Preliminary Research On The Biological Functions Ofβ-galactosidase (BgaC) In The Zoonotic Pathogen Streptococcus Suis Serotype2

Streptococcus suis (S. suis) is an important worldwide zoonotic pathogen associated with a wide range of diseases in pigs and humans, To date,thirty-three serotypes (types1/2,1-31and33) of S. suis have been described, of which S. suis2is the most frequently isolated and associated with disease. Of particular note, two big-scale endemics of human S. suis2infections had ever occurred in China (one in Jiangsu Province,1998, and the other in Sichuan Province,2005). A key feature of these two Chinese outbreaks is the prevalence of streptococcal toxic shock syndrome (STSS) manifesting itself as acute high fever, multiple organ failures, short course of disease and high lethality (more than80%).Surface proteins are thus far considered as the proven virulence factor playing key roles in the adhesion and invasion process in pathogensis. Exoglycosidases, which are one of the surface proteins, are recognized as receptors for invasion or as dietary substrates for the maintenance of colonial microflora. Our joint research group completed a comprehensive study of comparative genomics, decoding the whole genome sequences of the virulent S. suis2strain05ZYH33(isolated from Chinese STSS patients), and found that there is one β-Galactosidase encoding gene in it. We focused on β-Galactosidase named BgaC according to its code gene position, discovered that BgaC amino acid sequence of many kinds of streptococcus (S. pneumonia, S. dysgalactiae and so on) uniformity reaches as high as above63%. Notably, It has been reported that surface-associated exoglycosidase BgaC can hydrolyze and remove the host cellligand so that binding of pneumococci to the host is decreased, BgaC can remove galactose from β1,3-linked GlcNAc in lacto-N-tetraose of the glycolipid that could serve as a possible bindingsite for S. pneumoniae infection. However the reports of S. suis regarding the BgaC research have not been emerged.In this study, we characterized a functional BgaC from a Chinese S.suis2isloated,05ZYH33, and the following experiments are conducted:1. Cloning, prokaryotic expression and preparation of polyclonal antibodies of bgaC gene from β-galactosidase in Streptococcus suis2: The flanking DNA sequences to BgaC were amplified from the chromosomal DNA of05ZYH33and were cloned into the expression vecter. Thereafter, the gene was cloned into prokaryotic expression plasmid pET28a, and the recombinant plasmid pET28a-bgac was transformed into E.coli BL21. After induced expression by IPTG, the BgaC protein isolated was analyzed with SDS-PAGE and purified by chromatography. The mice anti-BgaC serum was obtained by immunizing mice with recombinant BgaC protein, and the titer of the serum was analyzed by ELISA. Finally the serum could be specifically reacted with the recombinant protein demonstrated by Western blot.2. Biological functions of the recombinant BgaC protein:Enzymatic activity was measured via the completely purified BgaC protein.The enzymatic activity analysis indicated the optimum temperature, action time, pH and the substrate concentration were42℃,30min,5.5and lOmM, respectively. Enzymatic activity of BgaC was about1615U/mL, specific activity was1076U/mg. On the basis of the above enzymatic reaction conditions we optimized, the S.suis2BgaC was proved to obviously possess the activity of β-galactosidase with Km of0.398mM, Kcat of5.63×102/s and Vm7.43×10-3mM/min. HPLC, immune electro-microscopy and the fluorescence-activated cell sorting (FACS) analysis showed that S.suis2BgaC specifically cleaved Galpl-3GIcNAc group and did act as a surface-anchored protein with the activity of β-galactosidase.3. Construction the AbgaC mutant:The Spc resistance (Spcr) gene cassette was inserted into a pUC18vector to create the recombinant plasmid pUC18-Spc. Two DNA fragments flanking the bgaC gene were cloned into pUC18-Spc to generate the knockout plasmid, pUC::bgaC. The pUC::bgaC plasmid was electroporated into05ZYH33competent cells. The fidelity of the double-crossover recombination was confirmed in the mutants by PCR using flanking primers lying outside the homologous regions, followed by direct DNA sequencing. The resulting mutant strain was further confirmed by a series of PCR, reverse transcription PCR and sequenceing.4. Biological functions of AbgaC mutant:After repeatedly serial subcultivation, the spectinomycin resistance phenotype was found to be stable in the AbgaC. The basic biological properties of the wild type strain05ZYH33and the AbgaC mutant were compared under the same conditions, colony and cell morphology, hemolytic activity and growth characteristics were examined but no significant differences between the wild type and mutant strain could be ascertained. However, the AbgaC mutant grew as much shorter chains of individual cells that different from S.suis2. Deletion of the bgaC gene in S.suis2increased its adhesion to Hep-2cells in vitro, and the ability of anti-phagocytosis was raised. RT-PCR results showed that bgaC gene could regulate partially its downstream PTS component. Mice-based experiments showed that an inactivation of bgaC failed to attenuate bacterial virulence, which is somewhat similar to the scenario seen with S. pneumoniae.To sum up, this study concluded that S. suis BgaC is an atypical surface-exposed protein within the involvement of bacterial adhesion. All above work lay the foundation for further developing the BgaC mechanism research.