Remodeling The Chain Length Of The O-polysaccharides In The N-glycosylation System In Escherichia Coli

As a kind of human gastroenteric pathogen, Campylobacter jejuni is one of the main causes of diarrhea. In 1999, Szymanski et al. found a pgl cluster which encodes the N-glycosylation pathway in the genome of C. jejuni. The N-glycosylation pathway of C. jejuni has significant similarities with the Wzy-dependent synthesis pathway of O-polysaccaride (O-PS) in Escherichia coli. In the cytoplasm, different kinds of glycotransferases sequentially add monosaccharides to a lipid carrier undecaprenol to form the oligosaccharide precursor. The flippase then translocates the oligosaccharide precursor from cytoplasm side to the periplasm side, where the bacteria oligosaccharyltransfase Pg1B transfers the oligosaccharide to the protein acceptor (in C. jejuni), or the oligosaccharide precursor is first polymerized into O-PSs and then transferred to lipid A core by the O-antigen ligase WaaL (in E. coli).It has been well documented that Pg1B could catalyze the transglycosylation reactions between the O-PSs of several serotypes and the protein accaptor AcrA in a WaaL deletion mutant E. coli. Therefore, the waaL gene in E. coli O86:K61:B7 was knocked out to obtain the recombinant strain B712, and then pglB and acrA were cloned into B712. We found that Pg1B transferred the O-PSs to AcrA to produce glycosylated AcrA with the O-PSs of O86:B7 (AcrA-O-PS). Thus, with the bacterial OST Pg1B, an N-glycosylation system has been established in E. coli O86:B7.In the Wzy-dependent synthesis of O-PS, the length of O-PS is regulated by the O-antigen length determinator Wzz. Each Wzz homologue individualizes its host with a specific length of O-PS. O86:B7 possesses O-PSs in short chain length, while O86:H2 has O-PSs in intermediate chain length. After WzzB7 being substituted with WzzH2, the chain length of O-PS linked to AcrA was remarkable increased as we had predicted. Therefore, we remodeled the chain length of the O-PS in this N-glycosylation system by introducing a Wzz homologue.The glycoprotein with O-PSs harbored in our N-glycosylation system could be used as sugar-conjugated vaccines to prevent bacterial infections. So next, the sugar acceptor was replaced from the model protein AcrA to the extensively used carrier proteins-tetanus toxin C fragment (TTc), dipheria toxoid mutant CRM197, and their mutants in which the Pg1B glycosylation sites were introduced. We called this new strategy of producing sugar conjugated vaccines "One-shot Biosynthesis Approach".The N-terminal of Pg1B is predicted to have 11 transmembrane (TM) a-Helixes by the software TMHMM. We constructed a series of truncated Pg1Bs (tPglBs), with the deletion of the 1st,1st-5th,1st-8th,1st-11th a-Helixes, respectively. Detected by our N-glycosylation system, all of these tPg1Bs lose the transglycosylation activity, indicating that the N-terminal domain has important roles to maintain Pg1B's function.In summary, our E. coli N-glycosylation system could be used as a new method to biosynthesize sugar-conjugated vaccines, which could effectively trigger TD immune response and prevent human from bacterial infections. Multifermentation and the high cost limit the application of the widespread chemosynthesis of sugar-conjugated vaccines, while the biosynthesis stratigy has certain advantages such as one pot fermentation and the high product quality. As a platform, our new system could be further improved to obtain N-glycoproteins which are linked with a variaty of O-PSs.