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Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae.

Cai Y, Yan W, Xu W, Yin Y, He Y, Wang H, Zhang X - Curr. Microbiol. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China.

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.

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DnaJ interacts with DnaK, SpxB, and Eno in XL1-Blue MRF’ Kan strain in vivo. A bacterial two-hybrid system was used to confirm the interaction between DnaJ and DnaK/SpxB/Eno in vivo. Interaction between DnaJ and DnaK/SpxB/Eno was monitored by the expression of HIS3 and aadA reporter genes on nonselective (a, d, g), selective (b, e, h) and dual selective (c, f, i) screening plates
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Fig3: DnaJ interacts with DnaK, SpxB, and Eno in XL1-Blue MRF’ Kan strain in vivo. A bacterial two-hybrid system was used to confirm the interaction between DnaJ and DnaK/SpxB/Eno in vivo. Interaction between DnaJ and DnaK/SpxB/Eno was monitored by the expression of HIS3 and aadA reporter genes on nonselective (a, d, g), selective (b, e, h) and dual selective (c, f, i) screening plates

Mentions: Bacteria co-expressing DnaJ and DnaK/Eno/SpxB were viable on nonselective, selective, and dual selective screening plates as the positive control (Fig. 3a–i). In sharp contrast, there were no colonies on selective and dual selective screening plates when the pBT and pTRG-dnaK/eno/spxB, pBT-dnaJ and pTRG, or pBT and pTRG fragments were co-transformed respectively (Fig. 3b, c, e, f, h, i), although they could be seen on nonselective screening plates (Fig. 3a, d, g), indicating the fitness of the bacterial two-hybrid system. These results demonstrated the interaction between DnaJ and DnaK also works in S. pneumoniae and Eno/SpxB interacts with DnaJ in S. pneumoniae.Fig. 3


Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae.

Cai Y, Yan W, Xu W, Yin Y, He Y, Wang H, Zhang X - Curr. Microbiol. (2013)

DnaJ interacts with DnaK, SpxB, and Eno in XL1-Blue MRF’ Kan strain in vivo. A bacterial two-hybrid system was used to confirm the interaction between DnaJ and DnaK/SpxB/Eno in vivo. Interaction between DnaJ and DnaK/SpxB/Eno was monitored by the expression of HIS3 and aadA reporter genes on nonselective (a, d, g), selective (b, e, h) and dual selective (c, f, i) screening plates
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824243&req=5

Fig3: DnaJ interacts with DnaK, SpxB, and Eno in XL1-Blue MRF’ Kan strain in vivo. A bacterial two-hybrid system was used to confirm the interaction between DnaJ and DnaK/SpxB/Eno in vivo. Interaction between DnaJ and DnaK/SpxB/Eno was monitored by the expression of HIS3 and aadA reporter genes on nonselective (a, d, g), selective (b, e, h) and dual selective (c, f, i) screening plates
Mentions: Bacteria co-expressing DnaJ and DnaK/Eno/SpxB were viable on nonselective, selective, and dual selective screening plates as the positive control (Fig. 3a–i). In sharp contrast, there were no colonies on selective and dual selective screening plates when the pBT and pTRG-dnaK/eno/spxB, pBT-dnaJ and pTRG, or pBT and pTRG fragments were co-transformed respectively (Fig. 3b, c, e, f, h, i), although they could be seen on nonselective screening plates (Fig. 3a, d, g), indicating the fitness of the bacterial two-hybrid system. These results demonstrated the interaction between DnaJ and DnaK also works in S. pneumoniae and Eno/SpxB interacts with DnaJ in S. pneumoniae.Fig. 3

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China.

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.

Show MeSH
Related in: MedlinePlus