The Role Of Fusobacterium Nucleatum Involved In The Microbial Dysbiosis And Visceral Hypersensitivity Of Irritable Bowel Syndrome

BackgroundIrritable bowel syndrome?IBS?is a common functional gastrointestinal disease.Its main clinical manifestations are abdominal pain,abdominal discomfort,change of defecation habits and abnormal stool characteristics.IBS affects more than 11%of the world's population.Although it is not a teratogenic or fatal disease,it still seriously affects the quality of life and brings serious economic burden.Although the pathogenesis of IBS is still not fully understood,studies have confirmed that there are multiple pathophysiological mechanisms involved in the occurrence and development of IBS.The intestinal microbial dysbiosis and visceral hypersensitivity have been found in many IBS patients,and it was found that fecal microbiota from IBS patients was observed to cause visceral hypersensitivity and motility dysfunction when inoculated into germ-free mice.Thus,microbial dysbiosis may be involved in the occurrence of visceral hypersensitivity in IBS.The intestinal epithelium receives the stimulation of intestinal microbiota.Enterochromaffin Cells?EC?,as an important neuroendocrine cell distributed in the intestinal epithelium,not only receive a variety of signals from intestinal microbiota and their metabolites,but also affect intestinal metabolism,motility and visceral sensation.Therefore,EC cells are likely to be an important bridge between the microbial dysbiosis and visceral hypersensitivity in IBS.However,the specific bacteria which plays a key role in the occurrence and development of IBS,and the specific mechanism of its action is still uncertain.It is of great clinical significance for the prevention and treatment of IBS to identify the key microorganisms involved in the occurrence and development of IBS.Therefore,this study was divided into three parts to explore the bacteria that might play an important role in IBS,its impact on microbial dysbiosis and visceral hypersensitivity,and its possible mechanisms.Part I:Characteristic changes of intestinal microbial dysbiosis in patients with Diarrhea predominant-irritable bowel syndromeAims:Intestinal microbial dysbiosis is considered to play an important role in the pathogenesis of IBS.There are 1013-14 microbes living in human intestine,which maintain the homeostasis of intestinal microbiota,while in disease condition,the homeostasis is broken.It is evidenced that intestinal microbial dysbiosis existed in many IBS patients,but the bacteria which plays a key role is still unclear.Therefore,we evaluated the characteristics of intestinal microbiota in patients with IBS.Methods:Patients with diarrhea predominant-IBS?IBS-D??diagnosed by the Rome ? criteria?and healthy controls aged 18-65 years were recruited from Qilu Hospital of Shandong University.The severity of symptoms was assessed by IBS symptom severity scale?IBS-SSS?.The Self-rating Anxiety Scale?SAS?and Self-rating Depression Scale?SDS?were used to evaluate anxiety and depression.Fecal samples of IBS-D patients and healthy controls were collected for 16S rRNA gene sequencing.Results:A total of 71 IBS-D patients and 39 healthy controls were recruited.Demographics between IBS-D patients and healthy controls were well balanced.The microbial diversity was compared by calculating the Shannon index.IBS-D patients showed a significantly lower Shannon index?P<0.05?.And the microbial community structure was evaluated by NMDS analysis of beta diversity.IBS-D patients showed different microbial structure from healthy controls?P<0.05?.The analysis of species composition showed that Fimicuites was the most dominant phylum,while Bacteroides,Blautia and Faecalibacterium were the dominant bacteria in genus level.The possible biomarkers associated with IBS-D were determined by LEfSe analysis.The genus Faecalibacterium,genus Ruminococcus,genus Subdoligranulum,etc.were highly enriched in healthy controls,and in IBS-D patients,the genus Prevotella,genus Streptococcus,genus Fusobacterium,etc.were significantly enriched.From these,all of the phylum Fusobacteria,class Fusobacteria,order Fusobacteriales,family Fusobacteriaceae and genus Fusobacterium were shown to be possible microbial biomarkers of IBS-D patients.Furthermore,as a single factor in distinguishing between IBS-D patients and healthy controls,the genus Fusobacterium had an area under the receiver operating curve?ROC?of 0.727?P<0.001?and the exhibited best cut-off value to discriminate IBS-D patients from healthy controls by ROC analysis,with a sensitivity and specificity of 57.7%and 84.6%,respectively.The abundance of Fusobacterium in the IBS-D patients was shown to remarkably higher than that observed in healthy controls?P<0.001?.In addition,the abundance of Fusobacterium in IBS-D patients was positively correlated with SDS values?P=0.034,rho=0.252?.Conclusions:1.IBS-D patients had characteristic intestinal microbial dysbiosis,and the diversity and species composition were significantly different from those of healthy controls2.Fusobacterium was a biomarker of IBS-D,and it might play an important role in the occurrence and development of IBS.Part ?:The mechanism of Fusobacterium nucleatum involved in microbial dysbiosis and visceral hypersensitivity in the animal model of irritable bowel syndromeAims:The intestinal microbial dysbiosis has been shown that it might be involved in the visceral hypersensitivity in IBS.In the Part ?,we found that Fusobacterium was a biomarker of IBS-D and had a positive correlation with SDS.Thus,Fusobacterium may be closely related to the visceral hypersensitivity associated with IBS.Fusobacterium nucleatum?F.nucleatum?,a typical strain of the genus Fusobacterium,is a gram-negative,obligate anaerobic bacterium that constitutively colonizes the oral mucosa.F.nucleatum has been suggested to contribute to the aetiology of some gastrointestinal disorders,such as appendicitis,colon cancer,and inflammatory bowel disease.In addition,F.nucleatum has been shown to be associated with pain and cold sensitivity in the oral cavity.Therefore,we explored whether F.nucleatum might affect the homeostasis of IBS intestinal microbiota and participate in the formation of visceral hypersensitivity.Methods:1.After modelling of maternal separation?MS?,the rats received F.nucleatum or normal saline once a week between week 4 to 8 by oral gavage,and then rested for 4 weeks.Fecal samples were collected at week 3,8 and 12.2.The fecal samples were used to 16S rRNA gene sequencing.3.The colorectal distension?CRD?experiment was performed on rats at week 12.The abdominal withdraw reflex?AWR?of the rats was observed and the score was recorded to evaluate the visceral hypersensitivity.4.PCR was used to detect F.nucleatum colonization.5.Western blotting was used to detect the specific IgA of F.nucleatum in the fecal supernatant of rats.6.Western blotting was used to detect the specific IgA of F.nucleatum in the fecal supernatant of IBS-D patients and healthy controls.The correlation between F.nucleatum-specific IgA and SAS or SDS were analyzed by Spearman correlation.Results:1.The microbial diversity showed that the Shannon index of control group was higher than the other three groups at week 8?P<0.05?,while group F.nucleatum had the highest Shannon index at week 12?P<0.05?.Similarly,Sobs and Chao₁ value also suggested that both MS and F.nucleatum-gavage could reduce the diversity of intestinal microbiota.The OTU data was clustered through an NMDS analysis.At week 3,no visible distinction was observed.However,control group had notably separated from the other three groups at week 8,although this separation was not obvious at week 122.The results of CRD showed that there were significant differences in AWR scores under the pressure of 0.4ml,0.6ml,0.8ml,1.0ml,1.2ml,1.4ml of balloon dilatation.The sum of the above AWR scores was used as the visceral hypersensitivity index.The visceral hypersensitivity index of group MS with F.nucleatum was higher than that of group MS?P<0.05?.3.The colonization of F.nucleatum was not detected by PCR.In order to further explore the mechanism of F.nucleatum,Western blotting was used to detect the specific IgA of F.nucleatum in the fecal supernatant of rats.Two strong bands with molecular masses of 40 and 130 kDa were detected at week 8 in most samples of group MS with F.nucleatum and group F.nucleatum.These two bands were also observed in most samples from group MS with F.nucleatum and group F.nucleatum at week 12.Further analysis showed that the proportional signals of the above two reaction bands were significantly different among groups at week 8 and 12.4.In order to verify whether the specific IgA of F.nucleatum was related to IBS,the fecal supernatant of 7 IBS-D patients and 5 healthy controls were detected by Western blotting.Compared with the healthy controls,the two strong response bands at 130 and 40 kDa were also observed in IBS-D patients,and the proportional signals were statistically different.In addition,the proportional signals of the above two bands were positively correlated with SAS and SDS scores.Conclusions:1.Both of MS and F.nucleatum could cause microbial dysbiosis.2.MS could cause visceral hypersensitivity in rats,F.nucleatum could exacerbate visceral hypersensitivity in a colonization-independent manner,and cause the increase of F.nucleatum specific IgA.Part ?:FomA participates in visceral hypersensitivity of irritable bowel syndrome through enterochromaffin cellsAims:EC receives the stimulations from bacterial components and their metabolic production,and affects intestinal metabolism,motility and visceral sensation.FomA protein is the main outer membrane protein of F.nucleatum,which is an agonist of toll like receptor 2?TLR2?with immune adjuvant function.The results of the part ? showed that F.nucleatum could exacerbate the visceral hypersensitivity of MS rats.In addition,when we analyzed a set of published transcriptome sequencing data,we found that the most abundant TLR receptor in EC cells was the TLR2 receptor.Therefore,we explored whether FomA was the key function protein of F.nucleatum and whether it might be involved in visceral hypersensitivity through EC cells.Methods:1.Through mass spectrometry,ecombination and Western blotting,the key protein which could bind to F.nucleatum specific IgA was screened and verified.2.According to Roman ? criteria,IBS-D patients and healthy controls were included.All patients showed no abnormalities under colonoscopy.The colon mucosa samples of the enrolled were collected,and the colon chromogranin A?ChgA,a universal marker of EC?was detected by immunohistochemistry to evaluate the expression of EC.3.The acute restraint stress?ARS?mouse model was constructed.The mice modeled with ARS were then received the recombinant FomA-positive E.coli BL21?DE3?or FomA-negative E.coli BL21?DE3?by oral gavage for 7 days,while the control mice received normal saline.4.After the gavage,the AWR score of CRD was used to evaluate the visceral hypersensitivity of mice.5.After CRD experiment,the mice were sacrificed,colon tissues were collected,and the immunohistochemistry was used to detect colon ChgA.6.The expression of TLR2 receptor and TPH1 in mouse colon was detected by Western blotting.7.The relative expression of Tlr2,Tphl,Tnf-?,Tlrl and Tlr4 in mouse colon was detected by quantitative Real-time PCR?qPCR?.Results:1.FomA protein was screened and verified as the key protein of F.nucleatum.2.The results of immunohistochemistry showed that the content of EC in the colon of IBS-D patients was significantly increased compared with the control group?P<0.05?.3.The AWR scores at 0.1 ml,0.15 ml,0.2 ml,and 0.25 ml all showed significant differences between groups.The sum of the above AWR scores was recorded as the visceral hypersensitivity index.And the visceral sensitivity of ARS mice receiving FomA-positive E.coli BL21?DE3?gavage was significantly higher than that of ARS mice receiving FomA-negative E.coli BL21?DE3?gavage?P<0.05?.4.The number of EC in the colon tissue of ARS mice was significantly higher than that of the control group?P<0.05?.5.Western blotting showed that the expressions of TLR2 receptor and TPH1 in ARS mice receiving FomA-positive E.coli BL21?DE3?gavage were significantly increased?P<0.05?.6.The results showed that the relative expressions of Tlr2,Tph1 and Tnf-? in the colon of ARS mice receiving FomA-positive E.coli BL21?DE3?gavage were significantly increased?P<0.05?,but the contents of Tlrl and Tlr4 were no different among groups.Conclusions:1.FomA protein was the key protein of F.nucleatum.2.The number of EC cells in colon tissues of IBS-D patients increased.3.FomA activated the expression of TLR2 receptors and upregulated the number and function of EC cells in colon tissues.