Biology subjects

Wall, D. M.

Publications and source records attributed to Wall, D. M..

13 recordsLinked to original sources

bioRxiv2025

Inhibition of systemic mammalian metabolism by carnitine mimics from the gut microbiota

BackgroundThe gut microbiota and microbiome-derived metabolites are implicated in various aspects of human health. Here we sought to determine the systemic effects, and mechanism of action, of microbiome-derived carnitine analogues in germ free and conventionally colonised mice. ResultsHere we report the systemic localization of the microbiome-derived carnitine analogues, 3-methyl-4-(trimethylammonio)butanoate (3M-4-TMAB) and 5-aminovalerate betaine (5-AVAB), post-administration to germ free mice, with systemic carnitine depletion and mitochondrial dysregulation, reflected in altered acylcarnitine profiles due to incomplete carnitine-mediated fatty acid oxidation. Studies on the inhibitory potency of 3M-4-TMAB at the enzymatic, cellular, and organism levels indicate that, in part, this is a result of inhibition of gammabutyrobetaine hydroxylase, which catalyses the final step in carnitine biosynthesis. Systemic administration of 13C- labelled 3M-4-TMAB to conventionally colonised animals to further investigate the physiological relevance of this inhibition, identified a significant reduction in systemic carnitine levels due to increased excretion in urine and faeces and disruption of carnitine-mediated metabolism across ten organs. ConclusionsThese results highlight the physiological relevance and significance of microbiome-derived metabolites in vivo. The depletion of carnitine and inhibition of its function have potentially long-term impacts on both mammalian energy generation and the protective, signalling and immune regulatory effects of this critical molecule.

microbiology↗

bioRxiv2025

Mapping gene expression across the microbiome-gut-brain axis of germ- and specific pathogen-free mice

Perturbation of the gut microbiota has been implicated in neurological diseases via communication across the microbiome-gut-brain axis. As a result, the discovery of mechanisms underlying interaction across this axis are becoming increasingly important. The germ-free (GF) mouse model has enabled an improved understanding of the influence of the gut microbiota on brain development and function. By utilising an advanced spatial profiling approach, we determined transcriptional changes in the brain, improving our understanding of how brain cells function and interact within their microenvironment in the absence of microbiome influence. Targeted regions of interest were selected based on brain regions implicated in neurological disease or reported structural differences between GF mouse brains and those of colonized mice. In the hippocampus 276 differentially expressed genes (DEGs) were identified, 345 DEGs in the thalamus, and 21 DEGs in pons. Contrastingly we identified only 2 DEGs in the midbrain and 4 in the medulla oblongata, with no DEGs in the cerebellum or corpus callosum. This data provides an overview of gut microbiota influence on gene expression in the brain, highlighting multiple genes of interest for further investigation in the context of microbiome influence on brain function and their potential relevance to neurological disease.

microbiology↗

bioRxiv2024

Spatial mapping of dextran sodium sulphate-induced intestinal inflammation and its systemic effects

Inflammatory bowel disease (IBD) is a multifactorial disease and patients frequently experience extraintestinal manifestations affecting multiple sites. Causes of systemic inflammation remain poorly understood but molecules originating from the intestine likely play a role, with microbial and host small molecules polarizing host immune cells towards a pro- or anti-inflammatory phenotype. Using the dextran sodium sulphate (DSS) mouse model, which mimics the disrupted barrier function, microbial dysbiosis and immune cell dysregulation of IBD, we investigated metabolomic and phenotypic changes at intestinal and systemic sites. Using spatial biology approaches we mapped distribution and relative abundance of molecules and cell types across a range of tissues revealing significant changes in DSS-treated mice. Molecules identified as contributing to the statistical separation of treated from control mice were spatially localized within organs to determine their effects on cellular phenotypes through imaging mass cytometry. This spatial approach identified both intestinal and systemic molecular drivers of inflammation, including several not previously implicated in inflammation linked to IBD or the systemic effects of intestinal inflammation. Metabolic and inflammatory pathway interplay underpins systemic disease and determining drivers at the molecular level may aid the development of new targeted therapies.

molecular biology↗

bioRxiv2024

Characterisation of prophages in Clostridium clostridioforme: an understudied component of the intestinal microbiome

Genome sequencing of Clostridium clostridioforme strain LM41 revealed the presence of an atypically high proportion of mobile genetic elements for this species, with a particularly high abundance of prophages. Bioinformatic analysis of prophage sequences sought to characterise these elements and identify prophage-linked genes contributing to enhanced fitness of the host bacteria in the dysbiotic gut. This work has identified 15 prophages, of which 4 are predicted to be intact, 2 are predicted to be defective, and 9 are unclassified. qPCR analysis revealed spontaneous release of four of the LM41 prophages into the culture supernatant, the majority of which had morphology akin to podoviruses when visualised using Transmission Electron Microscopy. We observed diversity in the lysogeny mechanisms utilised by the prophages, with examples of the classical {lambda}-like CI/Cro system, the ICEBs1 ImmR/ImmA-like system, and the Mu-like C/Ner system. Classical morons, such as toxins or immune evasion factors, were not observed. We did, however, identify a variety of genes with roles in mediating restriction modification and genetic diversity, as well as some candidate genes with potential roles in host adaptation. Despite being the most abundant entities in the intestine, there is a dearth of information about phages associated with members of the microbiome. This work begins to shed light on the contribution of these elements to the lifestyle of C. clostridioforme LM41.

microbiology↗

bioRxiv2024

Microbiome-derived metabolite effects on intestinal barrier integrity and immune cell response to infection

The gut microbiota exerts a significant influence on human health and disease. While compositional changes in the gut microbiota in specific diseases can easily be determined, we lack a detailed mechanistic understanding of how these changes exert effects at the cellular level. However, the putative local and systemic effects on human physiology that are attributed to the gut microbiota are clearly being mediated through molecular communication. Here we determined the effects of a number of gut microbiome-derived metabolites on the first line of defence in the gut. Using in vitro models of intestinal barrier integrity, and studying the interaction of macrophages with pathogenic and non-pathogenic bacteria, we could ascertain the influence of these metabolites at the cellular level at physiologically relevant concentrations. Many metabolites exerted competing influences on intestinal epithelial or immune cells, specific metabolite effects were noted on barrier function, polarised cytokine release and the lifespan of metabolite treated cells. Our findings reiterate the complexity of understanding microbiome effects on host physiology with many metabolites having contrasting effects on host cells. However, our results underline that microbiome metabolites are crucial mediators of barrier function and the innate response to infection. Understanding the effects of these metabolites at the cellular level will allow us to move towards a better mechanistic understanding of microbiome influence over host physiology, a crucial step in advancing microbiome research.

microbiology↗

bioRxiv2024

Genomic diversity of novel strains of mammalian gut microbiome derived Clostridium XIVa strains is driven by mobile genetic element acquisition

Despite advances in sequencing technologies that enable a greater understanding of mammalian gut microbiome composition, our ability to determine a role for individual strains is hampered by our inability to isolate, culture and study such microbes. Here we describe highly unusual Clostridium XIVa group strains isolated from the murine gut. Genome sequencing indicates that these strains, Clostridium symbiosum LM19B and LM19R and Clostridium clostridioforme LM41 and LM42, have significantly larger genomes than most closely related strains. Genomic evidence indicates that the isolated LM41 and LM42 strains diverge from most other Clostridium XIVa strains and supports reassignment of these groups at genus-level. We attribute increased C. clostridioforme LM41 and LM42 genome size to acquisition of mobile genetic elements including dozens of prophages, integrative elements, putative group II introns and numerous transposons including 29 identical copies of the IS66 transposase, and a very large 192 Kb plasmid. antiSmash analysis determines a greater number of biosynthetic gene clusters within LM41 and LM42 than in related strains, encoding a diverse array of potential novel antimicrobial compounds. Together these strains highlight the potential untapped microbial diversity that remains to be discovered within the gut microbiome and indicate that, despite our ability to get a top down view of microbial diversity, we remain significantly blinded to microbe capabilities at the strain level.

microbiology↗

bioRxiv2024

Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohns disease associated adherent-invasive Escherichia coli infection

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohns disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNF), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

microbiology↗

bioRxiv2022

Inhibition of proline tyrosine kinase 2 (Pyk2) phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohns Disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease through genome wide association studies. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while an inhibitor of Pyk2, PF-431396 hydrate, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

microbiology↗

bioRxiv2020

Microbiome-derived metabolites reproduce the mitochondrial dysfunction and decreased insulin sensitivity observed in type 2 diabetes

Diabetes is a global health problem that was estimated to be the 7th leading cause of death worldwide in 2016. Type 2 diabetes mellitus (T2DM) is classically associated with genetic and environmental factors, however recent studies have demonstrated that the gut microbiome, which is altered in T2DM patients, is also likely to play a significant role in disease development. Despite this, the identity of microbiome-derived metabolites that influence T2DM onset and/or progression remain elusive. Here we demonstrate that a serum biomarker for T2DM, previously of unknown structure and origin, is actually two microbiome-derived metabolites, 3-methyl-4-(trimethylammonio)butanoate (3M-4-TMAB) and 4-(trimethylammonio)pentanoate (4-TMAP). These metabolites are produced by the Lachnospiraceae family of bacteria, which are highly prevalent in the gut microbiome of T2DM patients and are associated with high dietary fat intake. Treatment of human liver cells with 3M-4-TMAB and 4-TMAP results in a distinct change in the acylcarnitine profile in these cells and significantly reduced their insulin sensitivity; both indicators of T2DM. These results provide evidence of a mechanistic link between gut microbiome derived metabolites and T2DM.

microbiology↗

bioRxiv2020

Monocytes mediate Salmonella Typhimurium-induced tumour growth inhibition

The use of bacteria as an alternative cancer therapy has been re-investigated in recent years. A number of bacterial strains for this purpose have been generated, one of which is SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential. Here we show that systemic administration of SL7207 induces melanoma tumour growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumour-infiltrating cells towards pro-inflammatory, with expression of the TH1 cytokines IFN-{gamma}, TNF-, and IL-12 significantly increased. Interestingly, Ly6C+MHCII+ monocytes were recruited to the tumours following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumour growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumour growth inhibition. This information highlights a novel role for monocytes in controlling tumour growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

immunology↗

bioRxiv2020

Mapping the influence of the gut microbiota on small molecules in the brain through mass spectrometry imaging

BackgroundThe gut microbiota is known to influence virtually all facets of human health. Recent work has highlighted a potential role for the gut microbiota in neurological health through the microbiome-gut-brain axis. Microbes can influence the brain both directly and indirectly; through neurotransmitter production, induction of host immunomodulators, or through the release or induction of other microbial or host molecules. MethodsHere we used mass spectrometry imaging (MSI), a label-free imaging tool, to map the molecular changes that occur in the murine gut and brain in germ-free, antibiotic-treated and control mice. ResultsWe determined the spatial distribution and relative quantification of neurotransmitters and their precursors across brain and gut sections in response to the microbiome. Using untargeted MSI of small molecules, we detected a significant change in the levels of four identified metabolites in the brains of germ-free animals compared to controls; vitamin B5, 3-hydroxy-3-methylglutaric acid, 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate. However, antibiotic treatment induced no significant changes in these metabolites in the brain after one week of treatment. ConclusionsThis work exemplifies the utility of MSI as a tool in determining the spatial distribution and quantification of bacterial and host metabolites in the gut and brain whilst also offering the potential for discovery of novel mediators of microbiome-gut-brain axis communication.

microbiology↗

bioRxiv2019

Regulatory T cells control the dynamic and site-specific T helper bias following Salmonella infection

FoxP3+ regulatory T cells (Tregs) control inflammation and maintain mucosal homeostasis, but their functions during infection are poorly understood. Th1, Th2 and Th17 cells can be identified by master transcription factors (TFs) T-bet, GATA3 and ROR{gamma}T; Tregs also express these TFs. While T-bet+ Tregs can selectively suppress Th1 cells, it is unclear whether distinct Treg populations can alter Th bias. To address this, we used Salmonella enterica serotype Typhimurium to induce non-lethal colitis. Following infection, we observed an early colonic Th17 response within total CD4 T cells, followed by a Th1 bias. The early Th17 response, which contains both Salmonella-specific and non-Salmonella-specific cells, parallels an increase in T-bet+ Tregs. Later, Th1 cells and ROR{gamma}T+ Tregs dominate. This reciprocal dynamic may indicate that Tregs selectively suppress Th cells, shaping the immune response. Treg depletion 1-2 days post-infection shifted the early Th17 response to a Th1 bias; however, depletion 6-7 days post-infection abrogated the Th1 bias. Thus, Tregs are necessary for the early Th17 response, and for a maximal Th1 response later. These data show that Tregs shape the overall tissue CD4 T cell response and highlight the potential for subpopulations of Tregs to be used in targeted therapeutic approaches.

immunology↗

bioRxiv2018

Propionic acid enhances the virulence of Crohn’s disease-associated adherent-invasive Escherichia coli

The short chain fatty acid propionic acid (PA) is a bacteria-derived human intestinal antimicrobial and immune modulator used widely in Western food production and agriculture. Here we examine the effect of PA on the pathogenicity of the Crohns disease-associated microbe, adherent-invasive Escherichia coli (AIEC). Passage of AIEC through a murine model, where the low intestinal PA levels were increased to replicate those of the human intestine, led to the recovery of AIEC post-infection that had significantly increased virulence. These phenotypic changes, including increased adhesion to intestinal epithelial cells and biofilm formation, could be replicated in AIEC in vitro through exposure to PA alone. This in vitro exposure of AIEC to PA fundamentally changed AIEC virulence, with strains exposed to PA in vitro subsequently persisting at 20-fold higher levels in a murine model compared to non-exposed strains. RNA-sequencing identified the transcriptional changes in AIEC in response to PA with upregulation of genes involved in biofilm formation, stress responses, metabolism, membrane integrity and alternative carbon source utilisation. These PA induced changes in virulence could be replicated in a number of E. coli isolates from Crohns disease patients. Finally, removal of the PA selective pressure was sufficient to reverse these phenotypic changes. Our data indicate that exposure of AIEC to PA evolves bacteria that are both resistant to this natural human intestinal antimicrobial and increasingly virulent in its presence.\n\nImportanceExposure to propionic acid, an intestinal short chain fatty acid and commonly used antimicrobial in Western food production, induces significant virulence associated phenotypic changes in adherent-invasive Escherichia coli (AIEC).

microbiology↗