Orlaith Keenan

Clostridioides difficile causes severe disease in adults but commonly colonizes infants asymptomatically. The consequences of early-life colonization on host development remain unknown. In a neonatal mouse model, C. difficile colonization drove proinflammatory and tissue repair responses in the intestinal epithelium, enriching injury-associated intestinal stem cell populations and skewing differentiation toward secretory lineages. Despite transient colonization, exposure to C. difficile early in life led to persistent changes into adulthood. Epithelial responses were toxin dependent, as colonization with nontoxigenic strains or maternal vaccination with a C. difficile –targeted messenger RNA–lipid nanoparticle vaccine protected neonates. Human infant intestinal epithelial cells were sensitive to C. difficile toxins, and biopsies from colonized infants exhibited altered intestinal stem cell behavior. This study redefines C. difficile as an underappreciated early-life pathogen with lasting effects on host development.

Clostridioides difficile is the leading cause of nosocomial infections and an urgent public health threat. This bacterial pathogen is challenging to treat due to antibiotic resistance and high recurrence rates, highlighting the need for additional therapeutic strategies. Host inflammatory responses are major drivers of disease severity during C. difficile infection (CDI), yet few therapeutic strategies target these pathways. Here, we show that the prostaglandin E ₁ (PGE ₁ ) analog misoprostol markedly reduces CDI severity by modulating host immune responses. During CDI, misoprostol decreases circulating neutrophils and limits infiltration into the colon, reducing epithelial damage, intestinal pathology, and infection severity. Additionally, misoprostol reduces serum granulocyte colony-stimulating factor (G-CSF), an important cytokine in neutrophil mobilization, controlling neutrophil levels during CDI. Together, these findings highlight neutrophil infiltration as a key driver of C. difficile- associated disease and identify innate immune modulation as a potential host-directed therapeutic strategy. IMPORTANCE: Clostridioides difficile remains a leading cause of antibiotic-associated diarrhea worldwide and a major healthcare burden. While antibiotics are commonly used to treat CDI, high antibiotic resistance and recurrence rates highlight the need for additional therapeutic approaches. Here, we demonstrate that modulation of the innate immune response can mitigate disease severity. Administration of the prostaglandin analog misoprostol reduces neutrophil availability during CDI, resulting in decreased inflammation and improved outcomes. These findings suggest that fine-tuning neutrophil responses may present a promising host-directed strategy to improve CDI outcomes.

Clostridioides difficile is the leading cause of nosocomial infections and an urgent public health threat. This bacterial pathogen is challenging to treat due to antibiotic resistance and high recurrence rates, highlighting the need for additional therapeutic strategies. The host inflammatory response is a major driver of C. difficile -associated disease and associated with worse clinical outcomes. Currently, few strategies targeting the inflammatory response have been leveraged to treat CDI. Here, we show that administration of the prostaglandin E ₁ (PGE ₁ ) analog misoprostol markedly reduces CDI severity by modulating host immune responses. During CDI, misoprostol decreases circulating neutrophils and limits infiltration into the colon, reducing epithelial damage, intestinal pathology, and infection severity. Additionally, misoprostol reduces serum granulocyte colony-stimulating factor (G-CSF), an important cytokine in neutrophil mobilization, controlling neutrophil levels during CDI. Together, these findings highlight neutrophil infiltration as a key driver of C. difficile- associated disease and identify innate immune modulation as a potential host-directed therapeutic strategy.

The gram-negative bacterium Kingella kingae is a leading cause of osteoarticular infections in young children and initiates infection by colonizing the oropharynx. Adherence to respiratory epithelial cells represents an initial step in the process of K. kingae colonization and is mediated in part by type IV pili. In previous work, we observed that elimination of the K. kingae PilC1 and PilC2 pilus-associated proteins resulted in non-piliated organisms that were non-adherent, suggesting that PilC1 and PilC2 have a role in pilus biogenesis. To further define the functions of PilC1 and PilC2, in this study we eliminated the PilT retraction ATPase in the ΔpilC1ΔpilC2 mutant, thereby blocking pilus retraction and restoring piliation. The resulting strain was non-adherent in assays with cultured epithelial cells, supporting the possibility that PilC1 and PilC2 have adhesive activity. Consistent with this conclusion, purified PilC1 and PilC2 were capable of saturable binding to epithelial cells. Additional analysis revealed that PilC1 but not PilC2 also mediated adherence to selected extracellular matrix proteins, underscoring the differential binding specificity of these adhesins. Examination of deletion constructs and purified PilC1 and PilC2 fragments localized adhesive activity to the N-terminal region of both PilC1 and PilC2. The deletion constructs also localized the twitching motility property to the N-terminal region of these proteins. In contrast, the deletion constructs established that the pilus biogenesis function of PilC1 and PilC2 resides in the C-terminal region of these proteins. Taken together, these results provide definitive evidence that PilC1 and PilC2 are adhesins and localize adhesive activity and twitching motility to the N-terminal domain and biogenesis to the C-terminal domain.