Interaction Of Enolase With Heat Shock Protein Dnaj In Streptococcus Pneumoniae

Objective:To determine whether the key glycolytic enzyme enolase (Eno) bind heat shock protein DnaJ directly and to analyze the binding sites between DnaJ and Eno in Streptococcus pneumoniae (S. pneumoniae), and conduct a preliminary investigate of the effect of DnaJ on Eno secretion.Methods:Recombinant Eno protein was obtained through prokaryotic expression system, and its α-Eno activity was analyzed. Kunming mice were immunized with the recombinant protein to prepare the polyclonal antibody to Eno protein and the titer of the antibody was determined by ELISA. The expression levels of Eno protein in S. pneumoniae with different serotypes were determined by western blot. The direct interaction between DnaJ and Eno was determined by biolayer interferometrvy (BLI) technology and direct binding assay. Truncated Eno protein was expressed and purified, and the binding sites of DnaJ on Eno were determined by direct binding assay. According to the binding sites, a series of truncated Eno were designed and cloned into the ectopic expression plasmid pJWv25. When the recombinant plasmids were transformed into S. pneumoniae R6 and induced by Zn2+, all the truncated Eno were ectopically expressed and tagged with N-terminal GFP for detection. Western blot was used to detect the expression of the truncations both in the whole cell and supernatant fraction to evaluate the effect of DnaJ on Eno secretion. Furthermore, DnaJ was ectopically over expressed in R6 strain and the expression of Eno in the whole cell and supernatant fraction was detected by western blot.Results:The recombinant protein Eno with high purity(about 90%) was obtained after purification of Ni affinity chromatography and exhibited α-enolase activity. The antibody titer of anti-Eno in the immunized mice serum reached to 7.68 × 106. Western blot showed the same band with Mr 47 000 presented in the supernatant of all the 7 serotypes of S. pneumoniae. The direct binding assay demonstrated that DnaJ bound Eno in a dose-dependent manner (r=0.920, P=0.000) and a concentration dependent binding of Eno to immobilized DnaJ was detected (KD=926 nmol/L) by BLI technology analysis. Binding experiments demonstrated that DnaJ bound more truncated Eno(aal-aa100) (2.25±0.38) than the other truncated Eno proteins (0.94±0.25), (1.08±0.09), (0.75±0.12) (P=0.000, P=0.001, P=0.000). Western blot showed that all the truncations were correctly expressed and detected at their predicted molecular mass in the whole cell fraction, but only Eno(aa1-aa317) and Eno(aa323-aa434) were detected in the supernatant fraction. When compared with the wild type and not induced strains, there was no significant difference of Eno expression in the whole cell fraction but its secretion was reduced in the supernatant of the DnaJ overexpression strain.Conclusions:DnaJ interacts with Eno directly in S. pneumoniae primarily via the binding sites, which are localized within 100 aa of the N-terminal domain of Eno. The extent of Eno binding DnaJ has no correlation with Eno secretion in S. pneumoniae. However, overexpressed DnaJ in S. pneumoniae R6 strain might reduce the secretion of Eno protein.