Biochemical Charaterization Of Glycosyltransferases Related To Capsular Polysaccharide Biosynthesis Of Streptococcus Pneumoniae Serogroup 18

Streptococcus pneumoniae（Sp.or S.pneumoniae）is a gram-positive bacterium and a major human pathogen.It is responsible for diseases such as pneumonia,meningitis,sepsis,bacteremia and otitis media,resulting in high morbidity and mortality worldwide,especially in population of children,the elderly and immunocompromised individuals.S.pneumoniae infections are mainly treated with antibiotics.However,the emergence of a large number of drug-resistant strains makes vaccines as the most effective manner to prevent pneumococcal infections.There are two types of pneumococcal vaccines including polysaccharide vaccine（PPV）and polysaccharide-protein conjugate vaccine（PCV）,in which the capsular polysaccharides（CPSs）are the main effective antigen component.The CPS presents on the outermost layer of the bacterial cell is the main virulence factor of S.pneumonia which plays an important role in the colonization and adhesion of bacteria and protects the bacteria against host immune phagocytosis.The diversity of the chemical structures of CPSs is one of the important factors for S.pneumoniae to escape the host immune system.At present,the capsular polysaccharides used in the pneumococcal vaccine are mainly separated from the bacterial culture;therefore,the purification steps are complicated and the contamination of cytotoxins during the production process also seriously affects the quality control of the vaccine.In addition,polysaccharide-conjugated vaccines involve chemical coupling between sugar chains and carrier proteins that cannot be fully controlled,so it is impossible to obtain components with a well-defined conjugate structure.In order to overcome the abovementioned problems,the chemical synthesis and enzymatic synthesis of CPSs are promising strategy to produce better reproducibility.lower manufacturing costs,more structurally defined epitopes,and improved the purity and safety of vaccines.In order to study the enzymatic synthesis of capsular polysaccharide,we first pay attention to elucidate the molecular mechanisms of pneumococcal capsular assembly and regulation.To date,98 immunochemically distinct CPS serotypes have been identified from S.pneumoniae,which belong to more than 40 pneumococcal serogroups.Except for serotypes 3 and 37,almost all of pneumococcal CPSs are synthesized by the Wzx/Wzy-dependent pathway.Studies have found that the genes involved in Wzx/Wzy-dependent pathway are located at the same positions in the chromosome between dexB and aliA.The conserved dexB and aliA genes on CPS biosynthetic（cps）loci have no function on capsule formation,and the remaining genes encode sugar nucleotide synthase,capsular polysaccharide synthesis regulator enzymes,polysaccharide synthetase,initial sugar transferases,glycosyltransferases（GTs）,acetyltransferase,polymerase and flipase,etc.In fact,the chemical structure of the capsular polysaccharide is closely related to the genes in the capsular polysaccharide gene cluster.Taking S.pneumoniae 18C serotype（ST18C）as an example,the upstream genes of the cps loci encode capsular polysaccharide synthesis regulator enzymes,which are involved in the regulation and output of CPS;the downstream genes encode sugar nucleotides,including dTDP-Rha synthesis enzymes and CDP-glycerol synthase,etc.;the middle position is the specific region that encodes glycosyltransferase,repeat unit polymerase（Wzy）and flippase（Wzx）.The assembly of the CPS from ST18C starts with the addition of a glucose-1-phosphate（Glc-1-P）onto undecaprenyl phosphate（Und-P）by membrane protein WchA to form Glc-PP-Und.Next,other glycosyl groups are sequentially added to Glc-PP-Und by glycosyltransferases followed by the modification by other enzymes until the complete CPS repeat unit（RU）is formed.Then,the RU-PP-Und is translocated to the periplasmic side of the inner membrane by the flippase Wzx and polymerized by the polymerase Wzy.Finally,the polymerized polysacchairde is connected to the peptidoglycan through an unknown enzyme.In addition to WchA,ST18C contains five putative glycosyltransferases,namely WchF,WciU,WciV,WciW and WciY.However,there is no definite biochemical evidence for the biochemical functions of these enzymes.S.pneumoniae serogroup 18 is one of the predominant invasive serogroups worldwide,which has four different serotypes 18F,18 A,18B and 18C.Among of these serotypes,ST18C has been embodied in all PPVs and PCVs.Therefore,we chose three enzymes WchF（Cps18CF）,WciU（Cps18CU）,and WciV（Cps18CV）,which involved in the synthesis of ST18C capsular polysaccharide repeat units,as the research object,and explored their enzyme activity and biochemical properties.Besides,considering that WciU of ST18A（Cps18AU）has different glycosylation function from other enzymes（Cps18BU,Cps18CU and Cps18FU）in 18 serogroup,we also focused on the characterization of biofunctional study of this enzyme.The main research content of this thesis includes the following four aspects:1.The cps18CF gene was derived from the ST18C cps loci,and four engineering strains including E.coli BL21（DE3）cps18CF-pGEX-6P-3,E.coli BL21（DE3）cps18CF-pET-22b,E.coli BL21（DE3）cps18CF-pET-32a and E.coli BL21（DE3）cps18CF-pET-28a were constructed for protein expression.Pure Cps18CF enzyme can be obtained from E.coli BL21（DE3）cps18CF-pET-28a with 10 mg in 1 liter culture.Cps18CF was identified as β-1,4 rhamnosyltransferase by use of Glcα-PP-O（CH₂）₁₁OPh GF-1 as acceptor substrate and dTDP-Rha as donor substrate,and showed the best catalytic activity under the conditions of 50 mM Pipes（pH 7.0）,2.5 mM Mn²⁺ and 37℃.The maximum reaction rates(V_max)of Cps18CF to dTDP-Rha and GF-1 were 39.05±3.06 μmol min^-1 mg^-1 and 146.3±6.389 μmol min^-1 mg^-1,respectively.The Michaelis constant values（K_m）of Cps18CF for dTDP-Rha and GF-1 were 82.83±31.64 μM and 670.4±93.18 μM,respectively.Compared with the K_m value（290±20 μM）of β-1,4 rhamnosyltransferase Cps23FT from S.pneumoniae 23F,Cps18CF showed a higher affinity for dTDP-Rha donor.Finally,Cps18CF catalyzed the monosaccharide GF-1 to form the disaccharide GF-2a with an amount of 6.6 mg in 60%yield.2.The cps18CU gene was derived from the ST18C cps loci,and five engineering strains including E.coli BL21（DE3）cps18CU-pET-28a,E.coli BL21（DE3）cps18CUpGEX-4T-1,Exoli BL21（DE3）cps18CU-pGEX-6P-3.E.coli BL21（DE3）cps18CUpET-32a and E.coli BL21（DE3）cps18CU-pET-22b were constructed for protein expression.Pure Cps18CU enzyme can be obtained from E.coli BL21（DE3）cps18CUpET-28a with 10.4 mg from 1 liter culture.Using Rhaβ1,4-Glcα-PP-O（CH₂）₁₁-OPh GF2a and UDP-Glc as substrates,Cps18CU was identified as α-1,3-glucosyltransferase and showed the best catalytic activity under the conditions of 50 mM Pipes（pH 7.0）,10 mM Mn²⁺ and 42℃.The V_max values of Cps18CU for UDP-Glc and GF-2a were 37.2±2.19 μmol min^-1 mg^-1 and 20.5±0.50 μmol min^-1 mg^-1,respectively,while the K_m values for UDP-Glc and GF-2a were 2 170 ± 310.50 μM and 600 ± 38.40 μM,respectively.The trisaccharide of Glcα1,3-Rhaβ1,4-Glcα-PP-O（CH₂）₁₁-OPh GF-3a was synthesized by Cps18CU from GF-2a in 2.6 mg with a yield of 72%.In addition,GF-3a could also be obtained by one-pot two-enzyme system from GF-1,which demostarted that the combination of Cps18CU with Cps18CF or Cps23FT could significantly improve the synthetic efficiency of trisaccharide GF-3a.A series of sugar nucleotide donors synthesized with glucose-1-phosphate transferase Cps23FL（dUDP-Glc,dTDP-Glc,dCDP-Glc,CDP-Glc,dGDP-Glc,GDPGlc,dADP-Glc,ADP-Glc,UDP-GlcNAc,UDP-Gal and UDP-Man）were investigated for donor substrate specificity recognized by Cps18CU.All of them but three（UDPGlcNAc,UDP-Gal and UDP-Man）could be well recognized by Cps18CU.Therefore,glucose reside in NDP-sugars is essential for donor recognition of Cps18CU.Moreover,we synthesized four receptor substrates including Rhaβ1,4-Glcβ-O（CH₂）₂-N₃ GF-2b,Rhaα1,4-Glcβ-O（CH₂）₂-N₃ GF-2c,Rhaβ-O（CH₂）₂-N₃ GF-2d and Rhaα-O（CH₂）₂-N₃ GF-2e for acceptor substrate specificity study.It has disclosed that only disaccharide GF-2b could be tolerated by Cps18CU and formed the trisaccharide Glcα1,3-Rhaβ1,4Glcβ-O（CH₂）₂-N₃ GF-3b with a 90%yield.Thus,Cps18CU could accepte such acceptor substrate containing Rhaβ1,4-Glc moiety in structure.In addition,GF-2b and GF-3b have better water solubility and their azide groups at the reducing end could offer potential active fuction group for the biological evaluation of these synthesized oligosaccharides.3.The cps18CV gene was derived from the ST18C cps loci,and four engineering strains including E.coli BL21（DE3）cps18CV-pET-21b,E.coli BL21（DE3）cps18CVpET-28a,E.coli BL21（DE3）cps18CV-pET-32a and E.coli BL21（DE3）cps18CVpGEX-6P-3 were constructed for protein expression.Pure enzyme Cps18CV can be obtained from E.coli BL21（DE3）cps18CV-pGEX-6P-3 with 8 mg in 1 liter culture.Cps18CV was identified as β-1,4 galactosyltransferase using GF-3a and UDP-Gal as substrates,and exhibits the best catalytic activity under the conditions of 50 mM TrisHCl（pH 8.0）,10 mM Mg²⁺and 25℃.For UDP-Gal and GF-3a,the apparent K_m values were 1163±56.91 μM and 242.3 μM,respectively,and the V_max values were 0.2867 ±0.005 μmol min-l mg^-1 and 0.1436 μmol min^-1 mg^-1,respectively.Tetrasaccharide Galβ1,4-Glcα1,3-Rhaβ1,4-Glcα-PP-O（CH₂）₁₁-OPh GF-4a could be synthesized with the catalysis of Cps18CV from GF-3a.Similarly,a one-pot three-enzyme synthetic strategy was developed for preparation of tetrasaccharide GF-4a with combination of Cps18CF,Cps18CU and Cps18CV,which afforded GF-4a in an overall yield of 73%from the starting material GF-1.4.The cps18AU gene was derived from the ST18A cps loci,and six engineering strains including E.coli BL21（DE3）cps18AU-pET-21b,E.coli BL21（DE3）cps18AUpET-22b,E.coli BL21（DE3）cps18AU-pET-28a,E.coli BL21（DE3）cps18AU-pET-32a,E.coli BL21（DE3）cps18A U-pGEX-4T-1 and E.coli BL21（DE3）cps 18A U-pGEX-6P-3 were constructed for protein expression.Pure Cps 18AU enzyme can be obtained from E.coli BL21（DE3）cps18AU-pET-32a with 2 mg in 1 liter culture.Cps18AU was identified as α-1,3-acetylglucosaminetransferase using GF-2a and UDP-GlcNAc as substrates,and exhibits the best catalytic activity under the conditions of 50 mM Pipes（pH 7.0）,5 mM Mn²⁺and 25℃.For UDP-GlcNAc and GF-2a,the apparent K_m values were 5850 μM and 857.9 μM,respectively,and the V_max values were 0.0.4874 μmol min^-1 mg^-1 and 0.2911 μmol min^-1 mg^-1,respectively.The trisaccharide GlcNAcα1,3Rhaβ1,4-Glcα-PP-O（CH₂）₁₁-OPh GF-3c was synthesized by Cps18CU from GF-2a in 3.5 mg with a yield of 71%.In addition,GF-3c could also be obtained by one-pot twoenzyme system（Cps18CF and Cps18AU）from GF-1 with the yield of 68%.Futhermore,tetrasaccharide Galβ1,4-GlcNAcα1,3-Rhaβ1,4-Glcα-PP-O（CH₂）₁₁-OPh GF-4b could be synthesized with the catalysis of Cps18CV from GF-3c or using a onepot three-enzyme synthetic strategy（Cps18CF,Cps18AU and Cps18CV）from the starting material GF-1,in an overall yield of 67%or 58%,repectively.In summary,this thesis mainly fouced on four glycosyltransferases that involved in biosynthesis of capsular polysaccharide repeat units of Streptococcus pneumonia 18 serogroup.The mainly significances of this thesis are as follows:（1）It is the first time to identify enzyme activities of 18CF,Cps18CU,Cps18CV and Cps18AU in vitro,which provides new tool enzymes for the enzymatic synthesis of the related CPS polysaccharide repeat units;（2）The structure of synthetic disaccharide GF-2a,trisaccharide GF-3a,and tetrasaccharide GF-4a were belonged to the capsular polysaccharide of ST18C,providing the solid material basis for the enzymatic synthesis of the other realted oligosaccharide analogues;（3）The function of α-1,3 acetylglucosaminyltransferase of Cps18AU was explored,its application in emzymatic synthesis of trisaccharide GF-3c and tetrasaccharide GF-4b demonstrated that glycosyltransferases from different serotypes could work well together.In view of the CPS RU structures of serotype 18B,18C,and 18F are identical and only differ in the third sugar residue from serotype 18A,our results provide a new thought for exploring the serotype evolution of serogroup 18 of Streptococcus pneumoniae.（4）The glycosyltransferases studied in this thesis are essential for the synthesis of capsular polysaccharides,thus they can be used as a drug target to design glycosyltransferase inhibitors and provide new points for the development of new antibacterial drugs.