title: Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae.
abstract: Streptococcus pneumoniae DnaJ is recognized as a virulence factor whose role in pneumococcal virulence remains unclear. Here, we attempted to reveal the contribution of DnaJ in pneumococcal virulence from the identification of its interacting proteins using co-immunoprecipitation method. dnaJ was cloned into plasmid pAE03 generating pAE03-dnaJ-gfp which was used to transform S. pneumoniae D39 strain. Then anti-GFP coated beads were used to capture GFP-coupled proteins from the bacterial lysate. The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. We finally obtained nine proteins such as DnaK, Gap, Eno, SpxB using this method. Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB. Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature. These results help to understand the role of DnaJ in the pathogenesis of S. pneumoniae.
authors.affiliation: Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China.
authors.forename: YingYing
authors.initials: Y
authors.lastname: Cai
authors.forename: WenJuan
authors.initials: W
authors.lastname: Yan
authors.forename: WenChun
authors.initials: W
authors.lastname: Xu
authors.forename: YiBing
authors.initials: Y
authors.lastname: Yin
authors.forename: YuJuan
authors.initials: Y
authors.lastname: He
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: XueMei
authors.initials: X
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism*; D047468:Immunoprecipitation; D018832:Molecular Chaperones / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D025941:Protein Interaction Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D050956:HSP40 Heat-Shock Proteins; D018832:Molecular Chaperones
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