Metabolomics Reveal Potential Natural Substrates of AcrB in Escherichia coli and Salmonella enterica Serovar Typhimurium

Abstract

In the fight against antibiotic resistance, drugs that target resistance mechanisms in bacteria can be used to restore the therapeutic effectiveness of antibiotics. The multidrug resistance efflux complex AcrAB-TolC is the most clinically relevant efflux pump in Enterobacterales and is a target for drug discovery. Inhibition of the pump protein AcrB allows the intracellular accumulation of a wide variety of antibiotics, effectively restoring their therapeutic potency. To facilitate the development of AcrB efflux inhibitors, it is desirable to discover the native substrates of the pump, as these could be chemically modified to become inhibitors. We analyzed the native substrate profile of AcrB in Escherichia coli MG1655 and Salmonella enterica serovar Typhimurium SL1344 using an untargeted metabolomics approach. We analyzed the endo- and exometabolome of the wild-type strain and their respective AcrB loss-of-function mutants (AcrB D408A) to determine the metabolites that are native substrates of AcrB. Although there is 95% homology between the AcrB proteins of S. Typhimurium and E. coli, we observed mostly different metabolic responses in the exometabolomes of the S. Typhimurium and E. coli AcrB D408A mutants relative to those in the wild type, potentially indicating a differential metabolic adaptation to the same mutation in these two species. Additionally, we uncovered metabolite classes that could be involved in virulence of S. Typhimurium and a potential natural substrate of AcrB common to both species. IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a global threat to human health. The AcrB efflux pump confers inherent and evolved drug resistance to Enterobacterales, including Escherichia coli and Salmonella enterica serovar Typhimurium. We provide insights into the physiological role of AcrB: (i) we observe that loss of AcrB function in two highly related species, E. coli and S. Typhimurium, has different biological effects despite AcrB conferring drug resistance to the same groups of antibiotics in both species, and (ii) we identify potential natural substrates of AcrB, some of which are in metabolite classes implicated in the virulence of S. Typhimurium. Molecules that inhibit multidrug efflux potentiate the activity of old, licensed, and new antibiotics. The additional significance of our research is in providing data about the identity of potential natural substrates of AcrB in both species. Data on these will facilitate the discovery of, and/or could be chemically modified to become, new efflux inhibitors.

Absence, loss-of-function, or inhibition of Escherichia coli AcrB does not increase expression of other efflux pump genes supporting the discovery of AcrB inhibitors as antibiotic adjuvants

“… 3 In previous publications, the acrB D408A missense mutation, causing AcrB to be expressed at normal levels but functionally inactivated, conferred increased susceptibility to AcrB substrates and had a different transcriptional response to deletion of the same gene. 23 , 32 In this study, single missense mutants were made in five other efflux pump genes in E. coli to investigate the hypothesis that bacteria with pump gene deletions may be responding to loss of a large membrane protein and not loss of efflux. The susceptibility of these mutants with missense mutations known or predicted to cause loss of function of the encoded efflux pump was determined for known efflux pump substrates.…”

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“… 134 − 136 A recent metabolomics study strongly suggests that oxidized fatty acids are the native substrates for AcrB in both Salmonella and E. coli . 137 Additionally, the ABC pump MacAB-TolC has been shown to protect S . Typhimurium from oxidative stress.…”

Antibiotic resistance: Time of synthesis in a post-genomic age

“…Besides genomic studies, transcriptomic, proteomic and, eventually, metabolomic analyses have been implemented with the aim of deciphering novel mechanisms of resistance [122] , [123] , [124] , [125] , or to detect the substrates, besides antibiotics, of known resistance determinants as multidrug efflux pumps [126] . The main drawback of these approaches is that, although the acquisition of AR usually implies changes in the level of expression of several genes/proteins, most of these changes are not associated with the mechanism of resistance, but with the fitness costs due associated with such resistance (see below).…”