Analysis Of The Biosynthetic Pathway Of The Key Group 2-acetamido-4-aminomethyl-fucose In Bordetella Pertussis Lipopolysaccharide

Bordetella pertussis is a Gram-negative bacteria that causes illnesses in the respiratory tract.Lipopolysaccharide can be used as an antigenic component in pertussis vaccines because it is a critical pathogenicity ingredient in B.pertussis.The LPS of B.pertussis consists of three main components:Lipid A,core polysaccharide and terminal trisaccharide.The presence or absence of the terminal trisaccharide is important for vaccinations that use LPS as antigen.However,biosynthesis pathway of FucNAc4NMe in the terminal trisaccharide is unknown and must be investigated further.In this study,the genes linked to the biosynthesis of FucNAc4NMe in the LPS of B.pertussis CS were studied and the synthesis of FucNAc4NMe in Escherichia coli(E.coli)was discovered using SDS-PAGE,LC-MS,HPAEC-PAD and NMR.In this way,a putative biosynthetic transfer pathway of FucNAc4NMe in B.pertussis was postulated.The following are the main findings:(1)Analysis of the terminal trisaccharide biosynthesis bpl gene cluster of B.pertussis CS by bioinformatics methods.Four genes may be associated to FucNAc4NMe synthetic transport,according to the results of protein homology comparison and conserved structural domains of enzyme proteins.BplF has a high homology with 4-aminotransferase SpsC of B.subtilis.BplL has high homology with dehydrase WbpM of P.aeruginosa PAO1 and isomerase PglF of C.jejuni.BplG was identified as a glycosyltransferase.The Amj enzyme protein,which belongs to the same superfamily as BplI,can compensate for the loss of the MurJ enzyme protein in E.coli.(2)Suitable host E.coli strains were selected for the biosynthesis of FucNAc4NMe.Exogenous expression of bplF,bplG,bplL and bpll in MDCO001/pW produced LPS with novel structures,indicating that the exogenously expressed products may be attached to Core-LipidA in engiengineering E.coli.Exogenous genes did not adversely affect growth of expressed strain.The monosaccharide composition analysis of LPS revealed a new product peak at 2.5 min.FucNAc4NMe structural analogue may be caused by interference of ECA and CA.As a result,E.coli MDCO020/pW,which was created by deleting CA and ECA biosynthesis-related genes from E.coli MDCO001/pW,was selected as the host strain for exogenous expression of B.pertussis CS FucNAc4NMe.(3)We created a series of transferase BplG expression plasmids containing one,two or three identified biosynthesis genes for expression in E.coli MDCO020/pW.SDS-PAGE analysis of the LPSs of all expressed strains revealed that BplL plays a key role in the production of FucNAc4NMe.FucNAc4NMe and its intermediates produced during biosynthesis are recognized and transferred by transferase BplG.LPS extracted from MDCO020/pWpBR 1 was closest to FucNAc4NMe.HPAEC-PAD analysis of MDCO020/pWpBR1 core oligosaccharide indicated that interference seen during structural analysis was due to ECA and CA,and that peak seen at 2.5 min was the exogenous expression product.NMR analysis was used to show that FucNAc4NMe is synthesized in E.coli.In summary,a biosynthetic transport pathway for FucNAc4NMe was summarized.