre, The Hospital for Sick Children , Toronto, Ontario, Canada , M5G 0A4.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Krieger
authors.affiliation: SPARC BioCentre, The Hospital for Sick Children , Toronto, Ontario, Canada , M5G 0A4.
authors.forename: Paul
authors.initials: P
authors.lastname: Taylor
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario, Canada , N1G 2W1.
authors.forename: Cezar M
authors.initials: CM
authors.lastname: Khursigara
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development*; D019295:Computational Biology; D000067128:Extracellular Vesicles / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D058977:Molecular Sequence Annotation; D010457:Peptidoglycan / Q000378:metabolism; D010619:Phenazines / Q000378:metabolism; D010933:Plankton / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D021381:Protein Transport; D020543:Proteome / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011710:Pyocyanine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D010619:Phenazines; D020543:Proteome; C000598831:phenazine; D011710:Pyocyanine
keywords: Pseudomonas aeruginosa; biofilms; outer membrane vesicles; quantitative proteomics; systems biology