Pareek, N. | bioRxiv Science

bioRxiv2025

CD38⁺ Endothelial Remodeling Defines Spatially Diverse Vasculopathy Programs in Rapidly Advancing Oral Inflammation

Oral inflammatory diseases affect nearly half of the global population. Among them, newly defined peri-implantitis and high-grade periodontitis represent rapidly advancing inflammatory disease types, marked by relatively rapid tissue destruction. Despite their prevalence, the cell mechanisms and spatial architecture driving this severity remain poorly understood. Focusing first on peri-implantitis versus low- and moderate-grade periodontitis, we applied microbial profiling, single-cell RNA sequencing (scRNA-seq), and spatial proteomics (sp-proteomics) to uncover shared pathogenic programs linked to accelerated niche breakdown. Furthermore, to preserve spatial fidelity, each tissue was anatomically orientated along the tooth- or implant- epithelial interface, analogous sites of disease origination. Laser capture microdissection followed by microbiome analysis of unique tissue compartments revealed reduced bacterial load and diversity in peri-implantitis stroma. We then expanded our version-1 Human Periodontal Atlas by integrating newly generated peri-implantitis scRNAseq data (36-total samples; 121395-cells), revealing widespread transcriptional alterations, including oxidative stress, hypoxic, and NAD+ metabolism-associated signatures, primarily in a subpopulation of TNFRSF6B+/ICAM1+post-capillary venules. We then performed high-resolution sp-proteomics (15-total samples; 337260-cells) and analyzed VEC states and associated neighborhoods via AstroSuite using newly developed tri-wise spatial analysis. This revealed CD34+-VEC loss and CD38+-VEC expansion almost exclusively in peri-implantitis. We extended this analysis to high-grade periodontitis. Mucosal biopsies from four lesion-affected and four unaffected sites within the same individuals (1:1 matched; 8-samples; 225137-cells) again demonstrated spatially restricted CD38+-VEC remodeling exclusively in affected tissues, with similar vasculopathy front patterning. The findings nominate spatially distinct vasculopathy patterning as a hallmark of rapidly advancing oral inflammation and a targetable therapeutic axis.

immunology↗

bioRxiv2021

c-di-GMP inhibits early sporulation in Clostridioides difficile

The formation of dormant spores is essential for the anaerobic pathogen Clostridioides difficile to survive outside of the host gastrointestinal tract. The regulatory pathways and environmental signals that initiate C. difficile spore formation within the host are not well understood. One bacterial second messenger signaling molecule, cyclic diguanylate (c-di-GMP), modulates several physiological processes important for C. difficile pathogenesis and colonization, but the impact of c-di-GMP on sporulation is unknown. In this study, we investigated the contribution of c-di-GMP to C. difficile sporulation. Overexpression of a gene encoding a diguanylate cyclase, dccA, decreased sporulation frequency and early sporulation gene transcription in both the epidemic R20291 and historical 630{Delta}erm strains. Expression of a dccA allele encoding a catalytically inactive DccA that is unable to synthesize c-di-GMP no longer inhibited sporulation, indicating that the accumulation of intracellular c-di-GMP reduces C. difficile sporulation. A null mutation in dccA slightly increased sporulation in R20291 and slightly decreased sporulation in 630{Delta}erm, suggesting that DccA may contribute to the intracellular pool of c-di-GMP in a strain-dependent manner. However, these data were highly variable, underscoring the complex regulation involved in modulating intracellular c-di-GMP concentrations. Finally, overexpression of dccA in known sporulation mutants revealed that c-di-GMP is likely signaling through an unidentified regulatory pathway to control early sporulation events in C. difficile. C-di-GMP-dependent regulation of C. difficile sporulation may represent an unexplored avenue of potential environmental and intracellular signaling that contributes to the complex regulation of sporulation initiation. IMPORTANCEMany bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. C-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile. However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogens life cycle, is unknown. Here, we demonstrate that c-di-GMP negatively impacts sporulation in two clinically relevant C. difficile strains, the epidemic R20291 and the historical 630{Delta}erm. The pathway through which c-di-GMP controls sporulation was investigated, and our results suggest that c-di-GMP is likely signaling through an unidentified regulatory pathway to control C. difficile sporulation. This work implicates c-di-GMP metabolism as a potential mechanism to integrate environmental and intracellular cues through c-di-GMP levels to influence C. difficile sporulation.

microbiology↗