Symbiotic bacteria produce non-lytic vesicles with nucleic acid cargo

Symbiotic bacteria in the gut have an important physiological impact on their hosts, but the mechanisms that underlie the exchange of molecular signals remain poorly understood. Membrane vesicles have been suggested to mediate the direct exchange of cytoplasmic content like nucleic acids (NAs), but their study is complicated by conflicting and imprecise reports of their type and composition. Here, we show that honeybee gut symbionts produce non-lytic membrane vesicles (MVs) enriched in NA, potentially explaining the RNAi activity of engineered Snodgrassella alvi in honeybees. Using cryogenic electron microscopy (cryo-EM), we developed a method to distinguish lytic from non-lytic MV production in Gram-negative bacteria and to differentiate outer membrane vesicles (OMVs) from outer-inner membrane vesicles (OIMVs) based on membrane ultrastructure. Among the strains studied, S. alvi, Gilliamella apicola, and Gilliamella apis exhibit clear OMV and OIMV budding, while Escherichia coli and Salmonella enterica show membrane debris and self-assembled vesicles, indicating lytic release. MVs from the symbionts carry significantly more NAs than non-symbionts. Assays on DNA and RNA contents confirm the cytoplasmic origin of MV cargo in S. alvi, suggesting a role in mediating NA delivery to the insect host. These findings enhance our understanding of symbiotic vesiculation and highlight the potential for engineering symbionts to boost honeybee immunity and deliver NA-based therapeutics via vesicular transport.