During the project, we aimed to answer two questions:
How does efflux pump redundancy affect antibiotic resistance? To investigate, we removed one-by-one multidrug efflux pump genes from the E. coli genome, generating strains with varying numbers of efflux pumps. These strains were then exposed to a panel of antibiotics, allowing us to measure how redundancy affects efflux activity and antibiotic resistance. Most single-gene deletions did not significantly increase antibiotic sensitivity, confirming their functional redundancy. The exception is the most potent efflux pump AcrAB, which plays a prominent role in antibiotic resistance. As additional genes were removed, we observed cases of increased sensitivity, indicating that efflux pump redundancy can mask the effects of gene loss.
How does genetic redundancy impact evolution of efflux pumps? Evolution relies on beneficial mutations, but most mutations are deleterious. We hypothesized that redundant genes, by masking deleterious effects, would increase the proportion of non-deleterious mutations and enhance adaptive potential. To test this hypothesis, we introduced precise mutations in the AcrAB efflux pump. We made over 6,000 mutations, targeting the protein region involved in drug binding and transport. We assessed these mutations in five bacterial strains with different gene content of efflux pumps and under five different classes of antibiotics, yielding more than 150,000 fitness estimates (6,000 mutations x 5 strains x 5 antibiotics). However, we found no evidence that the genetic redundancy of efflux pump genes increased the proportion of beneficial mutations.
Instead, we uncovered significant evolutionary correlations. Evolutionary correlations mean that an efflux pump adapting to export one class of antibiotics may improve its capability to export another class. This occurs because these antibiotics induce similar types of genotype-function relationship in this protein, even if they are not chemically similar, which explains how AcrAB has evolved to recognize and export a wide range of substrates, including antibiotics. Furthermore, we found substantial genetic interactions (epistasis), indicating that the same AcrAB mutations could be either beneficial or deleterious depending on the presence of particular efflux pump genes. To further explore these complex interactions, we conducted evolutionary simulations. Our results suggest that, while genetic redundancy may not universally enhance the adaptive evolution of efflux pumps, its impact is nuanced and depends on the specific antibiotic and set of redundant genes present.
