Kamboj, M. | bioRxiv Science

bioRxiv2023

Flagellar switch inverted repeat sequence variation impacts Clostridioides difficile RT027/MLST1 virulence.

Clostridioides difficile RT027 strains cause infections that vary in severity from asymptomatic to lethal, but the molecular basis for this variability is poorly understood. Through comparative analyses of RT027 clinical isolates, we determined that isolates that exhibit greater variability in their flagellar gene expression exhibit greater virulence in vivo. C. difficile flagellar genes are phase-variably expressed due to the site-specific inversion of the flgB 5UTR region, which reversibly generates ON vs. OFF orientations for the flagellar switch. We found that longer inverted repeat (IR) sequences in this switch region correlate with greater disease severity, with RT027 strains carrying 6A/6T IR sequences exhibiting greater phenotypic heterogeneity in flagellar gene expression (60%-75% ON) and causing more severe disease than those with shorter IRs (> 99% ON or OFF). Taken together, our results reveal that phenotypic heterogeneity in flagellar gene expression may contribute to the variable disease severity observed in C. difficile patients.

microbiology↗

bioRxiv2023

Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile

Clostridioides difficile (C. difficile), a leading cause of nosocomial infection, produces toxins that damage the colonic epithelium and results in colitis that varies from mild to fulminant. Variation in disease severity is poorly understood and has been attributed to host factors (age, immune competence and intestinal microbiome composition) and/or virulence differences between C. difficile strains, with some, such as the epidemic BI/NAP1/027 (MLST1) strain, being associated with greater virulence. We tested 23 MLST1(ST1) C. difficile clinical isolates for virulence in antibiotic-treated C57BL/6 mice. All isolates encoded a complete Tcd pathogenicity locus and achieved similar colonization densities in mice. Disease severity varied, however, with 5 isolates causing lethal infections, 16 isolates causing a range of moderate infections and 2 isolates resulting in no detectable disease. The avirulent ST1 isolates did not cause disease in highly susceptible Myd88-/- or germ-free mice. Genomic analysis of the avirulent isolates revealed a 69 base-pair deletion in the N-terminus of the cdtR gene, which encodes a response regulator for binary toxin (CDT) expression. Genetic deletion of the 69 base-pair cdtR sequence in the highly virulent ST1 R20291 C. difficile strain rendered it avirulent and reduced toxin gene transcription in cecal contents. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile strain without reducing colonization and persistence in the gut. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.

microbiology↗