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

Abstract

AbstractClostridioides difficileis a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors ofC. difficileare 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 ofC. difficile. We previously demonstrated that the genome of theC. difficilestrain 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 lysogenizedC. difficilestrain 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 ofptrXled to the upregulation of the expression of toxin genes, flagellar genes, andcsrA. In theptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor genesigD, possibly through an increased flagellar phase ON configuration ratio.Author SummaryClostridioides difficileis a Gram-positive, spore-forming anaerobic bacterium that can lead to antibiotic-associated diarrhea and pseudomembranous colitis. During theC. difficileinfection (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 ofC. difficilehas increasingly been research. We previously isolated aC. difficileclinical strain NCKUH-21, which carried a phage-like DNA sequence, and named it φNCKUH-21. However, whether this prophage could enhance the virulence ofC. difficileand 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 byNCKUH-21_03890gene) might influenceC. difficileflagellar phase variation to promote toxin production further. These findings are expected to clarify the mechanism for controlling the pathogenicity of φNCKUH-21-infectedC. difficile. Moreover, we also believe that the existence of hypervirulentC. difficilestrains carrying a prophage should be monitored proactively in hospitals to prevent severe CDI.