The Impact of Intestinal Microbiota and Toll-like Receptor 2 Signaling on α-Synuclein Pathology in Nontransgenic Mice Injected with α-Synuclein Preformed Fibrils.

Intestinal microbiota and Toll-like receptor 2 (TLR2), which can bind lipoteichoic acid produced by microbiota, might contribute to the pathogenesis of Parkinson's disease (PD), which is characterized by α-synuclein accumulation. Although the contribution of intestinal microbiota and TLR2 to PD pathology was validated in genetic PD models, evidence suggests that the effects of TLR2 signaling on proteinopathy might depend on the presence of a genetic etiology. We examined the impact of intestinal microbiota and TLR2 signaling on α-synuclein pathology in a nontransgenic mouse model of sporadic PD. While an α-synuclein preformed fibrils injection successfully reproduced PD pathology by inducing accumulation of α-synuclein aggregates, microglial activation and increased TLR2 expression in the brains of nontransgenic mice, antibiotic-induced reduction in the density of intestinal microbiota and TLR2 knockout had small impact on these changes. These findings, which are in contrast to those reported in transgenic mice harboring transgene encoding α-synuclein, indicate that the contribution of intestinal microbiota and TLR2 signaling to α-synuclein pathogenesis might be influenced by the presence of a genetic etiology. Additionally, these findings suggest that integrating insights from this experimental model and genetic models would further advance our understanding of the molecular mechanisms underlying sporadic PD.

Multiplatform metabolomic analysis of the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive, neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms. To investigate the metabolic alterations that occur in HD, here we examined plasma and whole-brain metabolomic profiles of the R6/2 mouse model of HD. Plasma and brain metabolomic analyses were conducted using capillary electrophoresis-mass spectrometry (CE-MS). In addition, liquid chromatography-mass spectrometry (LC-MS) was also applied to plasma metabolomic analyses, to cover the broad range of metabolites with various physical and chemical properties. Various metabolic alterations were identified in R6/2 mice. We report for the first time the perturbation of histidine metabolism in the brain of R6/2 mice, which was signaled by decreases in neuroprotective dipeptides and histamine metabolites, indicative of neurodegeneration and an altered histaminergic system. Other differential metabolites were related to arginine metabolism and cysteine and methionine metabolism, suggesting upregulation of the urea cycle, perturbation of energy homeostasis, and an increase in oxidative stress. In addition, remarkable changes in specific lipid classes are indicative of dysregulation of lipid metabolism. These findings provide a deeper insight into the metabolic alterations that occur in HD and provide a foundation for the future development of HD therapeutics.

Intestinal Clostridium species lower host susceptibility to enterohemorrhagic Escherichia coli O157:H7 infection.

Susceptibility to enterohemorrhagic Escherichia coli (EHEC) infection varies among humans. The intestinal microbiota seems to play an essential role in host defense against EHEC; thus, we hypothesized that indigenous bacteria, such as Clostridium ramosum and Clostridium perfringens, could influence the susceptibility to EHEC infection. To evaluate the effect of indigenous bacteria on EHEC infection, germ-free mice were precolonized with each indigenous bacterium, and then infected with EHEC O157:H7. Precolonization with C. ramosum or C. perfringens completely prevented death from EHEC infection througout a test period. Precolonization with C. ramosum also reduced the level of secreted Shiga toxin (Stx) 2 and prevented histopathological changes in the kidneys in a similar way to precolonization with Bifidobacterium longum, which is used as a model for preventing EHEC infection. In contrast, the mice precolonized with C. perfringens showed mild renal injuries. When evaluated using an in vitro co-culturing system, again C. ramosum inhibited the growth and Stx production of EHEC more potently than C. perfringens. These results indicate that C. ramosum and C. perfringens suppressed EHEC infection; however, the extent of their preventive effects differed. Therefore, the susceptibility to EHEC infection and its severity can depend on the functional bacteria present in the intestinal microbiota of individuals.

Effects of fermentation products of the commensal bacterium Clostridium ramosum on motility, intracellular pH, and flagellar synthesis of enterohemorrhagic Escherichia coli.

The flagellum and motility are crucial virulence factors for many pathogenic bacteria. In general, pathogens invade and translocate through motility and adhere to specific tissue via flagella. Therefore, the motility and flagella of pathogens are effectual targets for attenuation. Here, we show that the fermentation products of Clostridium ramosum, a commensal intestinal bacterium, decrease the intracellular pH of enterohemorrhagic Escherichia coli (EHEC) and influence its swimming motility. Quantifications of flagellar rotation in individual EHEC cells showed an increase in reversal frequency and a decrease in rotation rate in the presence of C. ramosum fermentation products. Furthermore, the C. ramosum fermentation products affected synthesis of flagellar filaments. The results were reproduced by a combination of organic acids under acidic conditions. Short-chain fatty acids produced by microbes in the gut flora are beneficial for the host, e.g. they prevent infection. Thus, C. ramosum could affect the physiologies of other enteric microbes and host tissues.

Inhibition of enterohemorrhagic Escherichia coli O157:H7 infection in a gnotobiotic mouse model with pre-colonization by Bacteroides strains.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been known to cause outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. We previously demonstrated that intestinal flora contribute to the prevention of EHEC infection in a mouse model. However, it has not yet been determined whether Bacteroides, a predominant genus in the human intestine, contributes to the prevention of EHEC infection. The aim of the present study was to investigate the effect of Bacteroides fragilis (B. fragilis) and Bacteroides vulgatus (B. vulgatus) on EHEC O157:H7 infection in vivo using gnotobiotic mice. These strains were inoculated into germ-free mice to create a gnotobiotic mouse model. EHEC was inoculated into the mice, which were then monitored for 7 days for any change in symptoms. The mice that had been pre-colonized with the Bacteroides strains did not develop lethal EHEC infection, although several inflammatory symptoms were observed in the B. vulgatus pre-colonized group. However, no inflammatory symptoms were identified in the B. fragilis pre-colonized group. Moreover, B. fragilis exerted an inhibitory effect on enterocyte-like cell apoptosis. B. fragilis protected HT29 cells from apoptosis caused by Shiga toxin. In conclusion, the findings of the present study demonstrated that colonization by Bacteroides strains can inhibit EHEC infection.

Immortalization of Fetal Bovine Colon Epithelial Cells by Expression of Human Cyclin D1, Mutant Cyclin Dependent Kinase 4, and Telomerase Reverse Transcriptase: An In Vitro Model for Bacterial Infection.

Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4), and human telomerase reverse transcriptase (TERT). The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1), TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection.