A Murine Model of Maternal Micronutrient Deficiencies and Gut Inflammatory Host-microbe Interactions in the Offspring.

BACKGROUND & AIMS: Micronutrient deficiency (MND) (ie, lack of vitamins and minerals) during pregnancy is a major public health concern. Historically, studies have considered micronutrients in isolation; however, MNDs rarely occur alone. The impact of co-occurring MNDs on public health, mainly in shaping mucosal colonization by pathobionts from the Enterobacteriaceae family, remains undetermined due to lack of relevant animal models. METHODS: To establish a maternal murine model of multiple MND (MMND), we customized a diet deficient in vitamins (A, B12, and B9) and minerals (iron and zinc) that most commonly affect children and women of reproductive age. Thereafter, mucosal adherence by Enterobacteriaceae, the associated inflammatory markers, and proteomic profile of intestines were determined in the offspring of MMND mothers (hereafter, low micronutrient [LM] pups) via bacterial plating, flow cytometry, and mass spectrometry, respectively. For human validation, Enterobacteriaceae abundance, assessed via 16s sequencing of 3-month-old infant fecal samples (n = 100), was correlated with micronutrient metabolites using Spearman's correlation in meconium of children from the CHILD birth cohort. RESULTS: We developed an MMND model and reported an increase in colonic abundance of Enterobacteriaceae in LM pups at weaning. Findings from CHILD cohort confirmed a negative correlation between Enterobacteriaceae and micronutrient availability. Furthermore, pro-inflammatory cytokines and increased infiltration of lymphocyte antigen 6 complex high monocytes and M1-like macrophages were evident in the colons of LM pups. Mechanistically, mitochondrial dysfunction marked by reduced expression of nicotinamide adenine dinucleotide (NAD)H dehydrogenase and increased expression of NAD phosphate oxidase (Nox) 1 contributed to the Enterobacteriaceae bloom. CONCLUSION: This study establishes an early life MMND link to intestinal pathobiont colonization and mucosal inflammation via damaged mitochondria in the offspring.

The Effect of Nutrient Deprivation on Early Life Small Intestinal Mucosal Microbiome and Host Proteome.

BACKGROUND: Nutrition plays a vital role in shaping the intestinal microbiome. However, many hospitalized children undergo periods of fasting during medical treatment. Changes to the small intestinal microbiota in early life in the setting of enteral deprivation have not been well described. OBJECTIVE: The aim of this study was to investigate the impact of enteral deprivation on the small intestinal mucosal microbiome and to identify factors that shape this interaction in infancy. METHODS: Intestinal biopsies were collected from proximal (fed) and distal (unfed) small bowel at the time of ostomy closure in children with a small intestinal enterostomy. Mucosal and luminal microbiome comparisons were performed including ß-diversity and differential abundance and correlations with clinical factors were analyzed. Host proteomics were compared between fed and unfed samples and correlated with microbiome parameters. Finally, microbial results were validated in another cohort of pediatric patients. RESULTS: Samples from 13 children (median age 84 d) were collected. Mucosal microbiome communities in the fed and unfed segments were strikingly similar [paired UniFrac distance (ß-diversity)], whereas luminal effluent differed significantly from fed samples (PERMANOVA, P = 0.003). Multivariate analysis revealed patient as the strongest predictor of the UniFrac distance. Environmental variables did not influence the intrapatient microbial dissimilarity. Host proteomics were similar intrapatient (paired fed-unfed Euclidian distance) and showed a correlation with the UniFrac distance (Spearman rho = 0.71, P < 0.001). Specific proteins and functional clusters were significantly different between paired samples, including lipid metabolism and intracellular trafficking, whereas no difference was seen in innate immune proteins. The microbiome results were validated in a different cohort with similar characteristics. CONCLUSION: We found the host to be the most dominant factor in the structure of the early life small intestinal mucosal microbiome. Nutrient deprivation was associated with specific changes in the host proteome. Further research is needed to better understand this host-microbe-nutrition interaction.

Gut microbes shape microglia and cognitive function during malnutrition.

Fecal-oral contamination promotes malnutrition pathology. Lasting consequences of early life malnutrition include cognitive impairment, but the underlying pathology and influence of gut microbes remain largely unknown. Here, we utilize an established murine model combining malnutrition and iterative exposure to fecal commensals (MAL-BG). The MAL-BG model was analyzed in comparison to malnourished (MAL mice) and healthy (CON mice) controls. Malnourished mice display poor spatial memory and learning plasticity, as well as altered microglia, non-neuronal CNS cells that regulate neuroimmune responses and brain plasticity. Chronic fecal-oral exposures shaped microglial morphology and transcriptional profile, promoting phagocytic features in MAL-BG mice. Unexpectedly, these changes occurred independently from significant cytokine-induced inflammation or blood-brain barrier (BBB) disruption, key gut-brain pathways. Metabolomic profiling of the MAL-BG cortex revealed altered polyunsaturated fatty acid (PUFA) profiles and systemic lipoxidative stress. In contrast, supplementation with an ω3 PUFA/antioxidant-associated diet (PAO) mitigated cognitive deficits within the MAL-BG model. These findings provide valued insight into the malnourished gut microbiota-brain axis, highlighting PUFA metabolism as a potential therapeutic target.

Effect of repeated seizures on spatial exploration and immediate early gene expression in the hippocampus and dentate gyrus.

Immediate early genes (IEGs) are coordinately activated in response to neuronal activity and can cause activation of secondary response genes that modulate synaptic plasticity and mediate long-lasting changes in behaviour. Excessive neuronal stimulation induced by epileptic seizures induce rapid and dramatic changes in IEG expression. Although the impact of acute seizure activity on IEG expression has been well studied, less is known about the long-term effects of chronic seizures on IEG induction during seizure free periods where behavioural and cognitive impairments are frequently observed in people with epilepsy and in animal models of epilepsy. The present study sought out to examine the impact of chronic pentylenetetrazole evoked seizures (PTZ kindling) on spatial exploration induced in IEG expression (c-Fos, ΔFosB, Homer1a, Egr1, Npas4, Nr4a1) in the hippocampus (CA1 and CA3 subfields) and dentate gyrus of rats. Male rats underwent two weeks of PTZ kindling (every 2 days) or received vehicle injections and were placed into a novel open field arena for 30 min either 24 hrs or 4 weeks after the last treatment. Although exploratory activity was similar between PTZ kindled and vehicle controls when examined 24 hrs after the last treatment, we observed a significant reduction in spatial exploration induced expression of c-Fos, Egr1, and ΔFosB in the hippocampus and dentate gyrus, and reduced expression of Nr4a1 in the dentate gyrus and Homer1a in the hippocampus only. When testing was conducted after a 4-week recovery period, only c-Fos continued to show reduced expression after exposure a novel environment in previously PTZ kindled animals. Interestingly, these animals also showed reduced activity in the center region of the open field suggestive of heightened anxiety-like behaviour. Collectively, these results suggest that repeated seizures may lead to longterm downregulation in hippocampal IEG expression that can extend into seizure free periods thereby providing a critical mechanism for the development of cognitive and behavioural deficits that arise during chronic epilepsy.

Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2.

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein-protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.

Here, there, and everywhere: How pathogenic Escherichia coli sense and respond to gastrointestinal biogeography.

Gastrointestinal (GI) pathogens enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), and related mouse pathogen Citrobacter rodentium, are referred to as attaching and effacing (AE) pathogens for the lesions they form upon colonisation of the host epithelium. EPEC, EHEC, and C. rodentium are well known to use a type III secretion system to intimately attach to intestinal cells and secrete bacterial effectors to manipulate host cell processes. Less well known is the ability of AE pathogens to overcome significant physiological and microbial barriers and target specific gut niches for initial colonisation of the host epithelium. This review considers recent work highlighting the biogeography of the GI tract as it applies to colonisation by enteric pathogens, including environmental barriers to enteric infection, signals sensed by AE pathogens for navigation of the GI tract, and the tools AE pathogens use to respond to the changing host environment.