Pathogenic Mechanisms Of TcdA/B In Clostridium Difficile Infection

Background and ObjectionClostridium difficile(C.difficile)is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis.The incidence and mortality rate of C.difficile infection have increased remarkably in both hospital and community settings during the last two decades.While some individuals are asymptomatic C.difficile carriers,symptomatic disease ranges from mild diarrhea to potentially lethal toxic megacolon.The wide disease spectrum has been attributed to the infected host’s age,underlying diseases,immune status,and microbiome composition.The relative virulence of different clinical C.difficile strains,although postulated to determine disease severity in patients,has been more difficult to consistently associate with mild versus severe colitis.C.difficile toxins are necessary for disease,and both toxin A(TcdA)and toxin B(TcdB)independently cause disease in animal models.Most toxinogenic strains produce both toxins(A+B+),but toxin A-negative,toxin B-positive(A-B+)variants also cause disease.A+B-strain variants,in contrast,have not been convincingly demonstrated to cause CDI,leading to a focus on toxin B as a diagnostic/therapeutic target.However,the treatment with TcdB monoclonal antibody cannot completely alleviate symptoms,which makes us reconsider the importance of TcdA in the pathogenesis of CDI.Many studies have found that the regulation of intestinal microecology is conducive to the prevention and treatment of CDI.Commensal microbiota is critically important in protecting against C.difficile colonization,termed colonization resistance.The direct mechanisms include direct killing of pathogens(phage,type 6 secretory system,bacteriocin,etc.)and secretory metabolites(short-chain fatty acids,secondary bile acids,etc.)to inhibit the growth of pathogenic bacteria.The indirect mechanisms are related to competition for limited nutrition and space,and enhancement of the immune response(type 3 natural lymphocytes,Th17,IgA and IgG antibodies).In addition to direct interaction with C.difficile,one study found that strains of C.difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive,and were associated with unusually abundant Enterococcus,which implies that the C.difficile toxins have different impacts on the faecal microbiota.Intestinal flora may inhibit GDI through direct interaction with toxins to maintain intestinal flora homeostasis.In this study,we aimed to investigate the toxin gene mutations of C.difficile clinical isolates in South China,screen for mutational sites with important clinic guidance meaning;determine fecal TcdA/B concentrations in clinical CDI patients and asymptomatic carriers to analyze the importance of TcdA in the pathogenesis of CDI;explore the possible interaction between C.difficile toxins and gut bacteria.This study is expected to obtain gene mutation sites related to C.difficile toxin with clinical guiding value,provid reference toxin gene sequences for the establishment of appropriate molecular biological detection in China and supporting evidence at gene level for the complex clinical manifestations caused by CDI.To provide clinical testing basis for TcdA/B toxin protein level;At the same time,add new ideas for the colonization resistance mechanism of intestinal symbiotic bacteria,and opens the way for new non-antibiotic prevention and treatment strategies of CDI.Methods1.A total of 100 clinical isolates of C.difficile were recovered from-80℃.The DNA was extracted from the identified colonies and used for amplification and sequencing of tcdA、tcdB、tcdC、tcdD、tcdE and cdtA、cdtB.Among them,whole genome of four representative strains were sequenced.The various parameters,such as age,sex,length of hospital stay,blood routine,biochemical test,and endoscopic and pathologic findings,were reviewed and joint analysed with the sequence mutation hot spots using SPSS 19.0 statistical software and a P value<0.05 was considered statistically significant.Another 83 C.difficile strains were used for confirmatory research.Cytotoxic effects of C.difficile isolates on NCM460 cell line were measured using a Cell Counting Kit-8(CCK-8).The effect of different isolates supernatants on tight junction(TJ)protein ZO-1 and occludin in the human intestinal Caco-2 cell line were assessed by Western blot.HUVEC cells were placed on Transwell filters and grown in a culture medium composed of DMEM with indicated culture supernatant of C.difficile for 24 h.Next,transepithelial electrical resistance(TEER)was measured.These strains were also tested in vivo in a CDI mouse model.Pathological manifestations of colon were observed by HE stains.Cellulose exudation was stained with modified MSB cellulose dry.2.Stool samples were collected from 187 CDI patients and 44 carriers at Beth Israel Deaconess Medical Center under IRB-approved protocols.Isolates were cultured from stool and presence of tcdA/B genes verified by PCR.For each isolate,toxin production/activity in vitro were assessed by culture and cytotoxicity assay(CTA);most were also tested in vivo in a CDI mouse model.An ultrasensitive,quantitative Single Molecule Array(Simoa)immunoassay was used to measure toxins A and B in each condition.3.The TcdA and TcdB protein sequences were analyzed by bioinformatics.The TcdA and TcdB toxin proteins were purified by FPLC system.Binding of purified toxin A and B(labeled with Alexa-555),along with wheat germ agglutinin(labeled with Alexa-488 to selectively stain Gram-positive bacteria),to formalin-fixed bacteria harvested from healthy C57BL6 mice was determined by flow cytometry.We used increasing doses of lipoteichoic acid(LTA)to compete with toxin A’s binding to gut microflora to test whether LTA mediates binding of toxins.The protective effect of LTA was studied by analysis the cytotoxic effects of toxins on 3T3 fibroblasts and NCM460 cells in vitro using cell-rounding assay.Closed ileal loops were prepared at laparotomy in mice to examine the ability of LTA to inhibit the enterotoxicity of toxins in vivo.Purified LTA was evaluated as prophylaxis and treatment of CDI in mice.Results1.53 C.difficile isolates were successfully sequenced,50(94.3%)were A+B+,3(5.7%)were A-B+.We found the mutations within five toxin gene is highly linked.When tcdA cannot be detected,the mutations of tcdR didn’t participate in mutations of other toxin genes.The results of whole genome sequencing and first-generation sequencing were highly consistent,15 strains exist some gene serial substitute,38 does not exist any mutations.Functional enrichment analysis in GO biological pathways database were carried out,we found differential target genes in the biological pathway mainly in terms of a variety of metabolic processes,single molecule transport,gene expression etc.In the molecular function of gene products,mainly concentrated in the catalytic activity of many enzymes;there were also significant differences in cell components,membrane structure.Enrichment analysis on the biological pathways KEGG pathway database found five relevant pathways within which different genes were significantly enriched,two glucose metabolism related aspects,one associated with homocysteine metabolism,and one transport-related and one correlation with flagellar assembly.Clinical data of 53 patients were retrospectively analyzed,between the two groups there were no significant difference about the contents of gender,age,length of stay,serum creatinine,urea nitrogen,albumin,blood glucose,blood lipids and the basic state of patients(P>0.05),while the incidence of pseudomembranous colitis in patients infected with strains exist gene serial substitute(P<0.001)and the percentage of cells of neutrophils(P<0.05)were significantly higher than the non-mutated strains.C.difficile Mutants show higher cytotoxicity in vitro and in vivo.Zo-1 and Occludin in Caco-2 cells were down-regulated after a 24 h incubation with supernatants of C.difficile Mutants.The TEER value of HUVEC cells after adding the supernatant filtrate of Mutants C.difficile was significantly deceased compared with No-Mutants(P<0.05).The colonic pathological staining indicated that the intestinal exudation of the mice infected by the mutant strain increased obviously.2.We found that 7 CDI patients and 6 carriers had stools with detectable toxin A(range 23-17,422 pg/mL)but with toxin B below the clinical detection limit(<20 pg/mL,range 2-12.5pg/ml,median TcdA/TcdB ratio 17.93,range 6.3 to 1914.5);all 12 recovered isolates had both tcdA and tcdB.Concentrations of toxin A far exceeded B in supernatants from in vitro cultures of all 12 isolates(A/B median ratio 26,range 11.1 to 3577.6)at 72 hours.All supernatants tested positive by CTA.Of eight A;B isolates tested in mice,4 caused diarrhea,and 3 of those 4 caused lethal disease.Mouse cecal toxin concentrations measured for one CDI isolate(BID-71)were>200,000 pg/ml toxin A and～100 pg/ml toxin B.Ribotyping analyses indicated that the isolates are not clonal but rather a diverse set of strains.3.Toxins A and B bind to over 70%of Gram-positive bacteria from mouse gut at 0.5 Rg/ml and reach～100%binding at higher toxin doses.LTA inhibited the cytotoxic effects of toxin A and B on both test cells,in a dose-dependent fashion with complete inhibition at the highest LTA concentrations.Purified free-form LTA reduced binding of toxin A to mouse gut flora dose-dependently and neutralized enterotoxicity of toxin A in vivo.70%of mice treated with LTA prophylactically(0.25 mg/ml)survived CDI compared to 0%of controls(P<0.0001);post-infection LTA treatment(30mg/kg/day)also demonstrated a trend of protection,though not statistically significant.Conclusion1."Linked Mutations" within Pathogenicity Locus of C.difficile associated with increased risk of pseudomembranous colitis.Further study will be performed with expanding strains,so as to find appropriate molecular detection methods,and help hospitals to prevent and control CDI.2.We here report the discovery of clinical pathogenic C.difficile strains that produce high levels of toxin A but minimal toxin B.This pattern of toxin production is not rare(>5%of isolates)and is consistently observed in vitro and in vivo both in humans and mice.Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis,treatment and vaccine development.3.LTA-mediated neutralization of C.difficile toxins by gut microbiota serves as a protective barrier against CDI.This work adds novel concepts to the current"colonization resistance" theory in the field,which hypothesizes that commensal bacteria compete against C.difficile the whole bacteria for colonization "niches"within the host.