title: Proteomic comparison of membrane and extracellular proteins from invasive (PAO1) and cytotoxic (6206) strains of Pseudomonas aeruginosa.
abstract: Strains of Pseudomonas aeruginosa can be phenotypically classified by their mode of pathogenicity as either invasive, where the bacterium is internalised by host cells, or cytotoxic, where the host cell is killed without internalisation through the expression of cytotoxicity factors. These phenotypes are thought to depend primarily on the interactions of pseudomonal membrane and secreted proteins with host cells. We report here comparisons of outer membrane and extracellular protein-enriched fractions from invasive (PAO1) and cytotoxic (6206) strains of P. aeruginosa separated by two-dimensional (2-D) gel electrophoresis. Gel image comparisons revealed the two strains express essentially identical membrane protein profiles under the conditions investigated. Membrane protein strain differences were typically the result of minor amino acid sequence variations resulting in small mass and isoelectric point shifts visible on 2-D gels. Analysis of extracellular proteins from stationary phase growth, however, revealed significantly different protein profiles. Extracellular fractions from the invasive PAO1 strain were dominated by extracellular proteases including elastase (LasB), LasA protease and chitin-binding protein, as well as several previously designated 'hypothetical' proteins. LasB appeared to be highly processed with 28 discrete mass and isoelectric point forms detected in this study. The significant number of active extracellular proteases (including LasB itself) may account for this processing. Conversely, extracellular fractions from strain 6206 consisted mainly of cellular and membrane exposed proteins including GroEL, DnaK and flagellar subunits. These are thought to result from cellular turnover during growth and the reliance on the secretory mechanisms of this strain to produce high levels of cytotoxicity factors, such as ExoU, which may be produced only upon specific interactions with host cells. These studies will aid in elucidating the differences between invasive and cytotoxic P. aeruginosa at the proteome level.
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D005408:Flagellin / Q000378:metabolism; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000737:chemistry; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000737:chemistry; D019897:Periplasm / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013045:Species Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D005408:Flagellin; D004247:DNA; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C066371:staphylolytic protease