Multiplicity of Type 6 Secretion System toxins limits the evolution of resistance

Abstract

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 eco-evolutionary modelling and experimental evolution to examine how toxin type and multiplicity in Acinetobacter baylyi attackers shape resistance evolution in susceptible Escherichia coli 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 often drive the evolution of cross-resistance, protecting bacteria against unfamiliar toxin combinations, even though such evolutionary pathways were inaccessible against multi-toxin attackers. Our findings indicate that, comparable to antimicrobial and anticancer combination therapies, multi-toxin 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 bacteria T6SS-armed encode functionally diverse anti-competitor toxins. SignificanceToxin secretion systems, such as T6SSs, are widely used by bacteria to inhibit competing microorganisms. Here, we show that the secretion of multiple toxins in combination can suppress the evolution of resistance to the T6SS, rationalising its continued widespread use across diverse communities. Our work shows that principles of combination therapy--well known in the context of antimicrobial, antiviral and anticancer therapies--also apply in the context of microbial warfare, helping to explain why many bacteria maintain diverse T6SS toxin arsenals. Resistance suppression is also a technologically useful property of T6SS toxin cocktails, which could be harnessed as part of future biocontrol or biotherapeutic applications, using live T6SS-armed bacteria to limit the growth of problem microorganisms.