The role of genetic redundancy in adaptive evolution of efflux pumps
This project addresses the broad problem of genetic redundancy and its role in evolution. Genetic redundancy refers to the fact that multiple genes in a genome can carry out similar or identical functions. Because these genes can substitute each other functionally, the loss of some genes often has no apparent effect on biological fitness. Despite this, genetic redundancy is widespread across many organisms, including bacteria, suggesting that having redundant genes is advantageous even for species with relatively small genomes. This raises fundamental questions: why have functionally replaceable genes persisted for long evolutionary time, and is genetic redundancy evolutionarily advantageous?
To address these questions, we employed gene editing, deep sequencing, and computational analysis. We focused on genes encoding multidrug resistance efflux pumps. Efflux pumps are membrane proteins that actively export specific chemicals, including antibiotics, out of cells. The efflux of antibiotics is one of the major mechanisms allowing pathogenic bacteria to resist antibiotic exposure and leading to treatment failure. Most pathogenic bacteria have multiple multidrug resistance efflux pumps, representing five protein families with distinct structures and mechanisms yet overlapping activities against antibiotics. This makes efflux pumps a good model to study genetic redundancy.
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.
We addressed a fundamental problem of redundancy using an empirical approach that combines manipulation of the genomic composition of efflux pumps, high-throughput gene editing, deep sequencing, and evolutionary analysis. We estimated how genetic redundancy impacts the effect of over 6,000 mutations on the efflux of different antibiotics. Our findings demonstrate that the evolution of redundant genes is shaped by evolutionary correlations and gene interactions, providing insights into genome evolution as an integrated system rather than isolated gene functions.
During the course of this research, we obtained biological material and unique data, which will be a valuable resource for researchers working on the molecular biology of efflux pumps. We engineered difficult-to-make serial deletion mutants of E. coli without inserting foreign integration cassettes or transposons. We established a highly efficient protocol to edit the clinically important pump AcrAB, generating one of the most comprehensive collections of mutations for a functionally important region called the switch loop. We found that many amino acid substitutions selectively impact AcrAB activity on specific antibiotics. This new functional information will help to better understand the molecular basis of substrate specificity and binding, potentially aiding in developing novel drugs, such as efflux pump inhibitors.
The results of this work enhance our understanding of the evolution of efflux pumps and, in particular, explain how efflux pumps have evolved to export chemically unrelated substrates. We also explored the limitations of their evolutionary potential, which can be exploited to guide new strategies for preventing the emergence of antibiotic resistance. Specifically, we found that structural mutations that increase resistance against multiple antibiotics are much rarer than those increasing resistance to single antibiotics, which supports the case for using combination antibiotic therapy.