Intervention in gut microbiota increases intestinal γ-aminobutyric acid and alleviates anxiety behavior: a possible mechanism via the action on intestinal epithelial cells.

The role of the gut microbiota in the gut-brain axis has attracted attention in recent years. Some gut microbiota produces γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in mammals, in vitro, but the correlation between gut microbiota composition and intestinal GABA concentration, as well as the action of intestinal GABA in vivo, are poorly understood. Herein, we found that the intestinal GABA concentration was increased in mice by the intervention of the gut microbiota with neomycin or Bifidobacterium bifidum TMC3115 (TMC3115). Administration of TMC3115 reduced anxiety without affecting serum levels of serotonin, corticosterone, or GABA. We further found that intestinal epithelial cells expressed GABA receptor subunits and mediated mitogen-activated protein kinase signaling upon GABA stimulation. In addition, administration of TMC3115 induced mitogen-activated protein kinase signaling in colonic epithelial cells but not in small intestinal epithelial cells in mice. These results indicate that GABA produced by the gut microbiota, mainly in the colon, may affect host behavioral characteristics via GABA receptors expressed in intestinal epithelial cells without being transferred to the blood. This study suggests a novel mechanism by which intestinal GABA exerts physiological effects, even in the presence of the blood-brain barrier.

Strain-level detection of Fusobacterium nucleatum in colorectal cancer specimens by targeting the CRISPR-Cas region.

IMPORTANCE: Fusobacterium nucleatum is one of the predominant oral bacteria in humans. However, this bacterium is enriched in colorectal cancer (CRC) tissues and may be involved in CRC development. Our previous research suggested that F. nucleatum is present in CRC tissues originating from the oral cavity using a traditional strain-typing method [arbitrarily primed polymerase chain reaction (AP-PCR)]. First, using whole-genome sequencing, this study confirmed an exemplary similarity between the oral and tumoral strains derived from each patient with CRC. Second, we successfully developed a method to genotype this bacterium at the strain level, targeting the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated system, which is hypervariable (defined as F. nucleatum-strain genotyping PCR). This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. The new methods have great potential for application in etiological studies of F. nucleatum in CRC.

Regulation of Gene Expression through Gut Microbiota-Dependent DNA Methylation in Colonic Epithelial Cells.

A huge number of commensal bacteria inhabit the intestine, which is equipped with the largest immune system in the body. Recently, the regulation of various physiological functions of the host by these bacteria has attracted attention. In this study, the effects of commensal bacteria on gene expression in colonic epithelial cells (CoECs) were investigated with focus on regulation of DNA methylation. RNA sequencing analyses of CoECs from conventional, germ-free, and MyD88-/- mice indicated that, out of the genes affected by commensal bacteria, those downregulated in a MyD88-independent manner were most frequently observed. Furthermore, when the 5' regions of genes downregulated by commensal bacteria in CoECs were captured using a customized array and immunoprecipitated with the anti-methyl cytosine Ab, a certain population of these genes was found to be highly methylated. Comprehensive analysis of DNA methylation in the 5' regions of genes in CoECs from conventional and germ-free mice upon pull-down assay with methyl-CpG-binding domain protein 2 directly demonstrated that DNA methylation in these regions was influenced by commensal bacteria. Actually, commensal bacteria were shown to control expression of Aldh1a1, which encodes a retinoic acid-producing enzyme and plays an important role in the maintenance of intestinal homeostasis via DNA methylation in the overlapping 5' region of Tmem267 and 3110070M22Rik genes in CoECs. Collectively, it can be concluded that regulation of DNA methylation in the 5' regions of a specific population of genes in CoECs acts as a mechanism by which commensal bacteria have physiological effects on the host.

Commensal microbiota-induced microRNA modulates intestinal epithelial permeability through the small GTPase ARF4.

The intestinal tract contains many commensal bacteria that modulate various physiological host functions. Dysbiosis of commensal bacteria triggers dysfunction of the intestinal epithelial barrier, leading to the induction or aggravation of intestinal inflammation. To elucidate whether microRNA plays a role in commensal microbiome-dependent intestinal epithelial barrier regulation, we compared transcripts in intestinal epithelial cells (IECs) from conventional and germ-free mice and found that commensal bacteria induced the expression of miR-21-5p in IECs. miR-21-5p increased intestinal epithelial permeability and up-regulated ADP ribosylation factor 4 (ARF4), a small GTPase, in the IEC line Caco-2. We also found that ARF4 expression was up-regulated upon suppression of phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4), which are known miR-21-5p targets, by RNAi. Furthermore, ARF4 expression in epithelial cells of the large intestine was higher in conventional mice than in germ-free mice. ARF4 suppression in the IEC line increased the expression of tight junction proteins and decreased intestinal epithelial permeability. These results indicate that commensal microbiome-dependent miR-21-5p expression in IECs regulates intestinal epithelial permeability via ARF4, which may therefore represent a target for preventing or managing dysfunction of the intestinal epithelial barrier.

α-Defensin 5 gene expression is regulated by gut microbial metabolites.

α-Defensin 5 is important to both maintenance of a gut microbiota and host immunity. While previous reports have shown that gut bacteria are able to upregulate α-defensin 5 through Toll-like receptor signaling, we demonstrate here that α-defensin 5 expression can also be regulated by microbial metabolites. Among these, lactate appeared to significantly suppress α-defensin 5 gene transcription. Actually, fractions of <3 kD compounds obtained from the ceca of SPF mice were suppressed α-defensin 5 gene transcription at specific concentrations. Our results also suggest that cecal content may include as yet unidentified factors that can enhance α-defensin 5 expression. Our data point to a novel function for the gut microbial metabolites in controlling the expression of antimicrobial peptides in the intestine.

Characterization of the Pathogenicity of Streptococcus intermedius TYG1620 Isolated from a Human Brain Abscess Based on the Complete Genome Sequence with Transcriptome Analysis and Transposon Mutagenesis in a Murine Subcutaneous Abscess Model.

Streptococcus intermedius is known to cause periodontitis and pyogenic infections in the brain and liver. Here we report the complete genome sequence of strain TYG1620 (genome size, 2,006,877 bp; GC content, 37.6%; 2,020 predicted open reading frames [ORFs]) isolated from a brain abscess in an infant. Comparative analysis of S. intermedius genome sequences suggested that TYG1620 carries a notable type VII secretion system (T7SS), two long repeat regions, and 19 ORFs for cell wall-anchored proteins (CWAPs). To elucidate the genes responsible for the pathogenicity of TYG1620, transcriptome analysis was performed in a murine subcutaneous abscess model. The results suggest that the levels of expression of small hypothetical proteins similar to phenol-soluble modulin ß1 (PSMß1), a staphylococcal virulence factor, significantly increased in the abscess model. In addition, an experiment in a murine subcutaneous abscess model with random transposon (Tn) mutant attenuation suggested that Tn mutants with mutations in 212 ORFs in the Tn mutant library were attenuated in the murine abscess model (629 ORFs were disrupted in total); the 212 ORFs are putatively essential for abscess formation. Transcriptome analysis identified 37 ORFs, including paralogs of the T7SS and a putative glucan-binding CWAP in long repeat regions, to be upregulated and attenuated in vivo This study provides a comprehensive characterization of S. intermedius pathogenicity based on the complete genome sequence and a murine subcutaneous abscess model with transcriptome and Tn mutagenesis, leading to the identification of pivotal targets for vaccines or antimicrobial agents for the control of S. intermedius infections.

Post-Transcriptional Regulation of Toll-Interacting Protein in the Intestinal Epithelium.

Immune responses against gut microbiota should be minimized to avoid unnecessary inflammation at mucosal surface. In this study, we analyzed the expression patterns of Toll-interacting protein (Tollip), an inhibitor of TLRs and IL-1 family cytokine-related intracellular signaling, in intestinal epithelial cells (IECs). Comparable mRNA expression was observed in murine small and large IECs (S-IECs and L-IECs). However, Tollip protein was only detected in L-IECs, but not in S-IECs. Similar results were obtained in germ-free mice, indicating that L-IEC-specific TOLLIP expression does not depend on bacterial colonization. Next, to understand the mechanisms underlying the post-transcriptional repression of Tollip, 3´-UTR-mediated translational regulation was evaluated. The region +1876/+2398 was responsible for the repression of Tollip expression. This region included the target sequence of miR-31. The inhibition of miR-31 restored the 3´-UTR-meditaed translational repression. In addition, miR-31 expression was significantly higher in S-IECs than in L-IECs, suggesting that miR-31 represses the translation of Tollip mRNA in S-IECs. Collectively, we conclude that the translation of Tollip is inhibited in S-IECs, at least in part, by miR-31 to yield L-IEC-specific high-level expression of the Tollip protein, which may contribute to the maintenance of intestinal homeostasis.

Isolation and identification of Enterobacteriaceae from raw horsemeat intended for human consumption (Basashi).

The status of Enterobacteriaceae contamination was investigated in a total of 131 samples of raw horsemeat (Basashi) intended for human consumption purchased from a general meat shop or by mail-order from October 2012 to December 2013. The bacteria were isolated from 105 of the 112 samples (93.8%). Prominent differences in the isolation rate due to the place of manufacture/sale or by the cut of the meat were not observed. Moreover, in a comparison between domestic (92.6%) and imported (100%) samples, the isolation rate was slightly higher in the imported samples. When Enterobacteriaceae isolated from raw horsemeat was identified, it was highly diverse, with 14 species identified in total. From among these species, Hafnia alvei was the most common, with 33 strains (19.8%),followed by 27 strains (16.2%) of Klebsiella pneumoniae and 26 strains (15.6%) of Enterobacter cloacae, indicating that these three species were dominant. A trend was observed, with the dominant strain differing depending on the place of manufacture/sale or the cut of the meat. H. alvei was isolated at an especially high frequency from imported samples. An investigation was carried out regarding raw horsemeat intended for human consumption from Yamanashi Prefecture and Canada, regularly purchased from one store in Kanagawa Prefecture. Enterobacteriaceae were isolated during five of nine (55.6%） trials, in which the isolated bacteria were H. alvei, K. pneumoniae, etc. Moreover, they were isolated at a very high isolation rate of seven among 10 trials for the Canadian meat, and H. alvei was the most commonly isolated bacteria. Accordingly, when an investigation was carried out regarding the differences in the strain level in the six isolates of H. alvei periodically isolated from raw horsemeat from Canada by the pulsed-field gel electrophoresis (PFGE) pattern using a restriction enzyme, SfiI, there was a possibility that these were the same H-38 strain (November 2013) and H-64 strain (April 2014) as well as the same H-104 strain (July 2014) and H-131 strain (December 2014). As mentioned above, it has been demonstrated that a variety of Enterobacteriaceae were isolated from raw horsemeat (Basashi) intended for human consumption, and at a high frequency. Moreover, based on the fact that the same species or strain was chronologically isolated, the possibility of contamination by the same contamination source at different times was suggested.

Transcription of the Tollip gene is elevated in intestinal epithelial cells through impaired O-GlcNAcylation-dependent nuclear translocation of the negative regulator Elf-1.

Intestinal epithelial cells (IECs) must be tolerant of the large number of commensal bacteria inhabiting the intestinal tract to avoid excessive inflammatory reactions. Toll-interacting protein (Tollip), a negative regulator of Toll-like receptor signaling, is known to be expressed at high levels in IECs, and to thereby contribute to the hyporesponsiveness of IECs to commensals. In this study, we analyzed the underlying mechanisms for elevated transcription of the Tollip gene in IECs using a human IEC line, Caco-2, and a human monocyte line, THP-1, as a control. Elf-1 was identified as a transcription factor that negatively regulates Tollip gene expression. The transcription factor Elf-1 was localized in the nucleus by O-linked N-acetylglucosamine (O-GlcNAc) modification, whereas the unmodified form was detected only in the cytoplasm. Comparison of Caco-2 and THP-1 cells revealed that O-GlcNAc modification of Elf-1 was significantly lower in IECs than in monocytes. Collectively, the results indicate that insufficient O-GlcNAc modification prevents Elf-1-mediated transcriptional repression and thereby upregulates Tollip gene expression in IECs.

Epigenetic control of the host gene by commensal bacteria in large intestinal epithelial cells.

Intestinal epithelial cells (IECs) are continuously exposed to large numbers of commensal bacteria but are relatively insensitive to them, thereby averting an excessive inflammatory reaction. We have previously reported that the hyporesponsiveness of a human IEC line to LPS was primarily the result of a down-regulation of TLR4 gene transcription through epigenetic mechanisms. In the present study we show that DNA methylation in the 5' region of the TLR4 gene is significantly higher in IECs than in splenic cells in vivo. The methylation was shown to be dependent on the differentiation state of the IECs, as the differentiated IEC population that expressed higher levels of intestinal alkaline phosphatase (IAP) also displayed greater methylation and lower expression of the TLR4 gene than the undifferentiated population. The IAP(high), differentiated population also showed less abundant expression of CDX2, the transcription factor required for the development of the intestine, than the IAP(low), undifferentiated population. Overexpression of CDX2 in an IEC line decreased the methylation level of the TLR4 gene, increased transcriptional promoter activity of the gene, and increased responsiveness to the TLR4 ligand. Furthermore, the methylation level of the TLR4 gene was significantly lower in IECs of the large intestine of germ-free mice than in those of conventional mice, whereas the level in IECs of the small intestine was almost equal between these mice, indicating that commensal bacteria contribute to the maintenance of intestinal symbiosis by controlling epigenetic modification of the host gene in the large intestine.

Epigenetic regulation of TLR4 gene expression in intestinal epithelial cells for the maintenance of intestinal homeostasis.

Intestinal epithelial cells (IECs) are continuously exposed to large numbers of commensal bacteria but are relatively insensitive to them, thereby averting an excessive inflammatory reaction. In this study, we show that the low responsiveness of human IEC lines to LPS was mainly brought about by a down-regulation of TLR4 gene transcription. Additionally, the presence of an IEC-specific repressor element in the 5' region of the TLR4 gene and binding of a NF to the element was shown. The transcription factor ZNF160, which was expressed more abundantly in a LPS-low responder IEC line than in a LPS-high responder IEC line, repressed TLR4 gene transcription. ZNF160 is known to interact with the scaffold protein KAP1 via its N terminus to recruit histone deacetylase. Histone deacetylation, as well as DNA methylation, at the 5' region of the TLR4 gene was significantly higher in LPS-low responder IEC lines than in a monocyte line or a LPS-high responder IEC line. It was demonstrated that TLR4 gene transcription was repressed by these epigenetic regulations, which were, at least in part, dependent on ZNF160. Down-regulaton of TLR4 gene expression by these mechanisms in IECs possibly contributes to the maintainance of homeostasis in the intestinal commensal system.