The Antibiotic Efflux Protein TolC Is a Highly Evolvable Target under Colicin E1 or TLS Phage Selection

Abstract

Bacteriophages and bacterial toxins are promising antibacterial agents to treat infections caused by multidrug resistant (MDR) bacteria. In fact, bacteriophages have recently been successfully used to treat life-threatening infections caused by MDR bacteria [1-3]. One potential problem with using these antibacterial agents is the evolution of resistance against them in the long term. Here, we studied the fitness landscape of the Escherichia coli TolC protein, an outer membrane efflux protein that is exploited by a pore forming toxin called colicin E1 and by TLS-phage [4-8]. By systematically assessing the distribution of fitness effects (DFEs) of ∼9,000 single amino acid replacements in TolC using either positive (antibiotics and bile salts) or negative (colicin E1 and TLS-phage) selection pressures, we quantified evolvability of the TolC. We demonstrated that the TolC is highly optimized for the efflux of antibiotics and bile salts. In contrast, under colicin E1 and TLS phage selection, TolC sequence is very sensitive to mutations. Finally, we have identified a large set of mutations in TolC that increase resistance of E. coli against colicin E1 or TLS phage without changing antibiotic susceptibility of bacterial cells. Our findings suggest that TolC is a highly evolvable target under negative selection which may limit the potential clinical use of bacteriophages and bacterial toxins if evolutionary aspects are not taken into account.

MDR Pumps as Crossroads of Resistance: Antibiotics and Bacteriophages

“…Just as bacteriophages use the host’s metabolic and biosynthetic systems, they use the bacteria’s need for MDR pumps which they utilize to bind to bacterial cells and then enter them. Although MDR pump systems are well optimized for pumping out xenobiotics and are rather conservative [ 157 ], they undergo many mutations under the action of bacteriophages [ 158 ], which leads to a decrease in the efficiency of antibiotic pumping and a decrease in the contribution of nonspecific protection to overall resistance. The study and understanding of these processes will allow us to either seriously delay the onset of the post-antibiotic era or even prevent it altogether in the future.…”

Evolutionary Dynamics between Phages and Bacteria as a Possible Approach for Designing Effective Phage Therapies against Antibiotic-Resistant Bacteria

“…The efflux pumps play a major role in antibiotic resistance and bacterial pathogenicity [ 201 ]. The tolC -deficient E. coli was more sensitive to antibiotics and bile salts than the wild-type strain [ 202 ]. The phage-resistant bacteria are deficient in drug efflux pumps, resulting from the evolutionary trade-offs [ 30 , 203 ].…”

Multidrug Resistance Pumps as a Keystone of Bacterial Resistance

“…The evolutionary conservatism of pumps is evidenced by the ubiquitous distribution of pumps of the MFS and RND families both among prokaryotes (including bacteria and archaea) and among eukaryotes [ 41 , 42 ], so the conserved structure of pumps within the same species is not surprising [ 43 ]. This is also supported by the fact that pump structures were chosen by bacteriophages as targets for their binding [ 44 , 45 ]. It seems that the ability to efflux drugs appeared only a few times in the course of evolution and was stably preserved, but the modulation of the substrate specificity of these systems occurred repeatedly [ 46 ].…”