At the interface of glycolipids and glycoproteins in the two gastro-intestinal pathogens Campylobacter jejuni and Helicobacter pylori

Campylobacter jejuni and Helicobacter pylori are phylogenetically related human gastro-intestinal pathogens. C. jejuni colonizes the intestine and is the leading cause of bacterial gastroenteritis worldwide. H. pylori colonizes the stomach of half the world’s population, causing gastritis, gastric ulcers and gastric cancer. Both pathogens express glycoproteins and glycolipids (capsule, lipooligosaccharide or lipopolysaccharide (LPS)) that are important in pathogenesis. Sugar units from capsule or LPS can modify proteins in several bacteria. We reasoned that this may occur in C. jejuni and H. pylori as means to diversify their surface glycosylation and aid in colonization, immune evasion and virulence. The putative sugar nucleotide dehydratase of C. jejuni NCTC 11168, Cj1319, has predicted roles in initiating the pathway for capsular heptose modification or participating in legionaminic acid synthesis for flagellin glycosylation. Alternatively, we propose that Cj1319 may use the GDP-manno-heptose precursor normally used for capsular heptose modification in a novel protein glycosylation pathway. We determined through enzymatic reactions with GDP-manno-heptose and compositional analysis of capsule from a cj1319 knockout mutant that Cj1319 does not use GDP-manno-heptose and does not affect capsule. Furthermore, our comprehensive mass spectrometry (MS) analysis of flagellin glycopeptides showed that flagellin glycosylation is highly heterogeneous and includes modification by legionaminic acid. We determined that the cj1319 mutant glycosylates flagellins with more legionaminic acid than WT, thus excluding the direct involvement of Cj1319 in legionaminic acid synthesis. This increase in legionaminic acid correlated with higher colonization of chickens but with lower virulence in Galleria mellonella larvae. H. pylori NCTC 11637 expresses Lewis Y (Ley) blood group mimics in the O-antigen of LPS which is important in immune evasion. We determined that the outer membrane protein HopE is glycosylated with Ley through a comprehensive MS analysis of outer membrane peptides. This novel level of host mimicry is likely important for colonization and pathogenicity

Breaking Barriers: A Blended Mosaic Model of Inclusion for English Language Learners

This organizational improvement plan (OIP) presents a holistic, authentic, and proactive inclusion plan for English language learners (ELLs) at Graus Secondary School (GSS). It is framed around a problem of practice (PoP) that emerged due to an influx of newcomers to Ontario in 2016. This OIP proposes a solution to the inevitable exclusion that ELLs experience, builds on existing initiatives and offers additional support to administrators and school staff that maximizes authentic inclusion in the reception and inclusion of incoming ELLs. This solution, the author’s blended mosaic model of inclusion (BMMI), transforms the view of diversity from a fixed mosaic to a blended one that allows space for critical dialogue and authentic connection. This OIP is guided by social justice leadership and the PoP is framed by critical race theory (CRT) and critical sociocultural theory. The leadership approaches to change adopted are for a culturally responsive, social justice, and transformative leadership. Combined, these leadership approaches bridge gaps in the required conditions for equitable learning opportunities that are inclusive of and responsive to ELLs’ needs. A combination of Kang et al.’s (2020) and Deszca et al.’s (2020) change path models is used to outline the process of change. The change implementation plan outlines goals and priorities through the BMMI to close the gap between the current and desired states for ELLs. The monitoring and evaluation plan combines assessments before, during, and after change implementation, and the PDSA model, and is guided by the chosen change path model and leadership approaches

Role of Gp120 Glycosylation in Sexual Transmission of HIV

Background: In chronic HIV patients, the viral populations are genetically diverse due to mutations introduced by the viral reverse transcriptase during HIV replication. However, more than 80% new infections result from single transmission founder (TF) viruses; therefore, targeting the TFs is key to control AIDS worldwide. Gp120 is a glycosylated envelope protein required for HIV infection, propagation, and transmission. Glycans on gp120 influence HIV infectivity through their interactions with lectins, the carbohydrate-binding immune proteins in the host mucosa. To transmit sexually, viruses must overcome the lectin traps to access more target T cells. Hypothesis: TF viruses are less likely to be trapped by host lectins due to their reduced gp120 glycosylation, thus more infectious. Methods: We aim to characterize and compare the gp120 glycosylation signatures in TF and chronic HIV strains, B4 and Q0 respectively, using mass spectrometry (MS), surface plasmon resonance (SPR), and capillary electrophoresis (CE). To date, we have established a work flow to purify gp120 glycoproteins, perform MS using ETHcD methods, and analyze raw MS data using the GlycoPAT software. We are currently analyzing MS data for three replicates of B4 and the first replicate of Q0. Then we will compare the glycosylation patterns between the two strains. CE and SPR will be performed to test the glycan enrichment and functional interactions between gp120 and lectins, respectively. Discussion: Our results will provide qualitative and quantitative details about gp120 glycosylation underlying the strong infectivity of TF viruses, shedding light on new strategies to develop HIV vaccines

Protein Glycosylation in Helicobacter pylori: Beyond the Flagellins?

Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac) previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac) which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori

Effect of the polysaccharide capsule and its heptose on the resistance of Campylobacter jejuni to innate immune defenses

Abstract Campylobacter jejuni is a commensal in many animals but causes diarrhea in humans. Its polysaccharide capsule contributes to host colonization and virulence in a strain‐ and model‐specific manner. We investigated if the capsule and its heptose are important for interactions of strain NCTC 11168 with various hosts and their innate immune defenses. We determined that they support bacterial survival in Drosophila melanogaster and enhance virulence in Galleria mellonella. We showed that the capsule had limited antiphagocytic activity in human and chicken macrophages, decreased adherence to chicken macrophages, and decreased intracellular survival in both macrophages. In contrast, the heptose increased uptake by chicken macrophages and supported adherence to human macrophages and survival within them. While the capsule triggered nitric oxide production in chicken macrophages, the heptose mitigated this and protected against nitrosative assault. Finally, the C. jejuni strain NCTC 11168 elicited strong cytokine production in both macrophages but quenched ROS production independently from capsule and heptose, and while the capsule and heptose did not protect against oxidative assault, they favored growth in biofilms under oxidative stress. This study shows that the wild‐type capsule with its heptose is optimized to resist innate defenses in strain NCTC 11168 often via antagonistic effects of the capsule and its heptose