Screening And Identification Of Heat Shock Protein DnaJ Interaction Proteins In Streptococcus Pneumoniae

ObjectiveStreptococcus pneumoniae DnaJ is recognized as avirulence factor whose role in pneumococcal virulence remains unclear.Here, we attempted to reveal the contribution of DnaJ in pneumococcalvirulence from the identification of its interacting proteins.Methods We mainly used co-immunoprecipitation and bacterialtwo-hybrid system to screen and demonstrate DnaJ interacting proteins.dnaJ was cloned into plasmid pAE03generating pAE03-dnaJ-gfp whichwas used to transform Streptococcus pneumoniae D39strain named withD39-dnaJ-gfp. Then anti-GFP antibody coated beads were used to captureGFP-coupled proteins from the bacterial lysates. The resulting proteinmixtures were subjected to SDS-PAGE and those differential bandsbetween negative control D39and D39-dnaJ-gfp were determined bymatrix-assisted laser desorption/ionization time of flight massspectrometry. Furthermore, to confirm the interactions between DnaJ andthese candidates, bacterial two-hybrid system was employed to reveal, forexample, the interactions between DnaJ and DnaK, Eno, SpxB. Further β-galactosidase report gene dectection and western blot experiments wereutilized to observe the effects of these interactions on Eno and SpxB whenheat shocked. At the same time, we constructed bait plasmid pBT-dnaJ andgenome library to screen DnaJ interacting proteins and confirm theinteraction by co-IP. Moreover, according to the bioinformaticscharacteristics of the candidate protein, minimum inhibitory concentrationexperiment was carried out to detect the influence of the interaction withDnaJ on bacrerial drug resistance.ResultsWe finally obtained nine DnaJ interacting proteins such asDnaK, Gap, Eno and SpxB after co-IP screening and MALDI-TOF MSidentification. And, the interactions between DnaJ and three of them-DnaK, Eno and SpxB were confirmed by bacterial two-hybrid system.Furthermore, the results of β-galactosidase report gene dectection andwestern blot suggested that DnaJ helped Eno and SpxB fold correctly andprotected them from degradation during heat shock. And, it also facilitatedthe extracellular transportation of Eno. At the same time, we discoveredone DnaJ interacting protein-SPD1264by bacterial two-hybrid system andthis interaction was confirmed by co-IP definitely. Moreover, throughliterature scanning and preliminary minimum inhibitory concentrationexperiments we found that there was some relationship between SPD1264and bacterial drug resistance for penicillin, ampicillin and kanamycin.Conclusion DnaJ is able to affect bacterial virulence by influencing the protein level and extracellular secretion of Eno and SpxB; throughaffecting translation-related proteins, DnaJ may involve in protein synthesisincluding some virulence factors, and it seems to be one of the mechanismsof bacterial virulence; besides, DnaJ may regulate SPD1264which isconcerned with drug resistance through their interaction, and contributes tobacterial drug resistance. All these results help to understand the roles ofDnaJ in the pathogenesis and drug resistance of Streptococcus pneumonia.