Abstract
Clostridium difficile remains a leading nosocomial pathogen, putting considerable strain on the healthcare system. The ability to form endospores, highly resistant to environmental insults, is key to its persistence and transmission. However, important differences exist between the sporulation pathways of C. difficile and the model Gram-positive organism Bacillus subtilis. Amongst the challenges in studying sporulation in C. difficile is the relatively poor levels of sporulation and high heterogeneity in the sporulation process. To overcome these limitations we placed Ptet regulatory elements upstream of the master regulator of sporulation, spo0A, generating a new strain that can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). We demonstrate that this strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. Induction of Spo0A is titratable and this can be used in the study of the spo0A regulon both in vitro and in vivo, as demonstrated using a mouse model of C. difficile infection (CDI). Insights into differences between the sporulation pathways in B. subtilis and C. difficile gained by study of the inducible strain are discussed, further highlighting the universal interest of this tool. The Ptet-spo0A strain provides a useful background in which to generate mutations in genes involved in sporulation, therefore providing an exciting new tool to unravel key aspects of sporulation in C. difficile.
Highlights
Clostridium difficile is a Gram-positive, anaerobic, spore-forming pathogen and the leading cause of healthcare associated diarrhea worldwide (Rupnik et al, 2009; Smits et al, 2016)
We demonstrate that spo0A expression can be induced in a dose-dependent manner and that the level of expression correlates with sporulation efficiency, which reaches values higher than those observed in an isogenic wild type (WT) strain
To exclude the possibility that ATc was exerting a deleterious effect on normal growth, WT and Ptet-spo0A cultures were incubated in the presence or absence of ATc and their growth rate was monitored over time by measuring optical density (OD600)
Summary
Clostridium difficile is a Gram-positive, anaerobic, spore-forming pathogen and the leading cause of healthcare associated diarrhea worldwide (Rupnik et al, 2009; Smits et al, 2016). It is the spore that is the primary infectious agent, as mutant strains defective in sporulation are unable to efficiently persist in the environment and transmit disease (Deakin et al, 2012). Due to their multi-layered structure, spores are extremely robust and resistant to both chemical and physical insults, enabling C. difficile to survive exposure to heat, oxygen, alcohol, noxious chemicals, and certain disinfectants. Despite its key relevance in CDI, many aspects of sporulation in C. difficile are still understudied, hampered by low sporulation efficiency and high heterogeneity in the sporulation process
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