Altered Stool Microbiota of Infants with Cystic Fibrosis Shows a Reduction in Genera Associated with Immune Programming from Birth.

Previous work from our group indicated an association between the gastrointestinal microbiota of infants with cystic fibrosis (CF) and airway disease in this population. Here we report that stool microbiota of infants with CF demonstrates an altered but largely unchanging within-individual bacterial diversity (alpha diversity) over the first year of life, in contrast to the infants without CF (control cohort), which showed the expected increase in alpha diversity over the first year. The beta diversity, or between-sample diversity, of these two cohorts was significantly different over the first year of life and was statistically significantly associated with airway exacerbations, confirming our earlier findings. Compared with control infants, infants with CF had reduced levels of Bacteroides, a bacterial genus associated with immune modulation, as early as 6 weeks of life, and this significant reduction of Bacteroides spp. in the cohort with CF persisted over the entire first year of life. Only two other genera were significantly different across the first year of life: Roseburia was significantly reduced and Veillonella was significantly increased. Other genera showed differences between the two cohorts but only at selected time points. In vitro studies demonstrated that exposure of the apical face of polarized intestinal cell lines to Bacteroides species supernatants significantly reduced production of interleukin 8 (IL-8), suggesting a mechanism whereby changes in the intestinal microbiota could impact inflammation in CF. This work further establishes an association between gastrointestinal microbiota, inflammation, and airway disease in infants with CF and presents a potential opportunity for therapeutic interventions beginning in early life.IMPORTANCE There is growing evidence for a link between gastrointestinal bacterial communities and airway disease progression in CF. We demonstrate that infants with CF ≤1 year of age show a distinct stool microbiota versus that of control infants of a comparable age. We detected associations between the gut microbiome and airway exacerbation events in the cohort of infants with CF, and in vitro studies provided one possible mechanism for this observation. These data clarify that current therapeutics do not establish in infants with CF a gastrointestinal microbiota like that in healthy infants, and we suggest that interventions that direct the gastrointestinal microbiota closer to a healthy state may provide systemic benefits to these patients during a critical window of immune programming that might have implications for lifelong health.

Gut bacterial phospholipase Ds support disease-associated metabolism by generating choline.

The essential nutrient choline is metabolized by gut bacteria to the disease-associated metabolite trimethylamine (TMA). However, most of the choline obtained via the diet and present in the human body is incorporated into larger metabolites, including the lipid phosphatidylcholine (PC). Here, we report that many choline-utilizing gut microorganisms can hydrolyse PC using a phospholipase D (PLD) enzyme and further convert the released choline to TMA. Genetic and in vitro characterization of the PLD from Escherichia coli MS 200-1 showed this enzyme is essential for bacterial hydrolysis of PC and prefers this substrate. PLDs are also found in gut bacterial isolates that are unable to convert choline to TMA, suggesting that additional members of the gut microbiota may influence access to this substrate. Unexpectedly, this PLD is only distantly related to characterized PLDs from pathogenic bacteria, suggesting a distinct evolutionary history. Together, these results reveal a previously underappreciated role for gut microorganisms in phospholipid metabolism and a potential target for inhibiting TMA production.

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity.

The discovery of enzymes responsible for previously unappreciated microbial metabolic pathways furthers our understanding of host-microbe and microbe-microbe interactions. We recently identified and characterized a new gut microbial glycyl radical enzyme (GRE) responsible for anaerobic metabolism of trans-4-hydroxy-l-proline (Hyp). Hyp dehydratase (HypD) catalyzes the removal of water from Hyp to generate Δ1-pyrroline-5-carboxylate (P5C). This enzyme is encoded in the genomes of a diverse set of gut anaerobes and is prevalent and abundant in healthy human stool metagenomes. Here, we discuss the roles HypD may play in different microbial metabolic pathways as well as the potential implications of this activity for colonization resistance and pathogenesis within the human gut. Finally, we present evidence of anaerobic Hyp metabolism in sediments through enrichment culturing of Hyp-degrading bacteria, highlighting the wide distribution of this pathway in anoxic environments beyond the human gut.

Metabolic, Epigenetic, and Transgenerational Effects of Gut Bacterial Choline Consumption.

Choline is an essential nutrient and methyl donor required for epigenetic regulation. Here, we assessed the impact of gut microbial choline metabolism on bacterial fitness and host biology by engineering a microbial community that lacks a single choline-utilizing enzyme. Our results indicate that choline-utilizing bacteria compete with the host for this nutrient, significantly impacting plasma and hepatic levels of methyl-donor metabolites and recapitulating biochemical signatures of choline deficiency. Mice harboring high levels of choline-consuming bacteria showed increased susceptibility to metabolic disease in the context of a high-fat diet. Furthermore, bacterially induced reduction of methyl-donor availability influenced global DNA methylation patterns in both adult mice and their offspring and engendered behavioral alterations. Our results reveal an underappreciated effect of bacterial choline metabolism on host metabolism, epigenetics, and behavior. This work suggests that interpersonal differences in microbial metabolism should be considered when determining optimal nutrient intake requirements.

The Plot Thickens: Diet Microbe Interactions May Modulate Thrombosis Risk.

Thrombosis plays an important role in cardiovascular disease (CVD). Platelet activation is an essential step in the genesis and propagation of atherothrombotic complications. In a recent publication, Zhu and colleagues report that gut microbe-derived TMAO enhances platelet responsiveness and thrombosis, providing a novel mechanistic connection between microbes and CVD (Zhu et al., 2016).

Characterization and detection of a widely distributed gene cluster that predicts anaerobic choline utilization by human gut bacteria.

UNLABELLED: Elucidation of the molecular mechanisms underlying the human gut microbiota's effects on health and disease has been complicated by difficulties in linking metabolic functions associated with the gut community as a whole to individual microorganisms and activities. Anaerobic microbial choline metabolism, a disease-associated metabolic pathway, exemplifies this challenge, as the specific human gut microorganisms responsible for this transformation have not yet been clearly identified. In this study, we established the link between a bacterial gene cluster, the choline utilization (cut) cluster, and anaerobic choline metabolism in human gut isolates by combining transcriptional, biochemical, bioinformatic, and cultivation-based approaches. Quantitative reverse transcription-PCR analysis and in vitro biochemical characterization of two cut gene products linked the entire cluster to growth on choline and supported a model for this pathway. Analyses of sequenced bacterial genomes revealed that the cut cluster is present in many human gut bacteria, is predictive of choline utilization in sequenced isolates, and is widely but discontinuously distributed across multiple bacterial phyla. Given that bacterial phylogeny is a poor marker for choline utilization, we were prompted to develop a degenerate PCR-based method for detecting the key functional gene choline TMA-lyase (cutC) in genomic and metagenomic DNA. Using this tool, we found that new choline-metabolizing gut isolates universally possessed cutC. We also demonstrated that this gene is widespread in stool metagenomic data sets. Overall, this work represents a crucial step toward understanding anaerobic choline metabolism in the human gut microbiota and underscores the importance of examining this microbial community from a function-oriented perspective. IMPORTANCE: Anaerobic choline utilization is a bacterial metabolic activity that occurs in the human gut and is linked to multiple diseases. While bacterial genes responsible for choline fermentation (the cut gene cluster) have been recently identified, there has been no characterization of these genes in human gut isolates and microbial communities. In this work, we use multiple approaches to demonstrate that the pathway encoded by the cut genes is present and functional in a diverse range of human gut bacteria and is also widespread in stool metagenomes. We also developed a PCR-based strategy to detect a key functional gene (cutC) involved in this pathway and applied it to characterize newly isolated choline-utilizing strains. Both our analyses of the cut gene cluster and this molecular tool will aid efforts to further understand the role of choline metabolism in the human gut microbiota and its link to disease.

Quality control test for sequence-phenotype assignments.

Relating a gene mutation to a phenotype is a common task in different disciplines such as protein biochemistry. In this endeavour, it is common to find false relationships arising from mutations introduced by cells that may be depurated using a phenotypic assay; yet, such phenotypic assays may introduce additional false relationships arising from experimental errors. Here we introduce the use of high-throughput DNA sequencers and statistical analysis aimed to identify incorrect DNA sequence-phenotype assignments and observed that 10-20% of these false assignments are expected in large screenings aimed to identify critical residues for protein function. We further show that this level of incorrect DNA sequence-phenotype assignments may significantly alter our understanding about the structure-function relationship of proteins. We have made available an implementation of our method at http://bis.ifc.unam.mx/en/software/chispas.

In Rhodobacter sphaeroides, chemotactic operon 1 regulates rotation of the flagellar system 2.

Rhodobacter sphaeroides is able to assemble two different flagella, the subpolar flagellum (Fla1) and the polar flagella (Fla2). In this work, we report the swimming behavior of R. sphaeroides Fla2(+) cells lacking each of the proteins encoded by chemotactic operon 1. A model proposing how these proteins control Fla2 rotation is presented.