Droplet Sequencing Reveals Virulence Gene Clusters in Oral Biofilm Extracellular Vesicles

Bacterial extracellular vesicles (BEVs), produced by a broad spectrum of bacteria, play a crucial role in enhancing intercellular communication through DNA transfer. A vital determinant of their gene transfer efficiency is the gene content diversity within BEVs, an aspect that conventional metagenomics fails to capture. Our study bridges this gap with a novel microdroplet-based sequencing technique that precisely details DNA content within hundreds of individual BEVs. This technique revealed a unique DNA profile in BEVs from the oral pathogen Porphyromonas gingivalis, pinpointing specific genomic regions related to DNA integration (e.g., DNA transposition and CRISPR-Cas systems). These enriched genes, overlooked by standard analyses that aggregate total read counts, indicate that our method offers a more focused view into the genetic contents of BEVs. Applying our technique to dental plaque-derived BEVs, we discovered a hundredfold higher prevalence of DNA encapsulation than previously estimated, with over 30% of BEVs containing DNA. Specifically, we identified a substantial presence of O-antigen biosynthesis genes, prominent hotspots of frequent horizontal gene transfer, from Alcaligenes faecalis. Given that O-antigens mediate host-bacterial interactions, this gene enrichment in the large fraction of BEVs suggests a potential novel pathway by which BEVs could influence pathogenicity within oral biofilms. Our research unveils critical insights into the potential functions of vesicular DNA in microbial communities, establishing a powerful platform for studying vesicular DNA in microbiomes. This technical breakthrough provides a foundational basis for future research in microbial communication and the development of potential therapeutic or diagnosis strategies. Significance StatementBEVs have been studied for decades, yet their roles in nature and disease are just beginning to be appreciated. Our study makes a significant leap in understanding the roles of BEVs as gene transfer vehicles. By developing a microdroplet-based sequencing technique, we have uncovered detailed DNA profiles within individual BEVs, a task beyond the capabilities of conventional metagenomic methods. This breakthrough highlights specific genomic regions enriched in BEVs from pure culture and human dental plaque. Furthermore, the high prevalence of biofilm BEVs enriched in O-antigen biosynthesis genes, suggests a potential impact on the pathogenicity of oral biofilms. This research establishes a new methodological platform for exploring the intricacies of BEV-mediated interactions in a complex microbial community.