Analysis of <em>Pseudomonas aeruginosa</em> Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

<div><p>We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen <em>Pseudomonas aeruginosa</em>. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on <em>P. aeruginosa</em>. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.</p> </div

Validation of NPs as magneto-capture tools of envelope structures.

<p>(A) Scheme of treatment of <i>P. aeruginosa</i> intact cells with activated NPs. Before treatment with activated NPs (dark purple circles), cells were washed by the culture medium to remove extracellular proteins. After treatment for 5 min, NPs were inactivated (pink circles) and cells disrupted. NPs were magnetically recovered and washed thoroughly. NPs that interact with cell surface can establish covalent bonds with free -NH₂ moieties (e.g. those of lysine of exposed proteins, red dots) and, upon cell lysis, envelope fragments that stick to NPs (NP-Env) can be magneto-captured. (B) Scheme of treatment with inactive NPs. Before treatment with inactive NPs (pink circles), cells were washed by the culture medium to remove extracellular proteins. Upon treatment for 5 min, cells were disrupted. NPs were magnetically recovered and washed thoroughly. Inactive NPs can interact with cell surface but no covalent bonding occurs and thus envelope fragments are not magneto-captured. (C) Reactive and inactive NPs, that had been used to treat <i>P. aeruginosa</i> intact cells as illustrated in (A) and (B), respectively, were loaded onto SDS-PAGE to analyze protein contents. M: protein molecular weight marker. (D) Fluorescence emission spectra (λ_ex: 390 nm; λ_em: 400–550 nm) of: unreacted NPs (NPs); NP-Env; NP-Env extensively washed with SDS at 60°C (NP-Env+SDS); total membrane preparation (Membranes). All spectra were taken in the presence of the hydrophobic fluorescent probe 0.1 mM 1-anilinonaphthalene-8-sulfonate, tracking the presence of lipids. Note the overlapping spectra of NP-Env and Membranes.</p

List of envelope-associated proteins not directly bound by NPs.

a<p>Class 1: Function experimentally demonstrated in <i>P. aeruginosa</i>; Class 2: Function of highly similar gene experimentally demonstrated in another organism; Class 3: Function proposed based on presence of conserved amino acid motif, structural feature or limited sequence similarity to an experimentally studied gene. Class 4: Homologs of previously reported genes of unknown function, or no similarity to any previously reported sequences.</p>b<p>CC: Cell compartment. OM: outer membrane; P: periplasm; E: extracellular; F: flagellar; IM: inner membrane; U: unknown; C: cytoplasmic.</p>c<p>Class 1: Subcellular localization experimentally demonstrated in <i>P. aeruginosa</i>; Class 2: Subcellular localization of highly similar gene experimentally demonstrated in another organism or to a paralog experimentally demonstrated in the same organism. BLAST expected value of 10e-10 for query within 80–120% of subject length. Class 3: Subcellular localization computationally predicted by PSORT.</p>d<p>Functional class and localization confidence is indicated according to the annotations in Pseudomonas Genome Database (<a href="http://www.pseudomonas.com" target="_blank">www.pseudomonas.com</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051062#pone.0051062-Winsor1" target="_blank">[31]</a>.</p>e<p>Lipoprotein, known or predicted <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051062#pone.0051062-Remans1" target="_blank">[24]</a>.</p

List of envelope-associated proteins covalently bound by NPs at cell surface (NP-CbP).

a<p>Class 1: Function experimentally demonstrated in <i>P. aeruginosa</i>; Class 2: Function of highly similar gene experimentally demonstrated in another organism; Class 3: Function proposed based on presence of conserved amino acid motif, structural feature or limited sequence similarity to an experimentally studied gene. Class 4: Homologs of previously reported genes of unknown function, or no similarity to any previously reported sequences.</p>b<p>CC: Cell compartment. OM: outer membrane; P: periplasm; E: extracellular; F: flagellar; IM: inner membrane; U: unknown; C: cytoplasmic.</p>c<p>Class 1: Subcellular localization experimentally demonstrated in <i>P. aeruginosa</i>; Class 2: Subcellular localization of highly similar gene experimentally demonstrated in another organism or to a paralog experimentally demonstrated in the same organism. BLAST expected value of 10e-10 for query within 80–120% of subject length. Class 3: Subcellular localization computationally predicted by PSORT.</p>d<p>Proteins identified also by trypsin digestion of NP-Env.</p>e<p>Functional class and localization confidence is indicated according to the annotations in Pseudomonas Genome Database (<a href="http://www.pseudomonas.com" target="_blank">www.pseudomonas.com</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051062#pone.0051062-Winsor1" target="_blank">[31]</a>.</p>f<p>Lipoprotein, known or predicted <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051062#pone.0051062-Remans1" target="_blank">[24]</a>.</p