The Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene () encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the gene has yet been characterized. We constructed a gene deletion mutant using Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 (CRISPR-Cpf1) system. The phenotypic and transcriptomic changes of the Δ mutant compared with the wild-type (WT) strain were studied. The deletion of the gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the Δ mutant is more sensitive than the WT strain to clindamycin, ampicillin, and erythromycin but more resistant than the latter to vancomycin and metronidazole. Moreover, mannitol utilization capability in the Δ mutant was lost. Comparative transcriptomic analyses indicated that (i) 22.90-fold upregulation of gene and unable to express gene were prominent in the Δ mutant; (ii) the gene was involved in vancomycin resistance of C. difficile by influencing the expression of d-Alanine-d-Alanine ligase; and (iii) the mannose/fructose/sorbose IIC and IID components were upregulated in Δ mutant. The present work deepens our understanding of the contribution of the gene to cell adhesion, stress tolerance, antibiotic resistance, and mannitol transportation of C. difficile. The cell wall protein 66 gene () encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the gene has yet been characterized. The current study provides direct evidence that the gene serves as a major adhesion in C. difficile, and also suggested that deletion of the gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the antibiotic resistance and carbon source utilization profiles of the Δ mutant were significantly changed. These phenotypes were detrimental to the survival and pathogenesis of C. difficile in the human gut and may shed light on preventing C. difficile infection.