Multiplicity of type 6 secretion system toxins limits the evolution of resistance.

The bacterial type 6 secretion system (T6SS) is a toxin-injecting nanoweapon that mediates competition in plant- and animal-associated microbial communities. Bacteria can evolve de novo resistance against T6SS attacks, but resistance is far from universal in natural communities, suggesting key features of T6SS weaponry may act to limit its evolution. Here, we combine ecoevolutionary modeling and experimental evolution to examine how toxin type and multiplicity in attackers shape resistance evolution in susceptible competitors. In both our models and experiments, we find that combinations of multiple distinct toxins limit resistance evolution by creating genetic bottlenecks, driving resistant lineages extinct before they can reach high frequency. We also show that, paradoxically, single-toxin attackers can drive the evolution of cross-resistance, protecting bacteria against unfamiliar toxin combinations, even though such evolutionary pathways were inaccessible against multitoxin attackers. Our findings indicate that, comparable to antimicrobial and anticancer combination therapies, multitoxin T6SS arsenals function to limit resistance evolution in competing microbes. This helps us to understand why T6SSs remain widespread and effective weapons in microbial communities, and why many T6SS-armed bacteria encode functionally diverse anticompetitor toxins.