A computational model of Pseudomonas syringae metabolism unveils a role for branched-chain amino acids in Arabidopsis leaf colonization.

Bacterial pathogens adapt their metabolism to the plant environment to successfully colonize their hosts. In our efforts to uncover the metabolic pathways that contribute to the colonization of Arabidopsis thaliana leaves by Pseudomonas syringae pv tomato DC3000 (Pst DC3000), we created iPst19, an ensemble of 100 genome-scale network reconstructions of Pst DC3000 metabolism. We developed a novel approach for gene essentiality screens, leveraging the predictive power of iPst19 to identify core and ancillary condition-specific essential genes. Constraining the metabolic flux of iPst19 with Pst DC3000 gene expression data obtained from naïve-infected or pre-immunized-infected plants, revealed changes in bacterial metabolism imposed by plant immunity. Machine learning analysis revealed that among other amino acids, branched-chain amino acids (BCAAs) metabolism significantly contributed to the overall metabolic status of each gene-expression-contextualized iPst19 simulation. These predictions were tested and confirmed experimentally. Pst DC3000 growth and gene expression analysis showed that BCAAs suppress virulence gene expression in vitro without affecting bacterial growth. In planta, however, an excess of BCAAs suppress the expression of virulence genes at the early stages of infection and significantly impair the colonization of Arabidopsis leaves. Our findings suggesting that BCAAs catabolism is necessary to express virulence and colonize the host. Overall, this study provides valuable insights into how plant immunity impacts Pst DC3000 metabolism, and how bacterial metabolism impacts the expression of virulence.

Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms.

BACKGROUND & AIMS: The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism. METHODS: We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites. RESULTS: Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells. CONCLUSIONS: Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Mucosal Genomics Implicate Lymphocyte Activation and Lipid Metabolism in Refractory Environmental Enteric Dysfunction.

BACKGROUND & AIMS: Environmental enteric dysfunction (EED) limits the Sustainable Development Goals of improved childhood growth and survival. We applied mucosal genomics to advance our understanding of EED. METHODS: The Study of Environmental Enteropathy and Malnutrition (SEEM) followed 416 children from birth to 24 months in a rural district in Pakistan. Biomarkers were measured at 9 months and tested for association with growth at 24 months. The duodenal methylome and transcriptome were determined in 52 undernourished SEEM participants and 42 North American controls and patients with celiac disease. RESULTS: After accounting for growth at study entry, circulating insulin-like growth factor-1 (IGF-1) and ferritin predicted linear growth, whereas leptin correlated with future weight gain. The EED transcriptome exhibited suppression of antioxidant, detoxification, and lipid metabolism genes, and induction of anti-microbial response, interferon, and lymphocyte activation genes. Relative to celiac disease, suppression of antioxidant and detoxification genes and induction of antimicrobial response genes were EED-specific. At the epigenetic level, EED showed hyper-methylation of epithelial metabolism and barrier function genes, and hypo-methylation of immune response and cell proliferation genes. Duodenal coexpression modules showed association between lymphocyte proliferation and epithelial metabolic genes and histologic severity, fecal energy loss, and wasting (weight-for-length/height Z < -2.0). Leptin was associated with expression of epithelial carbohydrate metabolism and stem cell renewal genes. Immune response genes were attenuated by giardia colonization. CONCLUSIONS: Children with reduced circulating IGF-1 are more likely to experience stunting. Leptin and a gene signature for lymphocyte activation and dysregulated lipid metabolism are implicated in wasting, suggesting new approaches for EED refractory to nutritional intervention. ClinicalTrials.gov, Number: NCT03588013. (https://clinicaltrials.gov/ct2/show/NCT03588013).

Increased Urinary Trimethylamine N-Oxide Following Cryptosporidium Infection and Protein Malnutrition Independent of Microbiome Effects.

Cryptosporidium infections have been associated with growth stunting, even in the absence of diarrhea. Having previously detailed the effects of protein deficiency on both microbiome and metabolome in this model, we now describe the specific gut microbial and biochemical effects of Cryptosporidium infection. Protein-deficient mice were infected with Cryptosporidium parvum oocysts for 6-13 days and compared with uninfected controls. Following infection, there was an increase in the urinary excretion of choline- and amino-acid-derived metabolites. Conversely, infection reduced the excretion of the microbial-host cometabolite (3-hydroxyphenyl)propionate-sulfate and disrupted metabolites involved in the tricarboxylic acid (TCA) cycle. Correlation analysis of microbial and biochemical profiles resulted in associations between various microbiota members and TCA cycle metabolites, as well as some microbial-specific degradation products. However, no correlation was observed between the majority of the infection-associated metabolites and the fecal bacteria, suggesting that these biochemical perturbations are independent of concurrent changes in the relative abundance of members of the microbiota. We conclude that cryptosporidial infection in protein-deficient mice can mimic some metabolic changes seen in malnourished children and may help elucidate our understanding of long-term metabolic consequences of early childhood enteric infections.

Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain.

The ends of coiled-coil tropomyosin molecules are joined together by nine to ten residue-long head-to-tail "overlapping domains". These short four-chained interconnections ensure formation of continuous tropomyosin cables that wrap around actin filaments. Molecular Dynamics simulations indicate that the curvature and bending flexibility at the overlap is 10-20% greater than over the rest of the molecule, which might affect head-to-tail filament assembly on F-actin. Since the penultimate residue of striated muscle tropomyosin, Ser283, is a natural target of phosphorylating enzymes, we have assessed here if phosphorylation adjusts the mechanical properties of the tropomyosin overlap domain. MD simulations show that phosphorylation straightens the overlap to match the curvature of the remainder of tropomyosin while stiffening it to equal or exceed the rigidity of canonical coiled-coil regions. Corresponding EM data on phosphomimetic tropomyosin S283D corroborate these findings. The phosphorylation-induced change in mechanical properties of tropomyosin likely results from electrostatic interactions between C-terminal phosphoSer283 and N-terminal Lys12 in the four-chain overlap bundle, while promoting stronger interactions among surrounding residues and thus facilitating tropomyosin cable assembly. The stiffening effect of D283-tropomyosin noted correlates with previously observed enhanced actin-tropomyosin activation of myosin S1-ATPase, suggesting a role for the tropomyosin phosphorylation in potentiating muscle contraction.

Protein- and zinc-deficient diets modulate the murine microbiome and metabolic phenotype.

BACKGROUND: Environmental enteropathy, which is linked to undernutrition and chronic infections, affects the physical and mental growth of children in developing areas worldwide. Key to understanding how these factors combine to shape developmental outcomes is to first understand the effects of nutritional deficiencies on the mammalian system including the effect on the gut microbiota. OBJECTIVE: We dissected the nutritional components of environmental enteropathy by analyzing the specific metabolic and gut-microbiota changes that occur in weaned-mouse models of zinc or protein deficiency compared with well-nourished controls. DESIGN: With the use of a 1H nuclear magnetic resonance spectroscopy-based metabolic profiling approach with matching 16S microbiota analyses, the metabolic consequences and specific effects on the fecal microbiota of protein and zinc deficiency were probed independently in a murine model. RESULTS: We showed considerable shifts within the intestinal microbiota 14-24 d postweaning in mice that were maintained on a normal diet (including increases in Proteobacteria and striking decreases in Bacterioidetes). Although the zinc-deficient microbiota were comparable to the age-matched, well-nourished profile, the protein-restricted microbiota remained closer in composition to the weaned enterotype with retention of Bacteroidetes. Striking increases in Verrucomicrobia (predominantly Akkermansia muciniphila) were observed in both well-nourished and protein-deficient mice 14 d postweaning. We showed that protein malnutrition impaired growth and had major metabolic consequences (much more than with zinc deficiency) that included altered energy, polyamine, and purine and pyrimidine metabolism. Consistent with major changes in the gut microbiota, reductions in microbial proteolysis and increases in microbial dietary choline processing were observed. CONCLUSIONS: These findings are consistent with metabolic alterations that we previously observed in malnourished children. The results show that we can model the metabolic consequences of malnutrition in the mouse to help dissect relevant pathways involved in the effects of undernutrition and their contribution to environmental enteric dysfunction.