Role of Glycogen Metabolism inClostridioides difficileVirulence

Abstract

AbstractGlycogen plays a vital role as an energy reserve in various bacterial and fungal species.Clostridioides difficilepossesses a glycogen metabolism operon that contains genes for both glycogen synthesis and utilization. In our investigation, we focused on understanding the significance of glycogen metabolism in the physiology and pathogenesis ofC. difficile. To explore this, we engineered aC. difficileJIR8094 strain lacking glycogen synthesis capability by introducing a group II intron into theglgCgene, the operon’s first component. Quantification of intracellular glycogen levels validated the impact of this modification. Interestingly, the mutant strain exhibited a 1.5-fold increase in toxin production compared to the parental strain, without significant changes in sporulation rate. Our analysis also revealed that wild-typeC. difficilespores contained glycogen, whereas spores from the mutant strain lacking stored glycogen showed increased sensitivity to physical and chemical treatments and had a shorter storage life. By suppressingglgPexpression, the gene coding for glycogen-phosphorylase, via CRISPRi, we demonstrated that glycogen accumulation but not the utilization is needed for spore resilience inC. difficile. Transmission Electron Microscopy (TEM) analysis revealed a significantly lower core/cortex ratio inglgCmutant strain spores. In hamster challenge experiments, both the parental andglgCmutant strains colonized hosts similarly; however, the mutant strain failed to induce infection relapse after antibiotic treatment cessation. These findings highlight the importance of glycogen metabolism inC. difficilespore resilience and suggest its role in disease relapse.ImportanceThis study on the role of glycogen metabolism inC. difficilehighlights its critical involvement in the pathogen’s energy management, its pathogenicity and resilience. Our results also revealed that glycogen presence in spores is pivotal for their structural integrity and resistance to adverse conditions, which is essential for their longevity and infectivity. Importantly, the inability of the mutant strain to cause infection relapse in hamsters post-antibiotic treatment pinpoints a potential target for therapeutic interventions, highlighting the importance of glycogen in disease dynamics. This research thus significantly advances our understanding ofC. difficilephysiology and pathogenesis, offering new avenues for combating its persistence and recurrence.