Surface-associated proteins play a key role in bacterial physiology and pathogenesis. Given these roles, identification and characterization of suchproteins may lead to novel antibacterial targets. In addition, since surface proteins face the host immune system, they may be basic elements of effective vaccines. We report on specific magneto-capturing followed by Multi-dimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of Pseudomonas aeruginosa. In this study, magnetic nanoparticles (NPs) were activated making them able to establish covalent bonds with proteins. Given their chemical composition, size (aver-age diameter = 70-90 nm), and negative charge, these NPs were expected to be atoxic for bacterial cells. Indeed, viable counts of bacterial cells incubated with and without NPs indicated no lethality. Transmission electron microscopy evidence also indicated that NPs were unable to get through thecell envelope and distributed instead over the cell surface. Furthermore, we demonstrated that only activated NPs could collect proteins from cells whereas no proteins were detected in controls experiments carried out with NPs inactivated prior to exposure to cells. These features supported the development of a method for magneto-capturing surface-exposed proteins that greatly improves sensitivity and specificity of previous methods such as surface “shaving” with proteases. Briefly, activated NPs were incubated with bacterial cells and, subsequently, their reactive groups were inactivated. Cells were disrupted, and NPs were magnetically separated from cell extracts, washed several times, and tested for protein and cell membrane content. Consequently the NPs bound to cell envelope structures (NP-Envelope) were digested by trypsin to shave protruding protein domains. Tryptic peptides shaved from NP-Envelope were identified by MudPIT analysis.Alternatively, heat and detergents were used to remove from NP-Envelope all envelope material but proteins covalently bound to NPs, which were identified by MudPIT analysis. Shedding of proteins and proteins released by spontaneous cell lysis were monitored through control experiments. As a result, 92 proteins were identified as associated to NP-Envelope and 63 proteins were those directly bound to NPs. A relevant number of proteins with high localization confidence in outer membrane, along with pilin and FliD (a flagellar hook-associated protein) or outer membrane porin family proteins, were identified. A feature of this approach that is relevant to vaccine development is the possibility to obtain information about protein topology by the direct identification with MudPIT of domains exposed from membrane layers. Therefore, our results might contribute to accelerating the process of vaccine target identification and development.
Analysis of cell envelope proteome of pseudomonas aeruginosa by capture of surface-exposed proteins on activated magnetic nanoparticles
M. Marengo;
2012-01-01
Abstract
Surface-associated proteins play a key role in bacterial physiology and pathogenesis. Given these roles, identification and characterization of suchproteins may lead to novel antibacterial targets. In addition, since surface proteins face the host immune system, they may be basic elements of effective vaccines. We report on specific magneto-capturing followed by Multi-dimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of Pseudomonas aeruginosa. In this study, magnetic nanoparticles (NPs) were activated making them able to establish covalent bonds with proteins. Given their chemical composition, size (aver-age diameter = 70-90 nm), and negative charge, these NPs were expected to be atoxic for bacterial cells. Indeed, viable counts of bacterial cells incubated with and without NPs indicated no lethality. Transmission electron microscopy evidence also indicated that NPs were unable to get through thecell envelope and distributed instead over the cell surface. Furthermore, we demonstrated that only activated NPs could collect proteins from cells whereas no proteins were detected in controls experiments carried out with NPs inactivated prior to exposure to cells. These features supported the development of a method for magneto-capturing surface-exposed proteins that greatly improves sensitivity and specificity of previous methods such as surface “shaving” with proteases. Briefly, activated NPs were incubated with bacterial cells and, subsequently, their reactive groups were inactivated. Cells were disrupted, and NPs were magnetically separated from cell extracts, washed several times, and tested for protein and cell membrane content. Consequently the NPs bound to cell envelope structures (NP-Envelope) were digested by trypsin to shave protruding protein domains. Tryptic peptides shaved from NP-Envelope were identified by MudPIT analysis.Alternatively, heat and detergents were used to remove from NP-Envelope all envelope material but proteins covalently bound to NPs, which were identified by MudPIT analysis. Shedding of proteins and proteins released by spontaneous cell lysis were monitored through control experiments. As a result, 92 proteins were identified as associated to NP-Envelope and 63 proteins were those directly bound to NPs. A relevant number of proteins with high localization confidence in outer membrane, along with pilin and FliD (a flagellar hook-associated protein) or outer membrane porin family proteins, were identified. A feature of this approach that is relevant to vaccine development is the possibility to obtain information about protein topology by the direct identification with MudPIT of domains exposed from membrane layers. Therefore, our results might contribute to accelerating the process of vaccine target identification and development.
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