Study On Gene Clusters Of Neisseria Meningitidis Capsular Antigen And Development Of Genotyping Method

INeisseria meningitidis is an obligate parasite to human and gramnegative. It causes meningitis and septicemia. It has long been a heavyburden to people’s health for the high rates of neural sequelae, mortalityand morbidity. It is necessary to carry out extensive research on Neisseriameningitidis in consideration of the importance. This bacterium transmitsthrough aerosol, droplet and close contact. The respiratory tract is usuallythe first to be exposed to this bacterium. About10percent of people carryNeisseria meningitidis asyptomatically in their throats. Once thebacterium gains access to blood and cerebral spinal fluid across thebarriers, it will cause severe infectious disease.The extracellular capsule surrounding Neisseria meningitidis is avirulent factor to the pathogenecity. Meningococcal capsularpolysaccharides have been classified into twelve distinct serogroups: A, B, C,29E, H, I, K, L, W-135, X, Y and Z based on capsularpolysaccharide components and immunological reactivity. Serogroups A,B, C, W-135, X and Y caused more than90%of the infections, belongingto common serogroups. Others are considered as rare serogroups thatonly cause sporadic infections or only in the carrier state. The commonserogroups have been better documented than the rare ones. The capsulargene sequences of serogroups A, B, C,29E, W-135, X and Y areavailable in the database of the GenBank. However, the capsular geneclusters of serogroups H, I, K, L and Z have not yet been characterizedtill now, to which extent, it badly impedes the vaccine design againstcapsule, serological detection for the prophylaxis or capsularevolutionary research. Therefore it is necessary to characterize thecapsular gene clusters of serogroups H, I, K, L and Z and analyze thegene functions, especially for the genes of transportation and biosynthesis.Moreover, the capsular sequences are also of importance of species and serogroup detection.In this study, the shot-gun bank was constructed. The capsular geneclusters of serogroups D, H, I, K, L and Z were characterized andsequences of23,488,24,676,27,709,18,453and21,871bp wereobtained respectively, with corresponding19,18,22,14and15orfsrespectively. The sequence identity of serogroups I and K was99%through the comparison of the capsular gene clusters by Clustal X,though the components of capsular polysaccharides varied. An isomerasegene was predicted to be outside the capsular gene cluster of serogroup I.The common Glycerol-3-PO4is present in the capsular polysaccharide ofserogroups H and Z and the capsular gene clusters are also similar inthese two serogroups. The present study also provided insight into theoverall evolutionary features of the capsular gene clusters by comparisonof the sequences and gene organization.In another part of this study was to develop a multiplex PCR assay to detect12serogroups, utilizing serogroup-specific and species-specificgene. Three multiplex PCRs were to be used consecutively to detect allserogroups and serologically nongroupable strains by means of thegenetic method. The rare serogroups were able to be effectivelydistinguished from the nongroupable in the process of detection. EachNeisseria meningitidis strain to be tested would able to produce threeamplicons: one was used for serogroup identification by thedistinguishable size; the other two were together utilized for speciesconfirmation to exclude the phylogenetically closed Neisseriagonorrhoeae and Neisseria lactamica.The property of multiplex PCR assay for serogroup detection wasevaluated with specificity and sensitivity.97Neisseria meningitidisstrains were used for specificity evaluation. The final result was100%inspecificity.46strains with unknown serogroup were used fordoubled-blind test in multiplex PCR assay. Finally the PCR results were compared with the serological serogrouping results and the specificitywas found to be98%.The sensitivity on genomic DNA and bacteria amount in thenoncultured mock clinical CSF sample was determined after numerousrepeat trials. The sensitivity of genomic DNA was determined at1ng/20μL for all the serogroups, equivalent to～4×105genomes. Thesensitivity was～3×105CFU/ml in the noncultured mock CSF sample.Therefore this assay was able to be used directly on the serogroupdetection with the clinical CSF sample or genomic DNA.IIEscherichia coli，a Gram-negative bacterium, is a representative ofEscherichia family. It distributes widely in the nature and acts as anormal bacteria population in the gut. Escherichia coli do not lead todisease in most of the cases or lead to disease conditionally. There are three forms of extracellular antigens of Escherichia coli, i.e. somatic (O),capsular (K) and flagellar (H) antigens. Because a variety oflipopolysaccharide structures is present externally, more than180serotypes of Escherichia coli can be detected only in the scheme of Oantigen.Escherichia coli cause intestinal tract, mucosa and urogenital tractinfections under the circumstance of low immunogenicity as well as thediseases of live stocks, such as pigs, cows and sheep. The extracellullarlipopolysaccharide is the primary surface components and plays animportant role in the pathogenicity, consisting of three covalently linkedparts: core oligosaccharide, O-specific side chain and lipid A. O-specificsaccharide chain is also called O antigen polysaccharide side chain withO antigen as the short form. O antigen is diverse, stable and specific. Thegenes responsible for the biosynthesis of O antigen generally locate onthe chromosome linearly. In E. coli, the gene cluster of O antigen synthesis commonly lies between the genes of galF and gnd, with theexception of an extreme minority of bacteria.Since O antigen is of importance in pathogenicity and evolutionaryresearch, the present study characterized the gene cluster sequence and Oantigen polysaccharide structure of E. coli O41.12open reading frameswere annoted from the sequence analysis, including GDP-L-Fucbiosynthesis genes (gmd, fcl, gmm, manC and manB), glycotransferasegenes (wfcV, wfcW, wfcX, wfcY and wfcZ) and O unit processing genes(wzy and wzx). The biosynthesis of O antigen is Wzy/Wzx-dependent.The polysaccharide structure is as follows:Through the comparison with the already known structures of O antigenpolysaccharides, the O antigen structure of E. coli O41is unique among the bacteria polysaccharides and no same style has ever been found.