Experimental Evolution of Diverse Strains as a Method for the Determination of Biochemical Mechanisms of Action for Novel Pyrrolizidinone Antibiotics

Abstract

The continuing rise of multidrug resistant pathogens has made it clear that in the absence of new antibiotics we are moving toward a "postantibiotic" world, in which even routine infections will become increasingly untreatable. There is a clear need for the development of new antibiotics with truly novel mechanisms of action to combat multidrug resistant pathogens. Experimental evolution to resistance can be a useful tactic for the characterization of the biochemical mechanism of action for antibiotics of interest. Herein, we demonstrate that the use of a diverse panel of strains with well-annotated reference genomes improves the success of using experimental evolution to characterize the mechanism of action of a novel pyrrolizidinone antibiotic analog. Importantly, we used experimental evolution under conditions that favor strongly polymorphic populations to adapt a panel of three substantially different Gram-positive species (lab strain Bacillus subtilis and clinical strains methicillin-resistant Staphylococcus aureus MRSA131 and Enterococcus faecalis S613) to produce a sufficiently diverse set of evolutionary outcomes. Comparative whole genome sequencing (WGS) between the susceptible starting strain and the resistant strains was then used to identify the genetic changes within each species in response to the pyrrolizidinone. Taken together, the adaptive response across a range of organisms allowed us to develop a readily testable hypothesis for the mechanism of action of the CJ-16 264 analog. In conjunction with mitochondrial inhibition studies, we were able to elucidate that this novel pyrrolizidinone antibiotic is an electron transport chain (ETC) inhibitor. By studying evolution to resistance in a panel of different species of bacteria, we have developed an enhanced method for the characterization of new lead compounds for the discovery of new mechanisms of action.

Keywords: antibiotic resistance; antibiotics; experimental evolution; mechanism of action; pyrrolizidinone.

Figure 1

Summary of approach to identify the mechanism of action of a novel antibiotic. Identifying an antibiotic’s mechanism of action can be challenging, as antibiotics can target a diverse range of processes in bacteria, such as cell wall synthesis, protein translation, and nucleic acid metabolism. Therefore, we developed an approach that can be used to efficiently develop a hypothesis for the mechanism of action of any novel antibiotic. (1) A panel of at least three different susceptible species of bacteria is selected on the basis of several different criteria. Ideally, the different strains should have well-annotated genomes that would allow mutated genes and pathways to be readily identified. Model organisms also allow experimental validation of the proposed mechanism. Thus, it is beneficial to include at least one model organism, such as a laboratory strain of Bacillus subtilis or Escherichia coli. Additionally, the panel should consist of phylogenetically diverse species, since distantly related organisms are more likely to adapt through different mechanisms and thus reveal more pathways and targets affected by the antibiotic. Finally, some of the selected strains should be clinically relevant species that the antibiotic would potentially be used against. (2) Replicate populations of each species are adapted to become resistant to the antibiotic under study. Including replicate populations of the same strain is important since it allows for the identification of genes and pathways that are mutated reproducibility across the different populations, which in turn reveals which targets are most important to resistance. During adaptation, conservative increases in antibiotic concentration should be used to favor genetically diverse populations. (3) Comparative WGS between the adapted resistant strains and the ancestral susceptible starting strains is used to determine what adaptive mutations were acquired. (4) The different adaptive mutations are compared across replicate populations and the different species to develop a hypothesis for the mechanism of action of the antibiotic. Loci and pathways mutated across the different replicate populations and species are more likely to be important to resistance and thus targets of the antibiotic. Experimental validation is then performed to confirm the proposed mechanism of action.

Figure 2

Structures of the natural product CJ-16,264 (left) and the synthetic analog KCN-AAS-35 (right). To demonstrate the effectiveness of our approach, an antibiotic with a unique structure and unknown mechanism of action was characterized. The selected antibiotic is a synthetic derivative of the natural pyrrolizidinone antibiotic CJ-16,264 (left) and is referred to as KCN-AAS-35 (right). The pyrrolizidinone structure of these compounds is not present in any other class of antibiotics and therefore indicates they likely have a unique mechanism of action. KCN-AAS-35 was selected for these studies since it has a simpler structure and was easier to synthesize.

Figure 3

Adaptation timeline for B. subtilis 168, S. aureus MRSA131, and E. faecalis S613 to resist KCN-AAS-35. 24 replicate populations of each strain were initially grown at subinhibitory concentrations of drug. A portion of each population was transferred daily to the same concentration and an elevated concentration. Once all of the populations could grow to a high cell density at the elevated drug concentration, the populations were transferred from that concentration to a higher concentration (see Methods). Transfers were continued until all of the populations could grow well at a concentration above their starting MIC. Therefore, if some of the populations were struggling to grow at the elevated drug concentration, all of the populations were maintained at a lower concentration until the slower adapting populations could also grow well at the higher concentration. The clinical strains, S. aureus MRSA131 and E. faecalis S613, struggled to adapt to KCN-AAS-35 and took 40 or more days to reach growth at their respective final concentrations. Additionally, many of the populations of S. aureus MRSA131 and E. faecalis S613 died during adaption, with only eight and five populations remaining viable by the end of adaptation for S613 and MRSA131, respectively. The lab strain, B. subtilis 168, achieved growth at an elevated concentration after 14 days.

Figure 4

Oxygen consumption assays in the three bacterial strains show that KCN-AAS-35 inhibits cellular respiration. Aerobic respiration in E. faecalis S613, S. aureus MRSA 131, and B. subtilis 168 was measured in an oxygen electrode chamber by recording the percentage of extracellular oxygen remaining in the chamber as a function of time after addition of cells. The intrinsic respiration rate of the cells was recorded in the absence of any compounds (orange trace). Oxygen consumption was then measured by the addition of 12.5 μL of DMSO (gray trace), 105 μg/mL tetracycline (yellow trace), 10 mM potassium cyanide (blue trace), or 62.5 μg/mL KCN-AAS-35 (green trace), with the addition being made when 60% oxygen remained in the chamber (represented by diamonds on the respective traces). All assays were done in triplicate. The addition of DMSO (vehicle) or tetracycline did not affect bacterial respiration. The addition of cyanide, a known respiratory inhibitor, aborted respiration in S. aureus and B. subtilis but not in E. faecalis, which uses a cyanide insensitive cytochrome bd terminal oxidoreductase. The addition of KCN-AAS-35 caused a decrease in the rate of respiration in all the bacterial strains, with the effect being more pronounced in B. subtilis.

Figure 5

Functional mitochondrial toxicity assay (XFe96 Seahorse) reveals that KCN-AAS-35 likely acts as an ETC inhibitor. The Cyprotex Company assayed for changes in the oxygen consumption rate (OCR), reserve capacity, and extracellular acidification rate (ECAR) of HepG2 liver mitochondria treated with vehicle and 0.4–400 μg/mL KCN-AAS-35 using solid-state sensors to simultaneously measure effects on oxidative phosphorylation (OXPHOS) and glycolysis as described in Eakins et al. The data is represented as the average ratio to the vehicle (DMSO) with the error bars showing the standard deviation between three replicates. Open circles were excluded due to data plateau. Dose dependent decreases in OCR and reserve capacity were observed, indicating that KCN-AAS-35 inhibits mitochondrial respiration and causes mitochondrial dysfunction. Additionally, a dose dependent increase in ECAR was observed, indicating the release of more protons into the media due to an increase in glycolysis. Combined, these results are consistent with known inhibitors of the ETC, such as rotenone, suggesting that KCN-AAS-35 is likely an inhibitor of the ETC.