Characterisation of a novel interaction partner for Atg16L1 and its role in regulating autophagy

Macroautophagy (hereafter referred to as autophagy) is a highly conserved eukaryotic cellular response to starvation, resulting in the formation of double-membrane vesicles known as autophagosomes. These sequester cytoplasmic contents to the lysosome, where they are degraded to generate amino acids which can be used for protein synthesis during periods of nutrient deprivation. Membrane targeting of LC3, the major structural protein of the autophagosome, is essential for autophagy. LC3 is processed from its cytosolic form (LC3-I) to its lipidated form (LC3-II) in a series of ubiquitin-like conjugation events. Of particular importance is Atg16-like-1 (Atg16L1), which through its interactions with Atg5-Atg12, specifies the site of LC3 incorporation into the expanding autophagosomal membrane. In this study tools have been generated for the study and characterisation of Atg16L1 domains in cell culture. Atg16L1 -/- MEFs have also been generated and used for reconstitution experiments, which confirm that the coiled-coil domain of Atg16L1 is sufficient to restore autophagy in response to starvation and to a surrogate pathogen. A novel interaction between Atg16L1 and the serine/threonine kinase MEKK4 has been characterised in this study. Existing research has established an interaction between the pathogen recognition receptors NOD1/2 and Atg16L1. The NOD receptors are activated by bacterial peptidoglycan (PG), which has been shown to drive autophagy. Interestingly, MEKK4 inhibits NOD2 signalling by binding the downstream adaptor protein RIP2, leading to the hypothesis that MEKK4 may play a role in autophagy. The interaction was verified by co-immunoprecipitation, with domain analysis indicating that MEKK4 binds to the linker region of Atg16L1. The kinase activity of MEKK4 was also required for the interaction. The complex between Atg16L1 and MEKK4 persisted during starvation induced autophagy. Lipidation of LC3 in response to starvation required expression of MEKK4, with a redistribution of LC3 to large perinuclear aggregrates that co-localised with p62 and ubiquitin in siRNA silenced cells and CRISPR/Cas MEKK4 knockout cells. The aggregates were also able to recruit LC3 I, indicating a role of aggresome-like induced structures (ALIS). This thesis documents an interaction between Atg16L1 and MEKK4 that is vital for LC3 processing. The potential role for MEKK4 in regulating autophagy through Atg16L1 is discussed and experiments proposed to further explore this interaction in the context of autophagy

Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus

Ruminococcus gnavus is a human gut symbiont which ability to degrade mucins is mediated by an intramolecular trans-sialidase (RgNanH). RgNanH comprises a GH33 catalytic domain and a sialic acid binding carbohydrate binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis, and binding assays to determine the structure and function of RgNanH_CBM40 (RgCBM40). RgCBM40 displays the canonical CBM40 b-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. RgCBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that RgCBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut

The role of the mucin-glycan foraging Ruminococcus gnavus in the communication between the gut and the brain

Ruminococcus gnavus is a prevalent member of the human gut microbiota, which is over-represented in inflammatory bowel disease and neurological disorders. We previously showed that the ability of R. gnavus to forage on mucins is strain-dependent and associated with sialic acid metabolism. Here, we showed that mice monocolonized with R. gnavus ATCC 29149 (Rg-mice) display changes in major sialic acid derivatives in their cecum content, blood, and brain, which is accompanied by a significant decrease in the percentage of sialylated residues in intestinal mucins relative to germ-free (GF) mice. Changes in metabolites associated with brain function such as tryptamine, indolacetate, and trimethylamine N-oxide were also detected in the cecal content of Rg-mice when compared to GF mice. Next, we investigated the effect of R. gnavus monocolonization on hippocampus cell proliferation and behavior. We observed a significant decrease of PSA-NCAM immunoreactive granule cells in the dentate gyrus (DG) of Rg-mice as compared to GF mice and recruitment of phagocytic microglia in the vicinity. Behavioral assessments suggested an improvement of the spatial working memory in Rg-mice but no change in other cognitive functions. These results were also supported by a significant upregulation of genes involved in proliferation and neuroplasticity. Collectively, these data provide first insights into how R. gnavus metabolites may influence brain regulation and function through modulation of granule cell development and synaptic plasticity in the adult hippocampus. This work has implications for further understanding the mechanisms underpinning the role of R. gnavus in neurological disorders

Chronic TNFα-driven injury delays cell migration to villi in the intestinal epithelium

The intestinal epithelium is a single layer of cells which provides the first line of defence of the intestinal mucosa to bacterial infection. Cohesion of this physical barrier is supported by renewal of epithelial stem cells, residing in invaginations called crypts, and by crypt cell migration onto protrusions called villi; dysregulation of such mechanisms may render the gut susceptible to chronic inflammation. The impact that excessive or misplaced epithelial cell death may have on villus cell migration is currently unknown. We integrated cell-tracking methods with computational models to determine how epithelial homeostasis is affected by acute and chronic TNFα-driven epithelial cell death. Parameter inference reveals that acute inflammatory cell death has a transient effect on epithelial cell dynamics, whereas cell death caused by chronic elevated TNFα causes a delay in the accumulation of labelled cells onto the villus compared to the control. Such a delay may be reproduced by using a cell-based model to simulate the dynamics of each cell in a crypt-villus geometry, showing that a prolonged increase in cell death slows the migration of cells from the crypt to the villus. This investigation highlights which injuries (acute or chronic) may be regenerated and which cause disruption of healthy epithelial homeostasis

Role of mucin glycosylation in the gut microbiota-brain axis of core 3 O-glycan deficient mice

Abstract Alterations in intestinal mucin glycosylation have been associated with increased intestinal permeability and sensitivity to inflammation and infection. Here, we used mice lacking core 3-derived O-glycans (C3GnT−/−) to investigate the effect of impaired mucin glycosylation in the gut-brain axis. C3GnT−/− mice showed altered microbial metabolites in the caecum associated with brain function such as dimethylglycine and N-acetyl-l-tyrosine profiles as compared to C3GnT+/+ littermates. In the brain, polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive granule cells showed an aberrant phenotype in the dentate gyrus of C3GnT−/− mice. This was accompanied by a trend towards decreased expression levels of PSA as well as ZO-1 and occludin as compared to C3GnT+/+. Behavioural studies showed a decrease in the recognition memory of C3GnT−/− mice as compared to C3GnT+/+ mice. Combined, these results support the role of mucin O-glycosylation in the gut in potentially influencing brain function which may be facilitated by the passage of microbial metabolites through an impaired gut barrier

Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut.

Sialic acid (N-acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus. R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans-sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan-cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that-in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains-the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease