title: Another turn of the screw in shaving Gram-positive bacteria: Optimization of proteomics surface protein identification in Streptococcus pneumoniae.
abstract: Bacterial surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either vaccines or antibodies. Proteomic analysis through "shaving" live cells with proteases has become a successful approach for a fast and reliable identification of surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to autolysis, and set up the conditions for maximizing the identification of surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with peptides derived from cytoplasmic proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the proteins described to induce protection against pneumococcal infection. In addition, we found other proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing antigens that can be used in serological tests for the diagnosis of pneumococcal disease.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Excelencia Internacional CeiA3, Instituto Maimónides de Investigación Biomédica de Córdoba, Córdoba, Spain.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Olaya-Abril
authors.forename: Lidia
authors.initials: L
authors.lastname: Gómez-Gascón
authors.forename: Irene
authors.initials: I
authors.lastname: Jiménez-Munguía
authors.forename: Ignacio
authors.initials: I
authors.lastname: Obando
authors.forename: Manuel J
authors.initials: MJ
authors.lastname: Rodríguez-Ortega
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D010447:Peptide Hydrolases / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods; D013296:Streptococcus pneumoniae / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D010447:Peptide Hydrolases