| Abstrakt: |
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. Author summary: Phase variation is a mechanism by which many bacteria introduce phenotypic heterogeneity into a population as a bet-hedging strategy to help ensure survival under detrimental conditions. In C. difficile, the intracellular signaling molecule c-di-GMP regulates production of flagella, toxins, adhesins, and other factors that impact virulence. C. difficile encodes multiple c-di-GMP synthases and hydrolases that modulate intracellular c-di-GMPs and control these processes. Here, we show that production of a c-di-GMP hydrolytic enzyme, PdcB, undergoes phase variation in C. difficile. We generated phase-locked mutants unable to phase vary and found that PdcB affects global intracellular c-di-GMP levels, swimming and surface motility, and biofilm formation. These findings suggest that phase variation of PdcB enables C. difficile to coordinately regulate the production multiple factors by generating heterogeneity in intracellular c-di-GMP levels among bacteria in the population. [ABSTRACT FROM AUTHOR] |
