Export Requirements Of Nocalssical Secretion Protein Gapdh And Its Interaction With Heat Shock Protein Dnaj In Streptococcs Pneumoniae

Objective:GAPDH is one of the noclassical proteins in streptococcus pneumoniae. There are two contrary hypothesis about noclassical secretion protein exportation in gram positive bacteria. One is that noclassical secretion protein is secreted in a cell lysis dependent manner, while the other one is absolutely contrary. The primary purpose in our study is to illuminate the relationship between GAPDH secretion and cell lysis and identify the secretion essential domain of GAPDH in streptococcus pneumoniae. Another question in this study is to study whether DnaJ could interact with GAPDH and contribute to its secretion.Methods:Recombinant GAPDH was expressed by a prokaryotic expression system and purified to prepare polyclonal antibody of GAPDH, which was used to test the conservation of GAPDH in streptococcus pneumoniae. Western blot was used to compare the expression of GAPDH in the whole cell and culture medium between wild type and LytA deficient strain of streptococcus pneumoniae. The conserved protein CodY, expressed only in the protoplasmic, was used as an internal reference to test cell lysis. The structural domain and three dimensional structure of GAPDH were analyzed with Jpred3 software and the Swiss-Model software, respectively. GAPDH truncation was designed according to its structural domain. All the truncates were cloned into the ectopic expression plasmid pJW-v25 under the control of a Zn2+ induced czcD promoter and transformed into streptococcus pneumoniae to ectopically express GFP tagged truncations. The secretion and surface display of GAPDH truncations were detected by western blot with GFP antibody. Structure replacement and amino acid mutation were used to further conform the importance of the secretion essential domain. To investigate whether the essential domain could lead the protein secretion, the essential domain was expressed with a GFP tag for detection in S. pneumoniae. The gap A gene was cloned onto the pJW-v25 plasmid and transformed into streptococcus pneumoniae to express GAPDH of bacillus subtilize 168. Western blot was used to detect its localization. Finally, BacterioMatch â…¡ two-hybrid system, direct binding assay and Bio-layer Interferometry (BLI) were used to conform the interaction between GAPDH and DnaJ.RESULTS:We successfully obtained the recombinant GAPDH with purity of 90% and prepared polyclonal antibody of GAPDH with higher titers. Conservation assay showed that GAPDH is highly conserved in streptococcus pneumoniae. In the early logarithmic growth phase, GAPDH was detected both in the cell and culture medium, while CodY was only detected in the protoplasmic fraction. Moreover, there was no significant difference of GAPDH expression in the LytA deficient strain when compared with the wild type. These result support the conclusion that GAPDH is secreted in a cell lysis independent manner. When the amino acid sequence of GAPDH submitted to the Jpred3 software, the result showed that GAPDH is composed by two domain. Domain â…  is consist of the N terminal 1-150aa and C terminal a helix and Domain â…¡ is the 151-316aa. We can also find that the N terminal and C terminal compose the domain â…  from the three dimensional structure of GAPDH. According to its structural domain, GAPDH was truncated and ectopically expressed with a GFP tag in streptococcus pneumoniae. Western bot showed that truncations no matter lack the N terminal 1-30aa or the C terminal a helix was only detected in the protoplasmic fraction and not detectable in the cell wall and culture medium fraction, which suggested that domain â…  is the secretion essential domain of GAPDH. Structure replacement in the N terminal conservative sequence 3-21aa and the C terminal a helix also shows the same results. Amino acid mutation further validated the indispensable role of the N terminal 1-21aa and the C terminal a helix in GAPDH secretion and surface display. Furthermore, the 3VKVGIN9 and 18GGG20 amino acid of N terminal and the 325RTLEYF330 of the C terminal a helix is the key amino acid for GAPDH secretion and surface display. The 10F is important to GADPH secretion but have no effect on its surface display, while the 322QLV324 is essential for GAPDH surface display and have no effect on its secretion. When the domain I was directly connected and expressed with a GFP tag in streptococcus pneumoniae, the fusion protein was only detected in the protoplasmic fraction. Once the domain I was connected by a flexible structure (GGS)2, the fusion protein was also detected only in the protoplasmic. The GAPDH of bacillus subtilis 168 strain was expressed in streptococcus pneumoniae but only detected in the protoplasmic not in the cell wall and culture medium fraction. Finally, BacterioMatch II two-hybrid system, direct binding assay and Bio-layer Interferometry (BLI) were confirmed that GAPDH could directly interact with heat shock protein DnaJ.Conclusion:In our study, all the results support the conclusion that the exportation of GAPDH is not due to cell lysis in streptococcus pneumoniae and domain I is the secretion essential domain. In addition, domain I is essential but not sufficient for GAPDH exportation, domain II is also needed to maintain its three dimensional structure. Furthermore, the exportation mechanism of GAPDH in bacillus subtilis is difference. Finally, we confirmed that GAPDH could directly interact with DnaJ. These result will make an important significance and direct us to investigate the exportation pathway of GAPDH deeper in streptococcus pneumoniae.