Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND (resistance-nodulation-cell division)-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated.
KEYWORDS: gepotidacin, zoliflodacin, drug efflux, TolC, AcrB, YhiV (MdtF), RND-type transporter, fluoroquinolones, clinical E. coli isolates
ABSTRACT
Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND (resistance-nodulation-cell division)-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated. The findings should reinforce efforts to develop efflux-bypassing drugs and provide AcrB targets with critical relevance for this purpose.
INTRODUCTION
The development of novel antibacterial drugs is essential in a world with continually increasing rates of multidrug-resistant (MDR) pathogens. Among promising candidates currently under clinical development are the topoisomerase type II inhibitors gepotidacin (GEP) (1) and zoliflodacin (ZOL) (2). Both GEP and ZOL belong to new drug classes, the triazaacenaphthylenes and the spiropyrimidinetriones, respectively, and both are active against Neisseria gonorrhoeae (3, 4). Like fluoroquinolones (FQs), they target the type II topoisomerases (DNA gyrase and topoisomerase IV), but by entirely different modes of action. Hence, mutations occurring in the quinolone resistance-determining regions (QRDRs) of the topoisomerase genes gyrA, gyrB, parC, and parE as a result of FQ treatment should not impair the efficacy of these drugs (4, 5). Activity of GEP against Escherichia coli and Gram-positive pathogens such as Staphylococcus aureus has also been shown (6), and activity of ZOL against fastidious Gram-negative pathogens has been demonstrated (2).
There has been evidence that the chemically unrelated compounds GEP and ZOL are substrates of the RND (resistance-nodulation-cell division)-type efflux pump MtrCDE in N. gonorrhoeae (7, 8). To our knowledge, nothing has been reported about the contribution of efflux to their resistance levels in E. coli and their putative substrate nature regarding the major E. coli RND-type MDR transporter AcrAB-TolC. To explore the role of efflux for the activities of the new topoisomerase inhibitors in MDR clinical E. coli, we selected highly FQ-resistant isolates from different sequence types and origins (Table 1) for tolC knockout experiments. Genetic engineering of MDR strains is challenging because of limited selection options (9, 10). We succeeded in constructing four tolC mutants from three different sequence types and with differing QRDR mutation patterns, including an isolate harboring the aminoglycoside/FQ resistance gene aac(6′)Ib-cr (Tables 1 and 2), by applying a phage λ-based homologous recombination method. A neomycin/kanamycin resistance cassette was amplified with homology flanks for recombination in tolC by using oligonucleotides given in Table S1 in the supplemental material and a cassette template from the Red/ET counterselection BAC modification kit (Gene Bridges, Heidelberg, Germany). Homologous recombination was performed by using a curable Red/ET plasmid with a chloramphenicol cassette (supplemental material). Selection of knockout mutants was possible with 40 µg/ml paromomycin, because we had recognized that the neomycin/kanamycin cassette mediates cross-resistance to this aminoglycoside to which, in contrast to kanamycin (MICs > 50 µg/ml), our clinical isolates show relative susceptibility (MICs ≤ 4 µg/ml). We additionally knocked out tolC from laboratory strain 3-AG100, a derivative of E. coli AG100, which overexpresses AcrAB-TolC (11) (Tables 1 and 2 and Fig. 1A).
TABLE 1.
E. coli strains and mutants used in this study
| E. coli strain or mutant | Description, MLSTa sequence type, and country |
Mutation(s) in QRDRb and episomal FQ resistance genesc |
Provider, source, or reference |
|---|---|---|---|
| KUN9180 | Clinical MDR E. coli isolate, ST 131, Japan |
GyrA: S83L, D87N; ParC: S80I, E84V; ParE: I529L |
Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan |
| 2012-0633 | Clinical MDR E. coli isolate, ST 1193, Japan |
GyrA: S83L, D87N; ParC: S80I; ParE: L416F |
Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan |
| SA128 | Clinical MDR E. coli isolate, ST 167, Sudan |
GyrA: S83L, D87N; ParC: S80R, E84V; aac(6′)Ib-cr |
Abha National Polyclinic, Abha, Saudi Arabia |
| FR11009 | Clinical MDR E. coli isolate, ST 167, Germany |
GyrA: S83L, D87N; ParC: S80I, E84G | Center for Microbiology, University Hospital Freiburg, Germany |
| KUN9180ΔacrB(::PGK-gb2-neo) | acrB knockout mutant | GyrA: S83L, D87N; ParC: S80I, E84V; ParE: I529L |
9 |
| KUN9180ΔtolC(::rpsL-neo) | tolC knockout mutant | GyrA: S83L, D87N; ParC: S80I, E84V; ParE: I529L |
This study |
| 2012-0633ΔtolC(::rpsL-neo) | tolC knockout mutant | GyrA: S83L, D87N; ParC: S80I; ParE: L416F |
This study |
| SA128ΔtolC(::rpsL-neo) | tolC knockout mutant | GyrA: S83L, D87N; ParC: S80R, E84V; aac(6′)Ib-cr |
This study |
| FR11009ΔtolC(::rpsL-neo) | tolC knockout mutant | GyrA: S83L, D87N; ParC: S80I, E84G | This study |
| ATCC 25922 | Reference E. coli strain | DSMZd, no. DSM-1103. | |
| AG100 | K-12 derivative, wild-type (wt) acrB |
22 | |
| 3-AG100 | AG100 derivative, wt acrB
overexpressed |
GyrA: D87G | 11 |
| 3-AG100ΔacrB |
acrB deletion mutant from 3-AG100 |
GyrA: D87G | 14 |
| 3-AG100ΔtolC(::rpsL-neo) |
tolC knockout mutant from 3-AG100 |
GyrA: D87G | This study |
| 2-DC14PS |
acrAB knockout mutant from AG100, acrF overexpressed |
GyrA: S83L | 23 |
| DKO |
acrAB_acrF double-knockout mutant from 2-DC14PS |
GyrA: S83L | 24 |
| DKO20/1 |
acrAB_acrF double-knockout mutant from 2-DC14PS, yhiV overexpressed |
GyrA: S83L, D87L; ParC: E84K | 24 |
| TKO |
acrAB acrF yhiV triple-knockout mutant from DKO20/1 |
GyrA: S83L, D87L; ParC: E84K | 24 |
| F136A, F178A, F610A, F615A, F617A, and F628A |
AcrB distal binding pocket mutants from 3-AG100 |
GyrA: D87G | 15 |
| V612F | AcrB distal binding pocket mutant from 3-AG100 |
GyrA: D87G | This study |
| G616N | AcrB distal binding pocket mutant from 3-AG100 |
GyrA: D87G | 17 |
| I38F/I671T | Entrance pathway mutant from 3-AG100 |
GyrA: D87G | 18 |
MLST, multilocus sequence typing.
QRDR, quinolone resistance-determining region. Only known quinolone resistance-determining mutations are shown.
FQ, fluoroquinolone. FQ resistance genes are given in italic.
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (https://www.dsmz.de).
TABLE 2.
Susceptibilities of E. coli strains and mutants to selected fluoroquinolones and new topoisomerase inhibitors
| E. coli strain or mutanta | MIC (µg/ml) forb: |
||||
|---|---|---|---|---|---|
| FQ drugs |
New topoisomerase inhibitors |
||||
| LVX | MXF | NDX | GEP | ZOL | |
| A. Clinical MDR E. coli isolates and derived mutants | |||||
| 2012-0633 | >32 | >8 | >512 | 16 | 16 |
| 2012-0633ΔtolC | 4 | 2 | 2 | 0.06 | 0.03 |
| FR11009 | 32 | >8 | 512 | 1 | 4 |
| FR11009ΔtolC | 4 | 2 | 2 | 0.03 | 0.01 |
| SA128 | >32 | >8 | >512 | 8 | 16 |
| SA128ΔtolC | 4 | 4 | 8 | 0.06 | 0.03 |
| KUN9180 | 32 | >8 | >512 | 2 | 4 |
| KUN9180ΔacrB | 4 | 2 | 32 | 0.06 | 0.25 |
| KUN9180ΔtolC | 4 | 2 | 8 | 0.03 | 0.06 |
| B. Laboratory strains and derived mutants | |||||
| ATCC 25922 (constitutive acrB expression) | 0.06 | 0.125 | 0.125 | 4 | 2 |
| AG100 (constitutive acrB expression) | 0.06 | 0.25 | 0.125 | 1 | 4 |
| 3-AG100 (acrB overexpressed) | 2 | 4 | 4 | 4 | 4 |
| 3-AG100ΔacrB | 0.06 | 0.06 | 0.03 | 0.06 | 0.06 |
| 3-AG100ΔtolC | 0.06 | 0.06 | 0.06 | 0.06 | 0.01 |
| 2-DC14PS (ΔacrAB, acrF overexpressed) | 2 | 4 | 16 | 2 | 16 |
| DKO (ΔacrAB ΔacrF) | 0.125 | 0.06 | 0.125 | 0.03 | 0.06 |
| DKO20/1 (ΔacrAB ΔacrF, yhiV overexpressed) | 32 | 32 | 256 | 0.06 | 8 |
| TKO (ΔacrAB ΔacrF ΔyhiV) | 8 | 4 | 8 | 0.02 | 0.03 |
| C. AcrB porter domain mutants derived from 3-AG100 | |||||
| F136A | 1 | 4 | 2 | 4 | 2 |
| F178A | 0.5 | 2 | 1 | 2 | 2 |
| F610A | 0.125 | 0.5 | 0.5 | 1 | 2 |
| V612F | 1 | 2 | 2 | 2 | 0.5 |
| F615A | 1 | 4 | 2 | 4 | 1 |
| G616N | 1 | 2 | 4 | 4 | 4 |
| F617A | 1 | 4 | 4 | 2 | 4 |
| F628A | 1 | 2 | 1 | 2 | 2 |
| I38F/I671T | 0.06 | 0.25 | 1 | 0.06 | 16 |
For sections A and B, the parental E. coli strain or isolate is shown first and the mutants derived from the strain or isolate are indented. Strains are listed in Table 1.
FQ, fluoroquinolone; LVX, levofloxacin; MXV, moxifloxacin; NDX, nadifloxacin; GEP, gepotidacin; ZOL, zoliflodacin. Values given in boldface type and underlined highlight MIC decreases by ≥4-fold and increases by ≥4-fold, respectively, with the following reference relationships: for section A, tolC and acrB mutants versus their respective parental clinical isolates; for section B, TKO versus DKO20/1, all other laboratory strains and mutants versus acrB overexpressing strain 3-AG100; for section C, AcrB mutants versus 3-AG100. >, MIC determination is limited due to drug precipitation at concentrations above the indicated values or to maximum concentrations in the commercial precast microdilution 96-well plates in the case of LVX and MXF.
FIG 1.
(A) BM-27 efflux initiated by the addition of glucose (black arrow). Assays were done at least in duplicate according to a published protocol (25). RFU, relative fluorescence units. (B) Expression of tolC and of TolC-dependent MDR transporter genes (shown if any expression levels of >0.4 were detected). Means plus standard deviations (SD) (error bars) from values normalized to the respective gyrB values (n ≥ 4) are shown. (C) Part of the AcrB porter domain (PDB accession no. 2HRT, chain B), side view from the periplasmic cleft (PC1, PC2, PN1, and PN2, subdomains) visualized using PyMOL (version 2.1.1; Schrödinger, LLC). Amino acid side chains are depicted as sticks, glycine as a sphere, and distal binding pocket residues with underlined labels. Blue-, green-, and red-colored sticks indicate impact by mutation on susceptibilities (no significant change, a significant increase, and significant decrease, respectively) regarding FQs (dashed circle, increase of NDX susceptibility only), GEP, and ZOL. (D) Efficacies of EPIs with GEP and ZOL given as ratios of the MIC in the absence and presence of an EPI (n ≥ 3).
Efflux disruption in the tolC mutants was assessed by standard MIC testing (Table 2) and real-time efflux assays in which bacterial cells de-energized with 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) were loaded with the piperazine arylideneimidazolone BM-27 to a final concentration of 10 µM. Efflux was started by reenergization with 1 mM glucose, and fluorescence of the intracellular remaining BM-27 was measured at an excitation and emission wavelength of 400 and 457 nm, respectively (Fig. 1A; see supplemental material also).
In an earlier study, we have shown that FQ susceptibilities significantly increase due to acrB inactivation in an E. coli isolate with QRDR mutations (9). As could be expected, we achieved similar results from the tolC mutants, including SA128ΔtolC harboring aac(6′)Ib-cr, but with an enhanced impact on the resistance to the more lipophilic nadifloxacin (NDX) (Table 2). The latter has been demonstrated to be least impaired by the outer membrane (OM) influx barrier (12), enabling the appearance of approximate net efflux. As reported previously, tolC disruption rarely increases FQ susceptibilities to clinical relevance in isolates with QRDR mutations and/or FQ resistance genes (10). However, with GEP and ZOL, MICs decreased by 32- to 512-fold, achieving values below 0.1 µg/ml (Table 2).
Even though AcrAB-TolC has been shown to play a major role for MDR in E. coli (13), the OM channel TolC could cooperate with further MDR efflux pumps, such as the RND-type transporters AcrF, YhiV (MdtF), and YegNO (MdtBC), the ABC (ATP-binding cassette)-type transporter MacAB, and the MFS (major facilitator superfamily) transporter EmrAB. To answer the question whether further TolC-dependent transporters contribute to efflux, we compared the tolC knockout mutants with the respective acrB knockout mutants available for E. coli 3-AG100 (14) and clinical isolate KUN9180 (9) from earlier studies. As just mentioned, remarkable differences between KUN9180ΔacrB and KUN9180ΔtolC were detected in susceptibilities to NDX but also to ZOL (Table 2) as well as in the BM-27 efflux of the KUN9180 mutants (Fig. 1A), indicating the involvement of at least one more TolC-dependent transporter. Interestingly, in the knockout mutants derived from laboratory strain 3-AG100, solely the ZOL susceptibilities were different (Table 2 and Fig. 1A). Results of gene expression studies suggested the relevance of YhiV within the clinical isolates KUN9180 (yhiV expression > acrB expression, P = 0.01) and 2012-0633 and FR11009 (similar expression of acrB and yhiV). In contrast, AcrB appeared to be the dominant exporter in SA128 and the laboratory strains (acrB expression > yhiV expression, P values < 0.01) (Fig. 1B).
In order to explore RND-type substrate pathway specificities of GEP and ZOL versus FQs, we examined the impact of AcrB porter domain mutations with known effects on the susceptibilities to other drugs. Among distal binding pocket (DB) mutations, the highest FQ susceptibility increases were achieved by F610A, which has been known to seriously impair resistance to almost all AcrB substrates except linezolid and sutezolid (14, 15). Remarkably, F610A also results in significant MIC decreases of GEP but not of ZOL. In contrast, DB mutation F615A exclusively affects resistance to ZOL among the drugs tested in this study, whereas DB mutation F178A showed significant effects with the FQs LVX and NDX, but none with the new topoisomerase inhibitors. No remarkable impact was found with G616N and F617A, which are substitutions at the so-called switch-loop (16, 17), as well as with F136A for all compounds tested, whereas F628A affects only resistance to the lipophilic FQ NDX (Fig. 1C). The most surprising effects were recognized from the double mutation I38F/I671T located in the lower porter domain and presumed to block a bottleneck in an entrance channel for predominantly smaller drug molecules (18). While this could also be demonstrated for GEP, ZOL appears more efficiently extruded in the I38F/I671T mutant than in the wild-type AcrB strain (Table 2 and Fig. 1C). Analogous results have been obtained with macrolides in an earlier study (18). The marginally larger size of ZOL (molecular weight [MW], 487.4) versus GEP (MW, 448.5) could explain pathway hindrance, but probably additional physicochemical factors play a role for the identified substrate route selectivity.
The experimental efflux pump inhibitors (EPIs) 1-(1-naphthylmethyl)-piperazine (NMP) and Phe-Arg-β-naphthylamide (PAβN) are known enhancers of FQ action (19, 20). Regarding the new topoisomerase inhibitors, both EPIs showed significant sensitizing with GEP but not with ZOL (Fig. 1D) when they were used as adjuvants in MIC assays (supplemental material). The efficacy of PAβN (used at 25 µg/ml) with GEP appeared more “strain dependent” than that of NMP (used at 100 µg/ml), presumably due to strain-specific cell envelope properties that might impair the permeabilizing activity of PAβN (21).
We additionally studied the substrate nature of GEP and ZOL regarding the AcrB homologs AcrF and YhiV by using mutants overexpressing these transporters while AcrAB and AcrAB_AcrF were knocked out, respectively. Both topoisomerase inhibitors appeared to be suitable substrates for AcrF, whereas MIC data suggest that ZOL, but not GEP, is extruded from YhiV (Table 2).
In conclusion, we have demonstrated a remarkable contribution of TolC-dependent efflux to resistance levels of the first-in-class topoisomerase inhibitors GEP and ZOL in FQ-resistant E. coli isolates. In addition to AcrB, YhiV was shown to be a putatively relevant substrate-specific transporter contributing to efflux in a percentage of clinical isolates. Critical sites in the AcrB efflux pathway of both structurally unrelated topoisomerase inhibitors were discovered and could inform the design of efflux-bypassing drugs. To obtain deeper insight into the requirements of those agents, further exploration of differences in the substrate nature of GEP and ZOL regarding other homologous RND-type transporters, including the Neisseria efflux pump MtrD, are needed.
Data availability.
ENA accession numbers of whole-genome sequencing data available from previous studies follow: for E. coli strain KUN9180, study accession no. PRJEB19331 and sample accession no. SAMEA100686418 (https://www.ebi.ac.uk/ena/browser/view/ERS1572363) (9); for E. coli strain 3-AG100, study accession no. PRJEB30347 and sample accession no. SAMEA5175928 (https://www.ebi.ac.uk/ena/data/view/ERS2983635) (21). ENA accession numbers of whole-genome sequencing data available from the present study follow: for E. coli strain 2012-0633, study accession no. PRJEB39933 and sample accession no. ERS4956175 (https://www.ebi.ac.uk/ena/browser/view/ERS4956175); for E. coli strain SA128, study accession no. PRJEB39933 and sample accession no. ERS4956177 (https://www.ebi.ac.uk/ena/browser/view/ERS4956177), and for E. coli strain FR11009, study accession no. PRJEB39933 and sample accession no. ERS4956176 (https://www.ebi.ac.uk/ena/browser/view/ERS4956176).
Supplementary Material
ACKNOWLEDGMENTS
This work was supported in part by the Innovative Medicines Initiative (IMI) Joint Undertaking project no. 115525 ND4BB Translocation (http://www.translocation.eu, contributions from the European Union seventh framework program and EFPIA companies).
We thank Yasufumi Matsumura from the Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, Japan, for kindly providing clinical isolates from Japan, Mutasim E. Ibrahim from the Abha National Polyclinic, Saudi Arabia (currently Department of Basic Medical Science, University of Bisha, Kingdom of Saudi Arabia) for clinical isolates from Sudan, and Jadwiga Handzlik from the Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Faculty of Pharmacy, Kraków, Poland, for compound BM-27.
We have no conflicts of interest to declare.
Footnotes
Supplemental material is available online only.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
ENA accession numbers of whole-genome sequencing data available from previous studies follow: for E. coli strain KUN9180, study accession no. PRJEB19331 and sample accession no. SAMEA100686418 (https://www.ebi.ac.uk/ena/browser/view/ERS1572363) (9); for E. coli strain 3-AG100, study accession no. PRJEB30347 and sample accession no. SAMEA5175928 (https://www.ebi.ac.uk/ena/data/view/ERS2983635) (21). ENA accession numbers of whole-genome sequencing data available from the present study follow: for E. coli strain 2012-0633, study accession no. PRJEB39933 and sample accession no. ERS4956175 (https://www.ebi.ac.uk/ena/browser/view/ERS4956175); for E. coli strain SA128, study accession no. PRJEB39933 and sample accession no. ERS4956177 (https://www.ebi.ac.uk/ena/browser/view/ERS4956177), and for E. coli strain FR11009, study accession no. PRJEB39933 and sample accession no. ERS4956176 (https://www.ebi.ac.uk/ena/browser/view/ERS4956176).