Expanding the repertoire of conservative site-specific recombination in Clostridioides difficile

Abstract

Recent genomic analysis of an epidemic ribotype 027 (RT027) Clostridioides difficile strain revealed the presence of several chromosomal site-specific invertible sites hypothesized to control the expression of adjacent genes in a bimodal on-off mode. This process, named phase variation, is thought to enhance phenotypic variability under homogeneous conditions ultimately increasing population fitness in un-predictable environmental fluctuations. The full extent of phase variation mediated by DNA-inversions in C. difficile is currently unknown. Here, we sought to expand our previous analysis by screening for site-specific inversions in isolates that belong to the rapidly emerging ribotypes RT017 and RT078. We report the finding of one novel inversion site for which we demonstrate the inversion potential and quantify inversion proportions during exponential and stationary growth in both historic and modern isolates of the same ribotype. We then employ a computational approach to assess the prevalence of all sites identified so far in a large collection of sequenced C. difficile isolates. We show that phase-variable loci are widespread with some sites being present in virtually all analyzed strains. Furthermore, in our small subset of RT017 and RT078 strains, we detect no evidence of gain or loss of invertible sites in historic versus modern isolates demonstrating the relative stability of those genomic elements. Overall, our results support the idea that C. difficile has adopted phase variation mediated by DNA inversions as its major generator of diversity which could be beneficial during the pathogenesis process.

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence

“…difficile R20291. Gray triangles represent inverted repeats flanking invertible elements, which are named according to previously defined nomenclature [29]. Downstream regulated genes are shown.…”

“…In C . difficile , eight invertible DNA sequences, or “switches,” have been identified, seven of which are present in the epidemic strain R20291 [28,29]. Two have been characterized: one controlling phase variation of cell wall protein V (CwpV) and the other controlling flagellar phase variation [19,30–32].…”

Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection

“…27,28 Eight DNA sequences that can undergo inversion have been identified in C. difficile, though individual strains may contain only a subset. 24,29 Three of these sequences have been experimentally demonstrated to modulate expression of adjacent genes leading to phase variation of the encoded factors: the cell wall protein CwpV, 22,30 flagella, 23,31 and the CmrRST signal transduction system. 26 C. difficile flagella are required for swimming motility and contribute to adherence to intestinal epithelial cells, colonization, and virulence in animal models of infection.…”

Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile

“…Two additional potential DNA inversions were identified upstream of genes involved in cyclic diguanylate metabolism [42]. Most recently, high-throughput sequencing was used to identify a total of seven invertible sequences flanked by inverted repeats in the C. difficile NAP1/B1/027 ribotype strain R20291, and an eighth in several 017 ribotype strains [43,44]. The identified sequences…”

“…Most recently, high-throughput sequencing was used to identify a total of seven invertible sequences flanked by inverted repeats in the C . difficile NAP1/B1/027 ribotype strain R20291, and an eighth in several 017 ribotype strains [ 43 , 44 ]. The identified sequences were shown to undergo inversion, and the sequence upstream of the cmrRST operon was confirmed to regulate expression of the downstream genes in a manner consistent with phase variation [ 44 , 45 ].…”