Capítulo II: Estructura Económica del Sector
2.3 Análisis Estructural del Sector Industrial
As discussed, C. jejuni NCTC 11168 has several glycosylation pathways that have been extensively studied. However, there are still missing links in these pathways, including the OTases for flagellin glycosylation, and many genes within the annotated glycosylation islands are of yet unknown functions. Cj1319 is a putative sugar nucleotide dehydratase which has potential roles in both protein glycosylation and capsule synthesis.
In H. pylori, we and others have shown that novel glycoproteins other than the well characterized flagellin glycoproteins exist (Champasa et al., 2013; Hopf et al., 2011). Additionally, the pathway for LPS synthesis has been recently deciphered and signifies an evolutionary connection between N-protein glycosylation and LPS biosynthesis (Hug et al., 2010). Combined with the fact that the H. pylori LPS O-antigen represents human Lewis blood group mimics and this O-antigen has an important role in both host evasion and immune modulation through molecular mimicry, it would not be surprising if H. pylori used these host mimicking structures for protein glycosylation as well.
Based on the above, we hypothesize that there are links between the machineries used for glycosylation of lipids and proteins in C. jejuni and H. pylori, whereby the sugar precursors of glycolipids or O-units can serve for protein glycosylation to generate glycoproteins that have not been identified to date. These novel glycoproteins may have important roles in virulence and would also contribute to the heterogeneity of glycosylated structures found on the cell surface.
To address this main hypothesis, we have two major objectives. Our first goal is to investigate a relationship between capsule synthesis and protein glycosylation in C. jejuni through the study of the enzyme Cj1319, a sugar nucleotide dehydratase, which has predicted roles in both protein glycosylation and capsule synthesis. We also aim to determine the contribution of this enzyme and its predicted glycosylation pathway in colonization and virulence. Secondly, our purpose is to show that H. pylori can glycosylate proteins with the O-antigen of its LPS by MS analysis of glycoprotein candidates to show that their glycopeptides contain O-antigen in addition to identifying the glycosylation site(s).
In chapter 3 of this thesis, sections 3.1-3.4 aim to examine the role of Cj1319 in C. jejuni and its contribution to colonization and virulence. Our hypothesis is Cj1319 is sugar nucleotide dehydratase participating in a novel protein glycosylation pathway with a link to capsule modified heptose synthesis through the use of a common precursor and contributes to the virulence of C. jejuni NCTC 11168. To address this specific hypothesis, we have three main objectives:
1) To determine the enzymatic function of Cj1319 by determining its substrate and product.
Determining the enzymatic function of Cj1319 is a key piece of information that will aid greatly in determining which glycosylation pathway(s) this enzyme functions within. We assessed its activity on GDP-manno-heptose to determine if it has a role in capsular heptose modification or if it shares the said precursor of this pathway. Additionally, we tested its activity on GDP-mannose, as there is functional similarity between heptose and hexose modifying enzymes. We controlled positively for its activity using GDP-GlcNAc which was described as the substrate of Cj1319 within the in vitro legionaminic acid synthesis pathway (Schoenhofen et al., 2009).
2) To show legionaminic acid decorates WT flagellins and investigate its presence in the cj1319::CAT mutant to determine if Cj1319 is involved in legionaminic acid synthesis.
Here we address the controversial role of Cj1319 in legionaminic acid synthesis through mass spectrometry analysis of flagellin glycopeptides from the WT, a cj1319::CAT mutant, and its complemented strain. Since limited flagellin glycopeptide data was available for our strain of interest at the onset of our work and legionaminic acid was only implied but not demonstrated to glycosylate the flagellins of C. jejuni, we performed a comprehensive flagellin glycopeptide analysis using LC-HCD-MS2 with an emphasis on determining the presence of legionaminic acid on the flagellins of our strains.
3) To determine phenotypes associated with the cj1319::CAT mutant to shed light on its biological activity.
In order to understand the importance of Cj1319 and the role of the glycosylation pathway this enzyme may participate in on C. jejuni and on its interactions with host cells, we examined the composition of the capsule and LOS of cj1319::CAT mutant, in addition to this mutant’s glycoprotein profile compared to WT. We further investigated functions of C. jejuni that are affected by glycosylated structures including motility. In addition, we studied the interaction of the WT and our mutant strain with several eukaryotic cells. Lastly, we examined the role of Cj1319 in vivo, specifically in chicken colonization and infection in an insect model.
Chapter 3, section 3.5 aims to identify a novel glycoprotein from H. pylori NCTC 11637 that is modified by a Lewis O-antigen. We hypothesize that H. pylori synthesizes the O- antigen Ley for protein glycosylation, specifically for outer membrane proteins which can interact with the host, in addition to incorporation into its LPS. For this we have three specific objectives:
1) To isolate Ley-modified peptide candidates from outer membranes.
Previous preliminary data from our laboratory indicates that at least one Ley- modified protein is found in the outer membrane. Since the outer membrane proteins copurify with LPS by ultracentrifugation during cell fractionation, we trypsinolyzed the outer membranes to release soluble peptides and glycopeptides into the supernatant. These peptides were passaged through a fucose binding lectin column to enrich for Ley containing peptides.
2) To analyze glycopeptides by mass spectrometry for the Ley modification.
In order to identify the Ley-modified protein, we analyzed the peptide samples with LC-HCD-MS2. Upon identification of the glycopeptide, we continued to analyze the sample with LC-CID/ETD-MS2 in order to map the glycosylation site(s).
3) To verify Ley-modified glycoproteins identified by mass spectrometry through
knockout mutagenesis and Western blot analysis using anti-Ley.
To verify that the glycoprotein identified in objective 2 is the Ley-modified protein candidate observed in our preliminary data, we constructed a knockout mutant of the gene encoding the glycoprotein identified. Anti-Ley Western blot was used to verify that the Ley glycoprotein candidate originally found in the WT is not present in the mutant.