Phi027 prophage shapes Clostridioides difficile virulence through the SinR/SinR’ axis and the flagellar switch network

Abstract

Clostridioides difficile is a major nosocomial pathogen whose virulence depends on toxin production and can be shaped by prophage carriage. Here, we dissect the molecular basis of phi027-dependent phenotypic differences between the clinical RT176 strain 500/12 and its prophage-free derivative CKH08. Comparative transcriptomic analysis showed that loss of phi027 is associated with reduced expression of the sinR / sinR ′ operon, broad downregulation of flagellar biosynthesis genes, and a shift of the flagellar switch towards the OFF orientation. In total, 166 genes were differentially expressed (|log2FC| ≥ 1, adjusted p  < 0.05), including 114 chromosomal host genes. These transcriptional changes align with previously observed reductions in adhesion, sporulation efficiency, and toxin-associated phenotypes in CKH08. To determine whether phi027-dependent differences extend to a whole-organism context, we compared the virulence of strains 500/12 and CKH08 in the Galleria mellonella infection model. Larvae infected with the phi027-lysogenic strain 500/12 showed significantly reduced survival ( p  < 0.001, log-rank test) and enhanced melanization relative to those infected with CKH08. Collectively, our findings indicate that phi027 carriage is associated with extensive rewiring of virulence-related regulatory pathways and implicate the SinR/SinR′-SigD-flagellar switch network as a key mediator of phi027-dependent phenotypic effects. These results support prophage-mediated regulation as an important contributor to the pathogenic potential of epidemic C. difficile lineages.