Study On The Regulation Mechanism Of Toxin Production Response To Cysteine In Clostridium Difficile Based On Transcriptomic Analysis

Clostridium difficile?C.difficile?is a Gram-positive,spore-forming,obligately anaerobic gastrointestinal bacterium that causes antibiotic-associated diarrhea and pseudomembranous colitis.Due to the widespread use of broad-spectrum antibiotics and the emergence of the hypervirulent C.difficile strains,the morbidity and mortality of C.difficile infection?CDI?have increased in the past two decades.CDI has become one of the most important infectious disease threats to global public health.The long-term or misuse of antibiotics could destroy the balance of intestinal microflora,which might provide a perfect condition for C.difficile to grow rapidly and produce a large number of TcdA and TcdB.These two exotoxins are the major virulence factors which could cause intestinal inflammation and then lead to CDI diseases.Early studies showed that toxin synthesis in C.difficile was impacted by environmental factors,such as carbon source,nitrogen source,temperature,and redox.However,the molecular regulation mechanism of toxin expression in C.difficile is not very clear.In this study,we investigated the effects of different amino acids on toxin levels in C.difficile,the transcriptome analysis of C.difficile response to cysteine,the role of CdsB on the regulation of toxin synthesis in the presence of cysteine,and the effect of CsrA on multiple virulence-associated processes in C.difficile.These results not only provide a huge amount of basic data for the future study on C.difficile and a reference to explore the regulation mechanism of toxin synthesis in C.difficile,but also provide theoretical basis for preventing CDI and developing new effective treatment.The major contents and conclusions are as follows:The effects of different amino acids?cysteine,proline,valine,tryptophan,arginine,leucine,and isoleucine?on the toxin levels were evaluated,and the results showed that cysteine could significantly inhibit toxin levels of C.difficile R20291.The RNA-Seq analysis of C.difficile cells cultured in the presence and absence of 5 mM cysteine for 4 h,8 h and 12h showed 216,469 and 756 differentially expressed genes?DEGs?,respectively?the fold ratio?2 times,and the p-value?0.01?.The response of C.difficile to cysteine is very complex and systematic and involves many cellular processes.We speculated that the iron transport,amino acids metabolism,energy metabolism,and flagellar synthesis may play important role in cysteine-dependent regulation of toxin production.C.difficile cdsB,eutW,eutVW,feoB1,feoB2,and feoB3 mutant strains were contructed using ClosTron system and the toxin levels of these strains cultured in TY or TYC medium were compared with that of the wild-type?WT?strain.The results showed that the toxin levels of all these strains had no significant difference compared with that of the WT strain in TY broth,while cysteine significantly repressed toxin production of all these strains except the cdsB mutant strain.The function of CdsB and its role on the cysteine-dependent repression of toxin production in C.difficile was further studied.When C.difficile was cultured in TYC broth,the cdsB gene was rapidly induced.The inactivation of cdsB altered the expression levels of intracellular cysteine-degrading enzymes and the production of hydrogen sulfide,which suggested that CdsB is a major inducible cysteine-degrading enzyme in C.difficile.The effects of Na₂S,pyruvate,and NH₄Cl on toxin production of C.difficile were further analyzed,and the results showed that Na₂S could also significantly repress toxin production.The inactivation of the cdsB gene failed to remove the Na₂S-dependent repression,which suggested that the cysteine-dependent repression of toxin production is closely associated with cysteine degradation and might be due to the accumulation of cysteine by-products.We also mapped a?⁵⁴?SigL?-dependent promoter upstream from the cdsB gene,and cdsB expression was not induced response to cysteine in the cdsR or sigL mutant strains.CsrA is a global post-transcriptional regulator that enables regulation of multiple virulence-associated processes in many pathogenic bacteria.We constructed C.difficile csrA overexpressing strain and found that overexpression of csrA in C.difficile could cause flagella defect and poor motility.The levels of toxin production and solvent production were increased in the csrA overexpressing strain.Moreover,csrA overexpression enhanced the adherence ability to Caco-2 cells.These results suggested that CsrA has an important role in the regulation of virulence-associated processes and carbon metabolism in C.difficile.