Carson, C. S. | bioRxiv Science

bioRxiv2026

Contact dependent suppression of Clostridioides difficile sporulation by enterococci requires the endocarditis and biofilm pilus

Clostridioides difficile is a healthcare-associated infection that arises when broad-spectrum antibiotic treatment disrupts the gut microbiota and is transmitted by highly resistant spores. Vancomycin-resistant Enterococcus faecium (VRE) is an opportunistic pathogen frequently co-isolated from C. difficile patients. We found that C. difficile sporulation is significantly reduced in VRE-C. difficile co-culture. Physical separation of C. difficile and VRE in transwell co-culture restored sporulation. Mixed macrocolony culture assays on solid agar confirmed physical contact is necessary for sporulation inhibition. We screened a panel of enterococci and found that most strains reduce sporulation, except Enterococcus saccharolyticus, which lacks predicted surface displayed virulence factors in its genome. We performed a candidate gene screen using an Enterococcus faecalis OG1RF transposon library and found that an insertion in the major pilin ebpC partially restored C. difficile sporulation in co-culture. These data were confirmed with in-frame deletions in the ebpABC pilus operon and a clinical isolate of E. feacalis lacking ebpABC. These findings suggest enterococci modulate C. difficile sporulation through a contact-dependent mechanism involving the Ebp pilus. ImportanceA characteristic of C. difficile infection is multiple episodes of acute disease. Spores are the transmission vector of C. difficile and are necessary for recurrence in models of disease. Our research demonstrates that C. difficile spore production is significantly reduced in the presence of enterococci, a common group of beneficial and pathogenic bacteria present in the gut microbiota. Physical contact with enterococci reduces C. difficile spore production. We attribute this effect to a protein structure on the surface of enterococci. This finding suggests a potential role for enterococci and the gut microbiota in general to uncover regulators of C. difficile spore formation. This may provide an avenue for innovative treatment strategies that reduce spore formation.

microbiology↗

bioRxiv2023

A Nanoparticle RIG-I Agonist for Cancer Immunotherapy

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving response to immune checkpoint inhibitors (ICI). However, the potency and clinical efficacy of 5-triphosphate RNA (3pRNA) agonists of RIG-I is hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of [~]100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to PD-1 ICI in an orthotopic EO771 model of triple negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined PD-1 + CTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with potential to enhance response to ICIs and other immunotherapeutic modalities.

bioengineering↗