N-linked protein glycosylation in Campylobacter and Helicobacter species

Abstract

N-linked protein glycosylation is the enzymatic transfer of a carbohydrate glycan toan asparagine residue of a polypeptide, catalysed by an N-oligosaccharyltransferase(OTase). Bacterial N-glycosylation is best understood in the foodborne pathogenCampylobacter jejuni, in which a heptasaccharide glycan is built at cytoplasmic face ofthe inner membrane, flipped to the periplasm and transferred to a polypeptide enbloc. C. jejuni encodes each of the proteins required for the N-glycosylation pathwayin a single genetic region, termed the pgl locus. Homologues of the gene encoding theC. jejuni OTase, PglB, are found in all Campylobacter species, three Helicobacterspecies, and more distantly related ε- and delta-Proteobacteria species such as Wolinellasuccinogenes, Desulfovibrio desulfuricans and Nitratiruptor tergarcus. A small numberof Campylobacter species and all three pglB-containing Helicobacter species have twodistinct pglB genes, pglB1 and pglB2, along with homologues of other C. jejuni pglgenes. The work presented in this thesis investigated the N-glycosylation system of abacterial species encoding two distinct PglBs, C. concisus. The roles of the two PglBenzymes in C. concisus were investigated using an in vitro OTase assay, and thestructure of a C. concisus N-glycan elucidated by mass spectrometry. The work in thisthesis also expands our knowledge of C. jejuni N-glycosylation by investigating the fullscope of N-glycosylation using an in silico method to predict the total C. jejuni Nglycoproteome.This was followed by experimental validation of these predictions, inwhich three novel C. jejuni N-glycoproteins were identified, bringing the number ofreported C. jejuni NCTC 11168 N-glycoproteins to 57. One of these novel Nglycoproteins,Cj0633, is the most extensively N-glycosylated bacterial glycoproteinreported to date, with the addition of up to eight N-glycans when expressed in thepresence of the C. jejuni pgl machinery. In the final investigation presented in thisthesis, a method to identify bacterial N-glycoproteins using anti-glycan antisera toimmunoprecipitate N-glycoproteins was developed. These data expand ourknowledge of C. concisus N-glycosylation and provide valuable insight into the fullscope of N-glycosylation in C. jejuni.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	James Linton (Supervisor) & Jeremy Derrick (Supervisor)