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

Vecchietti, Davide; Silvestre, Dario Di; Miriani, M.; Bonomi, Francesco; Marengo, Mauro; Bragonzi, Alessandra; Cova, Lara; Franceschi, Eleonora; Mauri, Pierluigi; Bertoni, Giovanni

doi:10.1371/journal.pone.0051062

Cited by 15 publications

(23 citation statements)

References 29 publications

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“…Proteomic analysis can provide further insights into the effects of nanomaterials on cells, such as their effects on membrane proteins after exposure to silver nanoparticles . Recently, high‐throughput liquid chromatography coupled with mass spectrometry methods have allowed efficient analysis of cell proteome and advancement in mass spectrometry has substantially increased the number of proteins that can be accurately identified and quantified . Such high‐throughput proteomic analysis represents major improvements over traditional methods and is expected to be more widely applied to study the antimicrobial mechanisms of nanomaterials.…”

Section: Genomics Transcriptomics and Proteomics Investigationsmentioning

confidence: 99%

Toxicity of Metal Oxide Nanoparticles: Mechanisms, Characterization, and Avoiding Experimental Artefacts

Djurišić

Leung

et al. 2014

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Metal oxide nanomaterials are widely used in practical applications and represent a class of nanomaterials with the highest global annual production. Many of those, such as TiO2 and ZnO, are generally considered non-toxic due to the lack of toxicity of the bulk material. However, these materials typically exhibit toxicity to bacteria and fungi, and there have been emerging concerns about their ecotoxicity effects. The understanding of the toxicity mechanisms is incomplete, with different studies often reporting contradictory results. The relationship between the material properties and toxicity appears to be complex and diifficult to understand, which is partly due to incomplete characterization of the nanomaterial, and possibly due to experimental artefacts in the characterization of the nanomaterial and/or its interactions with living organisms. This review discusses the comprehensive characterization of metal oxide nanomaterials and the mechanisms of their toxicity.

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Section: Genomics Transcriptomics and Proteomics Investigationsmentioning

confidence: 99%

Toxicity of Metal Oxide Nanoparticles: Mechanisms, Characterization, and Avoiding Experimental Artefacts

Djurišić

Leung

et al. 2014

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346

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“…Due to the relevant role of the envelope-associated proteins, in the last two decades different studies aimed to characterize the P. aeruginosa envelope proteome. Most of them focused on OM [7][8][9][10] and global membrane [11][12][13], while a lesser number analyzed proteins from periplasmic space [14] and IM [15]. Some of these studies were based on two-dimensional (2-D) gel electrophoresis protein separation [7,8,10,14].…”

Section: Introductionmentioning

confidence: 99%

The Landscape of Pseudomonas aeruginosa Membrane-Associated Proteins

Motta

Vecchietti

Martorana

et al. 2020

Cells

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Background: Pseudomonas aeruginosa cell envelope-associated proteins play a relevant role in infection mechanisms. They can contribute to the antibiotic resistance of the bacterial cells and be involved in the interaction with host cells. Thus, studies contributing to elucidating these key molecular elements are of great importance to find alternative therapeutics. Methods: Proteins and peptides were extracted by different methods and analyzed by Multidimensional Protein Identification Technology (MudPIT) approach. Proteomic data were processed by Discoverer2.1 software and multivariate statistics, i.e., Linear Discriminant Analysis (LDA), while the Immune Epitope Database (IEDB) resources were used to predict antigenicity and immunogenicity of experimental identified peptides and proteins. Results: The combination of 29 MudPIT runs allowed the identification of 10,611 peptides and 2539 distinct proteins. Following application of extraction methods enriching specific protein domains, about 15% of total identified peptides were classified in trans inner-membrane, inner-membrane exposed, trans outer-membrane and outer-membrane exposed. In this scenario, nine outer membrane proteins (OprE, OprI, OprF, OprD, PagL, OprG, PA1053, PAL and PA0833) were predicted to be highly antigenic. Thus, they were further processed and epitopes target of T cells (MHC Class I and Class II) and B cells were predicted. Conclusion: The present study represents one of the widest characterizations of the P. aeruginosa membrane-associated proteome. The feasibility of our method may facilitates the investigation of other bacterial species whose envelope exposed protein domains are still unknown. Besides, the stepwise prioritization of proteome, by combining experimental proteomic data and reverse vaccinology, may be useful for reducing the number of proteins to be tested in vaccine development.

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“…High-apparent affinity high capacity IgG were found to extensively cross-react with the closely related HK-EB. This indicated that these antibodies recognized epitopes present at high density and which seem similar in Escherichia and Salmonella , possibly due to their common features in LPS 40 – 42 . However, and as expected, less cross-reactivity (yet still a considerable amount) was found when these antibodies were assayed against HK-PA, an evolutionarily more distant Gram-negative bacteria (Fig.…”

Section: Resultsmentioning

confidence: 84%

“…4d ), making the immunodominant LPS a likely candidate antigen. LPS are strain-specific in their glycosylation, yet many variants also share similarities and conserved epitopes 40 – 42 . Consequently, we were interested in identifying whether the mice immunized with a specific bacteria species produced more specific or cross-reactive antibodies that might interact with other bacteria.…”

Section: Resultsmentioning

confidence: 99%

Deep phenotypic characterization of immunization-induced antibacterial IgG repertoires in mice using a single-antibody bioassay

et al. 2020

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Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. To characterize the anti-bacterial antibody repertoire, we developed an in-droplet bioassay with single-antibody resolution. The assay not only allowed us to identify whether the secreted antibodies recognized a bacterial surface antigen, but also to estimate the apparent dissociation constant (KD app) of the interaction and the density of the recognized epitope on the bacteria. Herein, we found substantial differences within the KD app/epitope density profiles in mice immunized with various species of heat-killed bacteria. The experiments further revealed a high cross-reactivity of the secreted IgG repertoires, binding to even unrelated bacteria with high affinity. This application confirmed the ability to quantify the anti-bacterial antibody repertoire and the utility of the developed bioassay to study the interplay between bacteria and the humoral response.

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

Cited by 15 publications

References 29 publications

Toxicity of Metal Oxide Nanoparticles: Mechanisms, Characterization, and Avoiding Experimental Artefacts

Toxicity of Metal Oxide Nanoparticles: Mechanisms, Characterization, and Avoiding Experimental Artefacts

The Landscape of Pseudomonas aeruginosa Membrane-Associated Proteins

Deep phenotypic characterization of immunization-induced antibacterial IgG repertoires in mice using a single-antibody bioassay

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