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.