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. These results indicate that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.

Multiple Regulatory Mechanisms Control the Production of CmrRST, an Atypical Signal Transduction System in Clostridioides difficile

“…The Cdi1 invertible element mediates phase variation of CwpV, which contributes to phage resistance ( 28 – 31 ). The Cdi2 element, or pdcB switch, modulates the production of a c-di-GMP phosphodiesterase and affects intracellular c-di-GMP and related phenotypes ( 32 , 33 ).…”

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

“… 24 , 29 Four 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 the CmrRST signal transduction system, 26 and the phosphodiesterase PdcB. 32 , 33 …”