Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile

Abstract

The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or ‘pdcB switch’, determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes, indicating that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.

Evolution of cyclic di-GMP signalling on a short and long term time scale

“…However, also phenotypes other than biofilm formation are subject to cyclic di-GMP signalling mediated regulation. As a reversible mechanism, phase variation in the promoter region of a cyclic di-GMP specific phosphodiesterase has been observed to lead to a hypo- versus hyper-sporulation phenotype of Clostridioides difficile [38, 39].…”

An intact S-layer is advantageous to Clostridioides difficile within the host

“…Regulation of toxin production is highly complex with multiple mechanisms of regulation reported to influence expression in response either directly (CodY, CcpA) or indirectly via interactions with tcd R (CodY, RstA, CcpA) in response to various stimuli including nutrient availability and quorum sensing [ 43 ]. More recently, c-di-GMP signalling and phase variation via seven invertible switches have been implicated in modulation of expression of genes associated with motility, toxin production and colony morphology [ 44 , 45 ]. Currently, it is unclear what mechanisms are involved in the observed altered toxin transcripts expression in FM2.5 and FM2.5 varB and reduced toxin production by FM2.5 during early growth in culture.…”

Clostridioides difficile-mucus interactions encompass shifts in gene expression, metabolism, and biofilm formation

“…E. coli strains were grown aerobically at 37°C in lysogeny broth (LB, Miller), and conjugations with C. difficile performed anaerobically. Methods for cloning and C. difficile mutagenesis, as previously applied 77,78 , are detailed in Text S1. Preparation of C. difficile cultures for growth assays are detailed in Text S1.…”

“…We also narrowed the list of genes applied to GSEAPreranked to only those meeting a differential expression threshold (Benjamini-Hochberg corrected p < 0.05, fold change > 2), using Log 2 fold change as the ranking metric. Select differentially expressed genes were validated using qRT-PCR as previously described 78,88,89 . Text S1 contains further RNA isolation, read processing, and qRT-PCR details.…”