Phage transcriptional regulator X (PtrX)-mediated augmentation of toxin production and virulence inClostridioides difficilestrain R20291

Abstract Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenic locus (PaLoc) genes of C. difficile . We previously demonstrated that the genome of the C. difficile strain RT027 (NCKUH-21) contains a prophage-like DNA sequence, which was found to be markedly similar to that of the φCD38-2 phage. In the present study, we investigated the mechanisms underlying the φNCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of φNCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three φNCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA . In the ptrX -overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD , possibly through an increased flagellar phase ON configuration ratio. Author Summary Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacterium that can lead to antibiotic-associated diarrhea and pseudomembranous colitis. During the C. difficile infection (CDI), the major virulence factor is the secretion of two exotoxins, toxin A and B, to destroy host intestinal epithelium cells. The investigation of bacteriophages affecting the toxicity of C. difficile has increasingly been research. We previously isolated a C. difficile clinical strain NCKUH-21, which carried a phage-like DNA sequence, and named it φNCKUH-21. However, whether this prophage could enhance the virulence of C. difficile and the mechanism for regulating the pathogenicity are still unclear. We successfully created a φNCKUH-21-lysogenized R20291 strain and showed that lysogenized R20291 performed stronger pathogenicity than the wild-type R20291. We found that a φNCKUH-21-specific protein (encoded by NCKUH-21_03890 gene) might influence C. difficile flagellar phase variation to promote toxin production further. These findings are expected to clarify the mechanism for controlling the pathogenicity of φNCKUH-21-infected C. difficile . Moreover, we also believe that the existence of hypervirulent C. difficile strains carrying a prophage should be monitored proactively in hospitals to prevent severe CDI.