There is increasing evidence that the intestinal microbiota may regulate behavior and neuronal function, affecting neurological disorders or possibly attenuating them. Goehler et al. reported that Campylobacter jejuni, a common food-poisoning bacterium, induces anxiety-like behavior and c-fos protein expression in the amygdala and nucleus of the solitary tract, which process visceral and autonomic information38. Hsiao et al. showed that 4-ethylphenylsulfate, the most markedly increased metabolite from the gut microbiota in maternal immune activation-induced autism mice, induced abnormal autism-like behaviors, whereas commensal Bacteroides fragilis treatment attenuated this behavior39. In the case of PD, a recent evidence reported that short chain fatty acids produced by gut microbiota promote α-synuclein-mediated gliosis and PD motor symptoms23. As shown in Fig. 9, we demonstrated that a specific gut bacterium, P. mirabilis, was involved in the pathogenesis in a mouse model of PD.

Figure 9 Schematic summary of P. mirabilis-induced PD pathogenesis in the colon and in the brain. Full size image

First, we observed that gut microbiota dysbiosis had occurred regardless of toxin type in the colons of neurotoxin-induced PD mice. The bacterial colonies of Enterobacteriaceae family were significantly higher than those of each corresponding normal group. Then, we determined the genus of increased bacterial colonies as Proteus and it was identified as P. mirabilis. Interestingly, this result is consistent with a recent report that Proteus was a remarkably increased genus of the bacteria in α-synuclein overexpressing mice with intestinal dysbiosis induced by fecal transplantation from PD patients23.

Thus, our result is consistent with the previous study that Enterobacteriaceae increased significantly in PD patients22. Although E. coli is the most representative bacterium in the Enterobacteriaceae family and its endotoxin increases intestinal permeability and α-synuclein levels17, it showed no change in PD mice, whereas P. mirabilis showed levels increased over 10-fold compared with the normal group in our study. Before now, there has been no clinical report on the quantification of E. coli and P. mirabilis in the intestine, but recently it was reported that P. mirabilis was significantly higher in the urine of PD patients than in controls40,41. In that study, it was suggested that P. mirabilis was a causative bacterium of ‘purple urine bag syndrome’, increasing urinary indoxyl sulfate, a bacteria-generated metabolite. It was also shown that P. mirabilis acts as an inducible bacterium in pathological colonic changes. P. mirabilis stimulates robust IL-1β production in response to dextran sulfate sodium-induced colitis, mediated by the NOD-like receptor family pyrin domain-containing 3 inflammasome activation42. It could also trigger pathological colonic changes, characterized by colonic barrier disruption and elevated TNF-α levels as a key feature in mice with T-bet−/−RAG2−/− ulcerative colitis26. Additionally, P. mirabilis was demonstrated to be one of the causative bacteria in brain infection, according to some case reports43,44,45. These previous studies suggest that P. mirabilis may induce pathological status in both the colon and the brain. Thus, we hypothesized that P. mirabilis may be pathogenic to dopaminergic neurons, and then we evaluated whether P. mirabilis treatment could exacerbate or induce PD-like pathological characteristics in mice. This bacterium worsened the motor symptoms and dopaminergic neuronal damage in mice at the premotor phase of PD induced by MPTP toxicity. Moreover, the treatment of this bacterium alone impaired motor function, induced severe dopaminergic neuronal damage, and activated glial cells in the SN and ST of normal mice. These results suggest that P. mirabilis may be involved in the pathogenesis of PD.

Next, we sought to examine how this bacterium affects brain damage. Because P. mirabilis is a gram-negative pathogenic bacterium that produces LPS endotoxin29, we considered that LPS derived from P. mirabilis in the colon could move to the periphery, and then induce neuroinflammation. We found that LPS levels in feces were elevated significantly at the 16th day after P. mirabilis treatment, and the levels in serum increased consistently. These results suggested the possibility that P. mirabilis-generated LPS may be transferred from the colon to the brain via blood circulation, showing our results that microglial activation was observed in the SN and ST of mice performed intra-rectal injections of LPS P. mirabilis . This is consistent with the report of Qin et al. that increased inflammatory cytokines by systemic injection of LPS transferred inflammatory reactions from periphery to brain inducing microglial activation and dopaminergic neuronal damage in the SN46.

It has been reported previously that LPS derived from bacteria impairs the colonic barrier by reducing tight junction proteins such as occludin, and contributes to the release of TNF-α, which is stimulated by T helper 1 cell differentiation following TLR4-mediated macrophage activation in the lamina propria of the colon47,48,49. In this study, colonic barrier disruption and elevated TNF-α levels via TLR4 activation were seen after P. mirabilis administration. Clairembault and colleagues reported that expression of colonic epithelial barrier tight junction proteins was significantly lower in biopsy tissues from PD patients compared to that of controls50. Pro-inflammatory cytokines, including TNF-α, were also overexpressed in colonic biopsies from PD patients18. Thus, these pathological changes induced by P. mirabilis may represent the condition in the colon of PD patients.

Then, we assessed whether P. mirabilis induced overexpression of α-synuclein, a pathological hallmark protein in PD, in the brain and in the colon. In an in vitro experiment, P. mirabilis treatment increased the mRNA levels of α-synuclein significantly in SH-SY5Y cells. Although we could not clarify whether the effect was induced by P. mirabilis itself or LPS generated from the microbiota, this finding indicates that P. mirabilis may be involved in the production of α-synuclein in dopaminergic neurons. We also demonstrated the increase of α-synuclein aggregates both in the colon and in the SN of mice after P. mirabilis treatment. Pan-Montojo and his colleagues reported that α-synuclein first appeared in enteric neurons, moved into the dorsal motor nucleus of the vagus, and was eventually detected in the SN of rotenone-induced PD-like mice37. According to Braak theory, α-synuclein migrates from the gastrointestinal tract to the brain and overexpression of α-synuclein starts in the intestine51,52. Our results in VGX mice showed the consistent with the previous results by Holmqvistet and his colleagues that aggregated α-synuclein that was injected directly into the intestine migrated from the intestinal wall to the brain via the vagal nerve16. It has been also reported that inflammatory factors such as LPS and pro-inflammatory cytokines may trigger aggregation and accumulation of α-synuclein in enteric nerves19,53. Based on these reports, we expect that the increased α-synuclein aggregates by P. mirabilis treatment in the distal colon may move to the SN where it triggered dopaminergic neuronal damage and neuroinflammation. Additionally, increased pro-inflammatory cytokine TNF-α due to LPS insult in the colon may move directly via the blood to the SN and trigger α-synuclein aggregation and dopaminergic neuronal damage. Several studies suggest that other virulence factors of P. mirabilis except LPS may involve in intestinal inflammation or increased intestinal permeability. For example, flagella of P. mirabilis allows invasion into colonic epithelial barrier, thereby induces the increase of permeability and P. mirabilis-generated mannose-resistant Proteus-like fimbriae, which is closely related to biofilm formation as an adhesion factor, exhibits cytotoxicity on eukaryotic epithelial cells54,55. Meanwhile, it is not possible to exclude the possibility that other gut microbiomes except P. mirabilis could be involved in PD pathology in P. mirabilis-treated mice. For example, Chen et al. indicated the causal relationship between E. coli-producing curli amyloid and α-synuclein pathology24. Further researches on the actions of other bacteria after P. mirabilis administration in mice are needed.

In conclusion, we demonstrated that P. mirabilis, which showed increased bacterial colonies in the feces of three PD mouse models, is pathogenic in these animal models. It induced PD-related pathological changes, including dopaminergic neuronal death, neuroinflammation, and α-synuclein aggregation in the brain. We suggest that these pathological changes may be due to LPS as a P. mirabilis-generated virulence factor in the colon. Here, our findings are meaningful in that they provide the experimental evidence that a specific gut bacterium, which is commonly increased in PD mice, may cause the phenotype and pathogenesis of PD. It is required that further exploration to clarify the relevance of P. mirabilis for PD pathology under the conditions with germ-free or P. mirabilis-specific antibiotics treatment.