Receptor Selectivity And Pathological Analysis Of Clostridioides Difficile Toxin B Variants

Clostridioides difficile(C.difficile)is a major pathogen causing antibiotic-associated diarrhea in hospitals and communities.It is widely found in human intestines,animals,residential areas and natural environments,and has been isolated and detected from meat products,seafood,vegetables and other foods.C.difficile can be transmitted between people,food and the environment.When antibiotic treatment destroys the inherent intestinal flora,spores from food or the environment can be ingested orally,ger minate in the gut and multiply,causing Clostridioides difficile infection(CDI).CDI can lead to C.difficile-associated diseases(CDAD)such as diarrhea,pseudomembranous colitis,and megacolon in the host.It is a worldwide health care problem with high morbidity and mortality.The elderly and hospitalized patients are extremely susceptible to infection.The pathogenicity of C.difficile mainly comes from three exotoxins it secreted,namely toxin A(TcdA),toxin B(TcdB)and C.difficile transferase toxin(CDT).Among them,TcdB is considered to be the most important virulence factor.Since 1978,when C.difficile infection was first reported to be associated with antibiotic overuse,researchers have isolated and identified a large number of clinically relevant strains,many of which have been found to contain some mutant TcdB.TcdB can be divided into 8subtypes or variants according to their sequence mutations.In this study,we expressed and purified the 8 TcdB subtypes from C.difficile,and then tested their binding ability to different receptors.The do minant strains(TcdB1,TcdB2,TcdB3 and TcdB4)expressed in the epidemic strains were also studied by structural and mechanistic functional analysis and pathological phenotype verification.By validation on different receptor knockout cells,we found that these subtypes showed a high degree of diversity in the preferences of known receptors chondroitin sulfate polysaccharide 4(CSPG4)and frizzled family proteins(FZDs,mainly FZD1/2/7): TcdB1 binds to two known receptors,CSPG4 and FZDs proteins.TcdB2 selectively binds to CSPG4,TcdB3 binds FZDs more easily,while TcdB4 binds neither CSPG4 nor FZDs at all.By constructing TcdB1-TcdB3 chimeric proteins and subsequent functional verification,we deter mined that the N-ter minal cysteine protease domain(CPD)was involved in the recognition of CSPG4.Through systematic mutation screening and residues switching between TcdB1 and TcdB3,we found the key a mino acid residues(G624,G626)of TcdB-CPD interacting with CSPG4.Combining the reported key a mino acid residues that bind to CSPG4 at the C-ter minal of TcdB,we found that although these sites are distributed in multiple non-adjacent domains,they are close to each other in spatial position,for ming a convergent functional region.We further evaluated the pathological phenotype caused by TcdB1～4 using a mouse rectal perfusion model,and the results showed that TcdB1 caused the most severe overall symptoms,followed by TcdB2 and TcdB3.When comparing the pathological phenotypes caused by TcdB2 and TcdB3,TcdB2 caused more edema,while TcdB3 caused more inflammatory cell infiltration.These findings together demonstrate the selectivity with a bias of the binding receptors of the TcdB subtypes from the epidemic strains and further lead to differences in the pathological phenotypes in their colons.In this study,we mainly researched the differences in host receptor recognition and pathological induction among the four prevalent subtypes of TcdB,and found the key domains and key a mino acid residues that affect the ability of different subtypes to bind receptors,suggesting that the differences in receptor binding preference among TcdB subtypes are related to the pathogenesis of CDAD.Therefore,our study provides new insights into the toxicology and pathology of C.difficile toxin variants,which are critical to the developing tests for C.difficile in food and effective treatments for C.difficile infections.