艰难拟梭菌PaLoc致病决定区基因座敲除突变株转录组学分析及其减毒验证

Transcriptomic analysis of theΔPaLoc mutant of Clostridioides difficile and verification of its toxicity

目的对艰难拟梭菌630(Cd630)菌株和致病决定区基因座(PaLoc)敲除突变株(ΔPaLoc)进行转录组测序和分析,获得Cd630野生型菌株和ΔPaLoc基因的差异性表达谱,并测定野生型Cd630菌株和ΔPaLoc突变株的细胞毒力,为构建艰难拟梭菌全菌减毒疫苗奠定基础。方法对Cd630菌株和ΔPaLoc敲除突变株进行转录组测序,接着对差异表达基因进行筛选,随后对差异基因进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析。最后在非洲绿猴肾细胞(Vero)和人克隆结肠腺癌细胞株(Caco-2)中进行Cd630野生型菌株和ΔPaLoc敲除突变菌株的细胞毒性验证实验。结果转录组数据表明ΔPaLoc突变株毒素基因tcdA、tcdB未转录。CD630_36010、CD630_020910、CD630_02080和cel等基因分别上调17.92、11.40、8.93和7.55倍;编码高丝氨酸脱氢酶的hom2基因、孢子形成蛋白基因CD630_15810、锌结合脱氢酶基因CD630_23230、1-磷酸半乳糖醇5-脱氢酶基因CD630_23240分别下调为野生型的0.06、0.075、0.133和0.183倍。GO和KEGG富集分析结果表明,ΔPaLoc突变株差异转录基因主要富集于密度感应系统、ABC运输系统、双组分系统、磷酸转移酶(PTS)系统、糖代谢通路以及万古霉素耐药相关的通路等。细胞毒力实验结果表明野生型Cd630菌株对Vero细胞和Caco-2细胞具有毒力,ΔPaLoc突变株丧失对Vero细胞和Caco-2细胞的毒力。结论通过对Cd630和ΔPaLoc突变株进行转录测序,表明毒素基因未转录,其他差异性基因可为进一步研究ΔPaLoc突变株生理生化特征提供指引。细胞毒力实验表明,ΔPaLoc突变株丧失了对Vero细胞和Caco-2细胞的毒力,ΔPaLoc突变株为艰难拟梭菌全菌减毒疫苗的构建奠定了基础。

Objective Comparative analyses of wild-type Clostridioides difficile 630(Cd630)strain and pathogenicity locus(PaLoc)knockout mutant(ΔPaLoc)by using RNA-seq technology.Analysis of differential expression of Cd630 wild-type strain andΔPaLoc mutant strain and measurement of its cellular virulence changes.Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioidesdifficile.Methods Analysis of Cd630 andΔPaLoc mutant strains using high-throughput sequencing(RNA-seq).Clustering differentially expressed genes and screening differentially expressed genes by DESeq software.Further analysis of differential genes using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment.Finally,cytotoxicity assays ofΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell(Vero)and the human colonic cell(Caco-2)lines.Results The transcriptome data showed that theΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed.Compared to the wild-type strain,CD630_36010,CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold,respectively.Whereas the hom2(high serine dehydrogenase),the CD630_15810(spore-forming protein),CD630_23230(zinc-binding dehydrogenase)and CD630_23240(galactitol 1-phosphate 5-dehydrogenase)genes were down-regulated by 0.06,0.075,0.133 and 0.183 fold,respectively.The GO and KEGG enrichment analyses showed that the differentially transcribed genes inΔPaLoc were enriched in the density-sensing system,ABC transport system,two-component system,phosphotransferase(PTS)system,and sugar metabolism pathway,as well as vancomycin resistance-related pathways.Cytotoxicity assays showed that theΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain.Conclusion Transcriptional sequencing analysis of the Cd630 andΔPaLoc mutant strains showed that the toxin genes were not transcribed.Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of theΔPaLoc mutant strain.Cytotoxicity assays confirmed that theΔPaLoc mutant lost virulence to Vero and Caco-2 cells,thus laying the foundation for constructing an toxin-attenuated vaccine strain against C.difficile.