Cui Guzhen, Zhou Qingshuai, Cheng Qinquan, Rao Fengqin, Cheng Yumei, Tian Yan, Zhang Ting, Chen Zhenghong, Liao Jian, Guan Zhizhong, Qi Xiaolan, Wu Qi, Hong Wei
Abstract
ObjectiveComparative 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 and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioidesdifficile.
MethodsAnalysis of Cd630 and ΔPaLoc 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 of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines.
ResultsThe transcriptome data showed that the ΔPaLoc 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 in ΔPaLoc 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 the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain.
ConclusionTranscriptional sequencing analysis of the Cd630 and ΔPaLoc 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 the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.
Key words:
Gene knockout techniques; Clustered regularly interspaced short palindromic repeats; Bacterial toxins; Clostridioides difficile; Attenuated mutants
Contributor Information
Cui Guzhen
Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou, Guizhou Medical University, Guiyang 550004, China
Zhou Qingshuai
Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education &
Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550001, China
Cheng Qinquan
Department of Clinical Laboratory, Shanghai 10th People′s Hospital of Tongji University, Shanghai 200072, China
Rao Fengqin
Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education &
Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550001, China
Cheng Yumei
General ICU of the Affiliated Hospital of Guizhou Medical University, Guiyang 550001, China
Tian Yan
Guizhou Polytechnic of Construction, Qingzhen 551400, China
Zhang Ting
Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education &
Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550001, China
Chen Zhenghong
Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou, Guizhou Medical University, Guiyang 550004, China
Liao Jian
Stomatological Hospital of Guizhou Medical University, Guiyang 550001, China
Guan Zhizhong
Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education &
Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550001, China
Qi Xiaolan
Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education &
Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550001, China
Wu Qi
Department of Clinical Laboratory, Shanghai 10th People′s Hospital of Tongji University, Shanghai 200072, China
Hong Wei
Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou, Guizhou Medical University, Guiyang 550004, China
Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education &
Key Laboratory of Medical Molecular Biology of Guizhou Province, Guizhou Medical University, Guiyang 550001, China
