Mutagenesis And Immunological Evaluation Of GAS C5a Peptidase As An Antigen For Vaccine And As A Carrier Protein For Glycoconjugate Vaccine And Study On GBS β-1,4-Galactosyltransferase

Streptococcus is a group of Gram-positive bacteria belonging to the Streptococcus Genus,Streptococcaceae Family.These bacteria divide along an axis,so they usually grow in pairs or chains.Because of these characteristics,they are called streptococcus,and are distinct from staphylococcus,which can divide along multiple axes and form a mass of cells.Streptococcus contains many species which widely exist in the environment and human body,and some of them are beneficial to human species whereas some are pathogenic,causing a number of diseases such as pink eyes,meningitis,bacterial pneumonia,endocarditis,erysipelas,necrotizing fasciitis,etc.Streptococcus can be classified into three hemolytic types,α,β and γ.The a hemolytic streptococcus can produce hydrogen peroxide and oxidize erythrocyte hemoglobin to produce methemoglobin.The β hemolytic streptococcus,also called complete hemolytic streptococcus,can secrete an exotoxin,the streptococcus hemolysin,which can completely rupture red blood cells.The γ streptococcus does not cause hemolysis.Clinically,the most important pathogenic bacteria are a hemolytic streptococci,Streptococcus pneumoniae and S.viridians,and β hemolytic streptococci,S.pyogenes and S.agalactiae,also known as group A and B streptococcus(GAS and GBS).In this thesis,we focus on GAS and GBS.GAS can cause four main types of diseases:superficial infection including pharyngotonsillitis,impetigo,erysipelas,vaginitis or post-partum infections;deep infections including bacteremia,cellulitis,myositis,necrotizing fasciitis,puerperal sepsis,pericarditis,meningitis,pneumonia or septic arthritis;toxin-mediated diseases including scarlatina and toxic shock-like syndrome;immunologically mediated diseases including rheumatic fever,post-streptococcal glomerulonephritis and reactive arthritis.It is estimated that more than 700 million people suffer from GAS infections each year,giving rise to over 500,000 annual deaths.The estimated cost of GAS pharyngitis among children is up to US$220-540 million every year in the United States alone.C5a peptidase is an important GAS virulence factor and has attracted significant attention in the development of GAS vaccines.In this thesis,a truncated form(amino acid residues 32-1032)of GAS C5a peptidase(ScpA)and a serial of its mutants,including H193A,D130A,S512A,N295A and D130A/S512A,were expressed and studied to find a suitable candidate for GAS vaccine development and as a carrier protein of glycoconjugate vaccines.Enzymatic studies on these recombinant proteins with a MALDI-TOF MS-based method to analyze the reaction showed that amino residues D130 and N295 may not play a critical role in the activity of ScpA.While S512 was proved to be important,it may not be absolutely required for the activity either.Since the △ScpAD130A,△ScpAN295A and △ScpAS512A mutants still retained some enzymatic activities,they were not suitable for being used as a vaccine or carrier protein.It was discovered,however,that a single mutation of amino acid residue H193 to Ala could completely abolish the enzymatic activity of ScpA.The △ScpAH193A mutant was therefore identified as a potential candidate to be subjected to immunological studies in mouse.It was revealed that △ScpAH193A elicited high titers of antigen-specific IgG1 antibodies and robust T cell-mediated immunities,suggesting its potential in the development of ScpA-based GAS vaccine.Moreover,it was discovered that conjugating the trisaccharide repeating unit of a GAS polysaccharide with △ ScpAH193A converted this immunologically inactive oligosaccharide into a highly immunogenic and T cell-dependent antigen,demonstrating the potential of △ScpAH193A as a carrier protein to help formulate robust glycoconjugate vaccines.In general,GBS is harmless and a part of the human microbiota colonizing the gastrointestinal and genitourinary tract.However,GBS can sometimes cause severe invasive infections,especially in pregnant women,newborns,and immunocompromised people.GBS is surrounded by a bacterial capsule composed of capsular polysaccharide(CPS)and exopolysaccharide.Depending on different structures of their CPSs,GBS is subclassified into ten serotypes(Ⅰa,Ⅰb,Ⅱ-Ⅸ).CPS is an important virulent factor of GBS.Moreover,they are located on the outer surface of bacterial cells,and their structures are conserved.Therefore,CPS is a potential target for the development of GBS vaccines.The biosynthesis of CPS is catalyzed by a series of synthases,glycosyltransferases,and polymerases.In many serotypes,the CPSs contain β-1,4-linked galactose,so β-1,4-galactosyltransferase is necessary to catalyze the synthesis of Galβ-1,4-Glc-pp-Und as the initial biosynthetic step of CPS.As a result,β-1,4-galactosyltransferase can be an excellent target for the development of enzyme inhibitor-based antibiotics or as a tool enzyme for the synthesis of various CPSs or their reapeting units useful for biological studies of PSs and vaccine design.In this research,we synthesized the whole gene of Cps4G,which has 542 bp,and the target Cps4G should contain 177 amino acid residues.Its theoretical pI and Mw are expected to be 5.64 and 20548.17,respectively.Cps4G was expressed in E.coli BL21(DE3)and isolated.The purified protein had two bands on SDS-PAGE with molecular weights of 20 kDa and 40 kDa.We analyzed these two bands by Western blot,MALDI-TOF-MS,and N-adman sequence.They were identified as target protein monomer and dimer,which could not be completely separated and could be transformed into each other.In addition,with the extension of time,the dimer tends to increase in proportion.The activity of the enzyme was aslso evaluated using glucose,4-aminophenyl b-D-glucose and N-acetyl-D-glucosamine as substrates.It was found that the protein had a high substrate specificity and the phospholipid moiety of the receptor Glc-pp-und might play a key role in the recognition of the substrate,as the enzyme could not accept substrates without this moiety.At the same time,we also probed the suitable conditions for this protein to crystallize.Under the optimized conditions,we obtained the protein crytals and performed X-ray diffraction analysis which gave 3 A resolution.In the next step,we will collaborate with other labs to resolve the 3D structure of the enzyme.This inofmration should be very useful for understanding the active center of this enzyme and optimize its catalytic activity,so as to help the design of enzyme inhibitors for the treatment of related diseases and help the development of a tool enzyme for the synthesis of CPSs and related oligosaccharides.