Butyrate's role in human health and the current progress towards its clinical application to treat gastrointestinal disease.

Butyrate is a key energy source for colonocytes and is produced by the gut microbiota through fermentation of dietary fiber. Butyrate is a histone deacetylase inhibitor and also signals through three G-protein coupled receptors. It is clear that butyrate has an important role in gastrointestinal health and that butyrate levels can impact both host and microbial functions that are intimately coupled with each other. Maintaining optimal butyrate levels improves gastrointestinal health in animal models by supporting colonocyte function, decreasing inflammation, maintaining the gut barrier, and promoting a healthy microbiome. Butyrate has also shown protective actions in the context of intestinal diseases such as inflammatory bowel disease, graft-versus-host disease of the gastrointestinal tract, and colon cancer, whereas lower levels of butyrate and/or the microbes which are responsible for producing this metabolite are associated with disease and poorer health outcomes. However, clinical efforts to increase butyrate levels in humans and reverse these negative outcomes have generated mixed results. This article discusses our current understanding of the molecular mechanisms of butyrate action with a focus on the gastrointestinal system, the links between host and microbial factors, and the efforts that are currently underway to apply the knowledge gained from the bench to bedside.

The mucosal-luminal interface: an ideal sample to study the mucosa-associated microbiota and the intestinal microbial biogeography.

BACKGROUND: Alterations in gastrointestinal microbial communities have been linked to human disease. Most studies use fecal samples as a proxy for the intestinal microbiota; however, the fecal microbiome is not fully representative of the mucosa-associated microbiota at the site of disease. While mucosal biopsies can be used instead, they often contain a high proportion of host DNA that can confound 16S ribosomal RNA (rRNA) gene sequencing studies. METHODS: To overcome these limitations, we sampled the mucosal-luminal interface (MLI) to study the mucosa-associated microbiota. We also employed a simple bioinformatics workflow to remove contaminants from 16S rRNA gene profiling results. RESULTS: Our results indicate that the microbial differences between individuals are greater than those between different microenvironments within the same individual. Moreover, biopsy samples frequently contained contaminants that could significantly impact biopsy profiling results. CONCLUSIONS: Our findings highlight the utility of collecting MLI aspirates to complement biopsies and stools for characterizing human microbial communities.

Metaproteomics reveals associations between microbiome and intestinal extracellular vesicle proteins in pediatric inflammatory bowel disease.

Alterations in gut microbiota have been implicated in the pathogenesis of inflammatory bowel disease (IBD), however factors that mediate the host-microbiota interactions remain largely unknown. Here we collected mucosal-luminal interface samples from a pediatric IBD inception cohort and characterized both the human and microbiota proteins using metaproteomics. We show that microbial proteins related to oxidative stress responses are upregulated in IBD cases compared to controls. In particular, we demonstrate that the expression of human proteins related to oxidative antimicrobial activities is increased in IBD cases and correlates with the alteration of microbial functions. Additionally, we reveal that many of these human proteins are present and show altered abundance in isolated free extracellular vesicles (EVs). Therefore, our study suggests that the alteration of intestinal EV proteomes is associated with the aberrant host-microbiota interactions in IBD.

Altered intestinal microbiota-host mitochondria crosstalk in new onset Crohn's disease.

Intestinal microbial dysbiosis is associated with Crohn's disease (CD). However, the mechanisms leading to the chronic mucosal inflammation that characterizes this disease remain unclear. In this report, we use systems-level approaches to study the interactions between the gut microbiota and host in new-onset paediatric patients to evaluate causality and mechanisms of disease. We report an altered host proteome in CD patients indicative of impaired mitochondrial functions. In particular, mitochondrial proteins implicated in H2S detoxification are downregulated, while the relative abundance of H2S microbial producers is increased. Network correlation analysis reveals that Atopobium parvulum controls the central hub of H2S producers. A. parvulum induces pancolitis in colitis-susceptible interleukin-10-deficient mice and this phenotype requires the presence of the intestinal microbiota. Administrating the H2S scavenger bismuth mitigates A. parvulum-induced colitis in vivo. This study reveals that host-microbiota interactions are disturbed in CD and thus provides mechanistic insights into CD pathogenesis.

Mucosa-Associated Ileal Microbiota in New-Onset Pediatric Crohn's Disease.

BACKGROUND: The composition of the intestinal microbiome seems relevant to the pathogenesis of Crohn's disease (CD), with differences in both diversity and composition of the gut microbiota in patients with CD compared with healthy individuals. However, there are still conflicting reports on the importance of various bacterial taxa in the pathogenesis of CD. The aim of this study was to characterize the composition of mucosa-associated intestinal microbiota in newly diagnosed pediatric patients with CD. METHODS: Mucosa-associated bacteria were identified from ileal biopsy specimens obtained at colonoscopy of 10 patients with either ileal or ileocolonic new-onset CD and 15 controls without mucosal inflammation. Microbial composition was performed by profiling the 16S rDNA V6 region using Illumina sequencing. Samples were analyzed for differences in alpha/beta diversity and also for differentially abundant taxa. RESULTS: Alpha diversity did not differ between the controls and CD cases or between CD subjects with localized ileal disease compared with those with more extensive disease. Controls also did not clearly separate from patients with CD by principal coordinate analyses; however, 117 operational taxonomic units were found to be differentially abundant between the two groups. In particular, numerous operational taxonomic units associated with Faecalibacterium prausnitzii species were increased in children with CD. CONCLUSIONS: These findings contribute to emerging evidence regarding dysbiosis in pediatric CD, and provide additional evidence challenging the protective role of F. prausnitzii in CD.

In Vitro Metabolic Labeling of Intestinal Microbiota for Quantitative Metaproteomics.

Intestinal microbiota is emerging as one of the key environmental factors influencing or causing the development of numerous human diseases. Metaproteomics can provide invaluable information on the functional activities of intestinal microbiota and on host-microbe interactions as well. However, the application of metaproteomics in human microbiota studies is still largely limited, in part due to the lack of accurate quantitative intestinal metaproteomic methods. Most current metaproteomic microbiota studies are based on label-free quantification, which may suffer from variability during the separate sample processing and mass spectrometry runs. In this study, we describe a quantitative metaproteomic strategy, using in vitro stable isotopically ((15)N) labeled microbiota as a spike-in reference, to study the intestinal metaproteomes. We showed that the human microbiota were efficiently labeled (>95% (15)N enrichment) within 3 days under in vitro conditions, and accurate light-to-heavy protein/peptide ratio measurements were obtained using a high-resolution mass spectrometer and the quantitative proteomic software tool Census. We subsequently employed our approach to study the in vitro modulating effects of fructo-oligosaccharide and five different monosaccharides on the microbiota. Our methodology improves the accuracy of quantitative intestinal metaproteomics, which would promote the application of proteomics for functional studies of intestinal microbiota.

Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur- and PerR-transcriptomes.

BACKGROUND: The genome of Campylobacter jejuni contains two iron activated Fur-family transcriptional regulators, CjFur and CjPerR, which are primarily responsible for regulating iron homeostasis and oxidative stress respectively. Both transcriptional regulators have been previously implicated in regulating diverse functions beyond their primary roles in C. jejuni. To further characterize their regulatory networks, RNA-seq was used to define the transcriptional profiles of C. jejuni NCTC11168 wild type, Δfur, ΔperR and ΔfurΔperR isogenic deletion mutants under both iron-replete and iron-limited conditions. RESULTS: It was found that 202 genes were differentially expressed in at least one mutant under iron-replete conditions and 331 genes were differentially expressed in at least one mutant under iron-limited conditions. The CjFur and CjPerR transcriptomes characterized in this study were compared to those previously identified using microarray profiling and found to be more extensive than previously understood. Interestingly, our results indicate that CjFur/CjPerR appear to co-regulate the expression of flagellar biogenesis genes in an opposing and iron-independent fashion. Moreover the ΔfurΔperR isogenic deletion mutant revealed that CjFur and CjPerR can compensate for each other in certain cases, suggesting that both regulators may compete for binding to specific promoters. CONCLUSIONS: The CjFur and CjPerR transcriptomes are larger than previously reported. In particular, deletion of perR results in the differential expression of a large group of genes in the absence of iron, suggesting that CjPerR may also regulate genes in an iron-independent manner, similar to what has already been demonstrated with CjFur. Moreover, subsets of genes were found which are only differentially expressed when both CjFur and CjPerR are deleted and includes genes that appear to be simultaneously activated by CjFur and repressed by CjPerR. In particular the iron-independent co-regulation of flagellar biogenesis by CjFur/CjPerR represents a potentially novel regulatory function for these proteins. These findings represent additional modes of co-regulation by these two transcriptional regulators in C. jejuni.

The transcriptional landscape of Campylobacter jejuni under iron replete and iron limited growth conditions.

The genome-wide Campylobacter jejuni transcriptional response under iron replete and iron limited conditions was characterized using RNA-seq. We have identified 111 novel C. jejuni 5'UTRs and mapped 377 co-transcribed genes into 230 transcriptional operons. In contrast to previous microarray results, the C. jejuni iron stimulon is less extensive than previously believed and consists of 77 iron activated genes and 50 iron repressed genes. As anticipated, the iron repressed genes are primarily those involved in iron acquisition or oxidative stress defense. Interestingly, these experiments have revealed that iron is an important modulator of flagellar biogenesis with almost all the components of the flagella found to be iron activated. Given that motility is a well-known C. jejuni colonization factor, this suggests that there is an important regulatory coupling of flagellar biogenesis and iron level in C. jejuni. In addition we have identified several consensus mutations in the C. jejuni NCTC11168 strain that are widespread in the Campylobacter research community and which may explain conflicting phenotypic reports for this strain. Comparative analysis of iron responsive genes with the known Fur regulon indicates that many iron responsive genes are not Fur responsive; suggesting that additional iron regulatory factors remain to be characterized in C. jejuni. Further analysis of the RNA-seq data identified multiple novel transcripts including 19 potential ncRNAs. The expression of selected ncRNAs was confirmed and quantified with qRT-PCR. The qRT-PCR results indicate that several of these novel transcripts are either Fur and/or iron responsive. The fact that several of these ncRNAs are iron responsive or Fur regulated suggests that they may perform regulatory roles in iron homeostasis.

Campylobacter jejuni ferric-enterobactin receptor CfrA is TonB3 dependent and mediates iron acquisition from structurally different catechol siderophores.

Campylobacter jejuni NCTC11168 does not produce any endogenous siderophores of its own yet requires the CfrA enterobactin transporter for in vivo colonization. In addition, the genome of C. jejuni NCTC11168 contains three distinct TonB energy transduction systems, named TonB1, TonB2, and TonB3, that have not been tested for their role in siderophore uptake or their functional redundancy. We demonstrate that C. jejuni NCTC11168 transports ferric-enterobactin in an energy dependent manner that requires TonB3 for full activity with TonB1 showing partial functional redundancy. Moreover C. jejuni NCTC11168 can utilize a wide variety of structurally different catechol siderophores as sole iron sources during growth. This growth is solely dependent on the CfrA enterobactin transporter and highlights the wide range of substrates that this transporter can recognize. TonB3 is also required for growth on most catechol siderophores. Furthermore, either TonB1 or TonB3 is sufficient for growth on hemin or hemoglobin as a sole iron source demonstrating functional redundancy between TonB1 and TonB3. In vivo colonization assays with isogenic deletion mutants revealed that both TonB1 and TonB3 are required for chick colonization with TonB2 dispensable in this model. These results further highlight the importance of iron transport for efficient C. jejuni colonization.

HADStress screen for posttraumatic stress: replication in ethiopian refugees.

Purpose was to assess whether a 4-symptom somatic screen, shown to correlate with current post-traumatic stress symptoms in 1 refugee group, could function as a screening instrument in another group of refugees. Sample consisted of 512 community-dwelling refugees from Ethiopia. Data collection included demography, types of torture and nontorture trauma experienced a decade earlier in Africa, and current posttraumatic stress symptoms. Somatic symptoms included headaches (H), appetite change (A), dizziness and faintness (D), and sleep problems (S), added with equal weighting into the HADStress Screen, ranging from 0 to 4. Results showed that age, gender, torture, and other trauma experiences from a decade ago, and current posttraumatic stress symptoms predicted current somatic symptoms on univariate analyses. On a negative binomial regression model, current posttraumatic stress symptoms, male gender, and number of torture types predicted a high HADStress score. Post hoc tests supported cut-off levels at 3 and at 4 symptoms. Conclusion is that the HADStress Screen can serve as an efficient, nonthreatening screen for posttraumatic stress symptoms among refugees.

Somali and Oromo refugees: correlates of torture and trauma history.

OBJECTIVES: This cross-sectional, community-based, epidemiological study characterized Somali and Ethiopian (Oromo) refugees in Minnesota to determine torture prevalence and associated problems. METHODS: A comprehensive questionnaire was developed, then administered by trained ethnic interviewers to a nonprobability sample of 1134. Measures assessed torture techniques; traumatic events; and social, physical, and psychological problems, including posttraumatic stress symptoms. RESULTS: Torture prevalence ranged from 25% to 69% by ethnicity and gender, higher than usually reported. Unexpectedly, women were tortured as often as men. Torture survivors had more health problems, including posttraumatic stress. CONCLUSIONS: This study highlights the need to recognize torture in African refugees, especially women, identify indicators of posttraumatic stress in torture survivors, and provide additional resources to care for tortured refugees.