Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis
- Department of Molecular Biosciences, The University of Texas at Austin, United States
- Centre for Bacterial Resistance Biology, Department of Life Sciences, Imperial College London, United Kingdom
- Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, United Kingdom
- Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Spain
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore
- Lee Kon Chian School of Medicine, Nanyang Technological University, Singapore
- Department of Biology, Indiana University, United States
- Laboratoire de Microbiologie, Institut de Biologie, Université de Neuchâtel, Switzerland
- John Ring LaMontagne Center for Infectious Diseases, The University of Texas at Austin, United States
Figures
Figure 1 with 1 supplement
The function of species-specific cysteine-containing β-lactamases from cystic fibrosis-associated pathogens depends on DsbA-mediated oxidative protein folding.
β-lactam MIC values for E. coli MC1000 expressing diverse disulfide-bond-containing β-lactamases (Ambler classes A, B, and D) are substantially reduced in the absence of DsbA (MIC fold changes: >2; fold change of 2 is indicated by the black dotted lines). No changes in MIC values are observed for the aminoglycoside antibiotic gentamicin (white bars), confirming that absence of DsbA does not compromise the general ability of this strain to resist antibiotic stress. Minor changes in MIC values (≤2 fold) are observed for strains harboring the empty vector control (pDM1) or those expressing the class A β-lactamases L2-1 and LUT-1, which contain two or more cysteines (Supplementary file 6, supplementary table 1), but no disulfide bonds (top row). Graphs show MIC fold changes for β-lactamase-expressing E. coli MC1000 and its dsbA mutant from three biological experiments each conducted as a single technical repeat; the MIC values used to generate this figure are presented in Supplementary file 2A (rows 2–7 and 9–20).
Figure 1—figure supplement 1
Complementation of dsbA restores the β-lactam MIC values for E. coli MC1000 dsbA expressing β-lactamase enzymes.
Re-insertion of dsbA at the attTn7 site of the chromosome restores representative β-lactam MIC values for E. coli MC1000 dsbA harboring (A) pDM1-blaBEL-1 (ceftazidime MIC), (B) pDM1-blaCARB-2 (cefuroxime MIC), (C) pDM1-blaAIM-1 (ceftazidime MIC), (D) pDM1-blaOXA-50 (ampicillin MIC), (E) pDM1-blaBPS-1m (ceftazidime MIC), and (F) pDM1-blaBPS-6 (ceftazidime MIC). Graphs show MIC values (µg/mL) and are representative of two biological experiments, each conducted as a single technical repeat.
Figure 2
Absence of DsbA results in degradation or misfolding of species-specific cysteine-containing β-lactamases.
(A) The protein levels of most tested disulfide-bond-containing Ambler class A, B, and D β-lactamases are drastically reduced when these enzymes are expressed in E. coli MC1000 dsbA; the amount of the control enzyme L2-1, containing three cysteines but no disulfide bonds, is unaffected. An exception to this is the class A enzyme BPS-1m, for which no decrease in abundance is observed in the dsbA mutant (compare lanes 11 and 12). Protein levels of StrepII-tagged β-lactamases were assessed using a Strep-Tactin-AP conjugate. A representative blot from three biological experiments, each conducted as a single technical repeat, is shown; molecular weight markers (M) are on the left, DnaK was used as a loading control and solid black lines indicate where the membrane was cut. Full immunoblots and SDS-PAGE analysis of the immunoblot samples for total protein content are shown in Supplementary file 3. (B) The hydrolysis of the chromogenic β-lactam nitrocefin by cysteine-containing β-lactamases is impaired when these enzymes are expressed in E. coli MC1000 dsbA. The hydrolytic activities of strains harboring the empty vector or expressing the control enzyme L2-1 show no dependence on DsbA. The ‘Enzyme stability’ column informs on the abundance of each enzyme when it is lacking its disulfide bond(s); this was informed from the immunoblotting experiments in panel (A) The ‘Nitrocefin hydrolysis’ column shows the amount of nitrocefin hydrolyzed per mg of bacterial cell pellet in 15 min. n=3, table shows means ± SD, significance is indicated by *=p < 0.05, ns = non-significant.
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Figure 2—source data 1
Original files of the full raw unedited immunoblots used to prepare Figure 2A.
‘Left’, ‘Middle’, and ‘Right’ in the file names refer to the part of the immunoblot to the left, in-between, or to the right of the vertical black lines shown in the final figure, respectively.
- https://cdn.elifesciences.org/articles/91082/elife-91082-fig2-data1-v1.zip
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Figure 2—source data 2
Uncropped immunoblots used to prepare Figure 2A.
The figure included in the paper is shown on the left and relevant bands used for each part of the figure are marked with red boxes on the uncropped immunoblots on the right. ‘Left’, ‘Middle’, and ‘Right’ in the file names refer to the part of the immunoblot to the left, in-between, or to the right of the vertical black lines shown in the final figure, respectively.
- https://cdn.elifesciences.org/articles/91082/elife-91082-fig2-data2-v1.zip
Figure 3 with 1 supplement
Absence of the principal DsbA analogue (DsbA1) allows treatment of multidrug-resistant Pseudomonas aeruginosa clinical isolates with existing β-lactam antibiotics.
(A) Deletion of dsbA1 in the AIM-1-expressing P. aeruginosa G4R7 clinical isolate sensitizes this strain to ceftazidime and results in reduction of the piperacillin/tazobactam MIC value by 192 μg/mL. (B) Deletion of dsbA1 in the AIM-1-expressing P. aeruginosa G6R7 clinical isolate sensitizes this strain to piperacillin/tazobactam and ceftazidime. (C) 100% of the G. mellonella larvae infected with P. aeruginosa G6R7 (blue curve) or P. aeruginosa G6R7 dsbA1 (light blue curve) die 18 hr post-infection, while only 52.5% of larvae infected with P. aeruginosa G6R7 and treated with piperacillin (red curve) survive 28 hr post-infection. Treatment of larvae infected with P. aeruginosa G6R7 dsbA1 with piperacillin (pink curve) results in 77.5% survival, 28 hr post-infection. The graph shows Kaplan-Meier survival curves of infected G. mellonella larvae after different treatment applications; horizontal lines represent the percentage of larvae surviving at the indicated time point (a total of 40 larvae were used for each curve). Statistical analysis of this data was performed using a Mantel-Cox test. The most relevant comparison is noted on the figure. Full statistical analysis is as follows: n=40; p=0.3173 (non-significance; P. aeruginosa vs P. aeruginosa dsbA1), p<0.0001 (significance; P. aeruginosa vs P. aeruginosa treated with piperacillin), p<0.0001 (significance; P. aeruginosa dsbA1 vs P. aeruginosa treated with piperacillin), p=0.0147 (significance; P. aeruginosa treated with piperacillin vs P. aeruginosa dsbA1 treated with piperacillin). (D) Deletion of dsbA1 in the GES-19/GES-26-expressing P. aeruginosa CDC #769 clinical isolate sensitizes this strain to piperacillin/tazobactam and aztreonam. (E) Deletion of dsbA1 in the GES-19/GES-20-expressing P. aeruginosa CDC #773 clinical isolate sensitizes this strain to piperacillin/tazobactam, aztreonam, and ceftazidime. (F) 100% of G. mellonella larvae infected with P. aeruginosa CDC #773 (blue curve), P. aeruginosa CDC #773 dsbA1 (light blue curve) or larvae infected with P. aeruginosa CDC #773 and treated with ceftazidime (red curve) die 21 hr post-infection. Treatment of larvae infected with P. aeruginosa CDC #773 dsbA1 with ceftazidime (pink curve) results in 96.7% survival, 24 hr post-infection. The graph shows Kaplan-Meier survival curves of infected G. mellonella larvae after different treatment applications; horizontal lines represent the percentage of larvae surviving at the indicated time point (a total of 30 larvae were used for each curve). Statistical analysis of this data was performed using a Mantel-Cox test. The most relevant comparison is noted on the figure. Full statistical analysis is as follows: n=30; p<0.0001 (significance; P. aeruginosa vs P. aeruginosa dsbA1), p>0.9999 (non-significance; P. aeruginosa vs P. aeruginosa treated with ceftazidime), p<0.0001 (significance; P. aeruginosa dsbA1 vs P. aeruginosa treated with ceftazidime), p<0.0001 (significance; P. aeruginosa treated with ceftazidime vs P. aeruginosa dsbA1 treated with ceftazidime). For panels (A), (B), (D), and (E), the graphs show MIC values (μg/mL) from three biological experiments, each conducted as a single technical repeat; red dotted lines indicate the EUCAST clinical breakpoint for each antibiotic.
Figure 3—figure supplement 1
Assessment of off-target effects for clinical strains of P. aeruginosa and S. maltophilia that are deficient in oxidative protein folding.
(A) P. aeruginosa CDC #769 and its mutant lacking dsbA1 have identical gentamicin MIC values, confirming that absence of DsbA does not compromise the general ability of the strain to resist antibiotic stress. (B) Re-insertion of the dsbA1 gene from P. aeruginosa PAO1 at the attTn7 site of the chromosome restores representative antibiotic MIC values for P. aeruginosa CDC #769 dsbA1 (left, piperacillin/tazobactam MIC; right, aztreonam MIC). (C) S. maltophilia AMM and its mutant lacking dsbA and dsbL have near-identical gentamicin MIC values, confirming that absence of DsbA and DsbL does not compromise the general ability of the strain to resist antibiotic stress. (D) Re-insertion of the dsbA1 gene from P. aeruginosa PAO1 at the attTn7 site of the chromosome restores representative antibiotic MIC values for S. maltophilia AMM dsbA dsbL (left, ceftazidime MIC; right, colistin MIC). (E) Changes in MIC values observed using the DSB system inhibitor (compound 36) are due solely to inhibition of the DSB system. The gentamicin MIC value of S. maltophilia AMM remains unchanged upon addition of the inhibitor (left), and the same is observed for the colistin MIC value of S. maltophilia AMM dsbA dsbL in the presence of the compound (right). This indicates that the chemical compound used in this study only affects the function of the DSB system proteins. For all panels, graphs show MIC values (µg/mL) and are representative of three biological experiments. β-Lactam MICs were conducted as a single technical repeat and colistin MICs were conducted in technical triplicate; red dotted lines indicate the EUCAST clinical breakpoint for each antibiotic, where applicable. In the absence of EUCAST clinical breakpoints for S. maltophilia, the black dotted lines indicate the EUCAST clinical breakpoint for each antibiotic for the related pathogen P. aeruginosa, where applicable.
Figure 4 with 1 supplement
Impairment of disulfide bond formation allows treatment of Stenotrophomonas maltophilia with β-lactam and colistin antibiotics while also inhibiting cross-protection between S. maltophilia and Pseudomonas aeruginosa in mixed communities.
(A–D) Impairment of disulfide bond formation allows the treatment of Stenotrophomonas maltophilia clinical strains with β-lactam and colistin antibiotics. (A, B) Deletion of dsbA dsbL in the S. maltophilia AMM and S. maltophilia GUE clinical isolates results in drastic decrease of their ceftazidime MIC values. (C) Deletion of dsbA dsbL in the S. maltophilia AMM clinical strain results in a drastic decrease of its colistin MIC value. (D) Use of a small-molecule inhibitor of DsbB against the S. maltophilia AMM clinical strain results in a decrease of its ceftazidime and colistin MIC values. For panels (A–D), graphs show MIC values (μg/mL) from three biological experiments; for β-lactam MIC assays, each experiment was conducted as a single technical repeat, whereas for colistin MIC assays, each experiment was conducted in technical triplicate. In the absence of EUCAST clinical breakpoints for S. maltophilia, the black dotted lines indicate the EUCAST clinical breakpoint for each antibiotic for the related pathogen P. aeruginosa. (E) Protection of P. aeruginosa by S. maltophilia clinical strains is dependent on oxidative protein folding. The susceptible P. aeruginosa strain PA14 can survive exposure to ceftazidime up to a maximum concentration of 4 μg/mL when cultured in isolation (white bars). By contrast, if co-cultured in the presence of S. maltophilia AMM, which can hydrolyze ceftazidime through the action of its L1-1 β-lactamase enzyme, P. aeruginosa PA14 can survive and actively grow in concentrations of ceftazidime as high as 512 μg/mL (dark pink bars). This protection is abolished if P. aeruginosa PA14 is co-cultured with S. maltophilia AMM dsbA dsbL (light pink bars), where L1-1 is inactive (as shown in (A) and Furniss et al., 2022). The graph shows P. aeruginosa PA14 colony-forming unit (CFU) counts for each condition; three biological replicates were conducted in technical triplicate, and mean CFU values are shown. The black dotted line indicates the P. aeruginosa PA14 inoculum. The mean CFU values used to generate this figure are presented in Supplementary file 2B.
Figure 4—figure supplement 1
Protection of P. aeruginosa by S. maltophilia clinical strains is dependent on oxidative protein folding.
(A) Comparison of the colony forming units (CFUs) of S. maltophilia AMM with the CFUs of S. maltophilia AMM dsbA dsbL after 6 hr of growth, prior to P. aeruginosa PA14 addition. The two S. maltophilia strains display equivalent growth. (B) Complementary analysis to Figure 4E; here the CFUs of all P. aeruginosa and S. maltophilia strains were enumerated in isolation and in mixed culture conditions for a more limited set of antibiotic concentrations. Equivalent trends to Figure 4E are observed. The susceptible P. aeruginosa strain PA14 can survive exposure to ceftazidime up to a maximum concentration of 4 μg/mL when cultured in isolation (white bars). By contrast, if co-cultured in the presence of S. maltophilia AMM (dark blue bars), which can hydrolyze ceftazidime through the action of its L1-1 β-lactamase enzyme, P. aeruginosa PA14 (dark pink bars) can survive and actively grow in higher concentrations of ceftazidime (see 128 μg/mL of ceftazidime). This protection is abolished if P. aeruginosa PA14 (light pink bars) is co-cultured with S. maltophilia AMM dsbA dsbL (light blue bars). In this case, L1-1 is inactive (as shown in Figure 4AB and Furniss et al., 2022), resulting in killing of S. maltophilia AMM and, in turn, eradication of P. aeruginosa PA14 (see 128 μg/mL of ceftazidime, absence of light pink bars). Three biological replicates were conducted in technical triplicate, and mean CFU values are shown. The gray line indicates the P. aeruginosa PA14 inoculum. The mean CFU values used to generate this figure are presented in Supplementary file 2C. Notably, for certain high-OD₆₀₀ conditions in the competition assay, transient aminoglycoside tolerance in these clinical isolates results in gentamicin selection underperforming at the plating (enumeration) step. This, in turn, yields artificially elevated apparent CFU counts (>10¹²). These inflated values occur reproducibly under the same conditions and do not affect the relative comparisons presented.
Figure 5 with 2 supplements
Inhibition of oxidative protein folding counters antibiotic resistance and inter-species interactions in CF-associated pathogens.
(Left) After Sec translocation to the periplasm and DsbA-assisted folding, cysteine-containing species-specific β-lactamase enzymes from recalcitrant pathogens, like P. aeruginosa or S. maltophilia, are active and can hydrolyze β-lactam antibiotics. However, in the absence of their disulfide bonds, DsbA-dependent β-lactamases either degrade or misfold, and thus can no longer confer resistance to β-lactam compounds. (Right) In multispecies bacterial communities, bacteria that degrade antibiotics, for example species producing β-lactamases, can protect antibiotic-susceptible strains. Targeting disulfide bond formation impairs interbacterial interactions that are reliant on the activity of DsbA-dependent β-lactamase enzymes, allowing eradication of both bacterial species.
Figure 5—figure supplement 1
The activity of additional species-specific β-lactamases depends on disulfide bond formation.
(A) β-Lactam MIC values for E. coli MC1000 expressing disulfide-bond-containing β-lactamases from P. otitidis (left, POM-1; Supplementary file 6, supplementary table 1) and Serratia spp. (right, SMB-1; Supplementary file 6, supplementary table 1) are reduced in the absence of DsbA (MIC fold changes:>2; fold change of 2 is indicated by the black dotted lines). No changes in MIC values are observed for the aminoglycoside antibiotic gentamicin (white bars), confirming that absence of DsbA does not compromise the general ability of this strain to resist antibiotic stress. Graphs show MIC fold changes for β-lactamase-expressing E. coli MC1000 and its dsbA mutant. MIC assays were performed in three biological experiments each conducted as a single technical repeat; the MIC values used to generate this figure are presented in Supplementary file 2A (rows 22–25). (B) Protein levels of disulfide-bond-containing β-lactamases are either unaffected (POM-1) or drastically reduced (SMB-1) when these enzymes are expressed in E. coli MC1000 dsbA. Protein levels of StrepII-tagged β-lactamases were assessed using a Strep-Tactin-AP conjugate. A representative blot from three biological experiments, each conducted as a single technical repeat, is shown; molecular weight markers (M) are on the left, DnaK was used as a loading control and solid black lines indicate where the membrane was cut. Full immunoblots and SDS PAGE analysis of the immunoblot samples for total protein content are shown in Supplementary file 3, supplementary table 1. (C) The hydrolytic activities of both tested β-lactamases are significantly reduced in the absence of DsbA. The hydrolytic activities of strains harboring the empty vector or expressing the control enzyme L2-1 show no dependence on DsbA; the same data for the control strains are also shown in Figure 2B. n=3 (each conducted in technical duplicate), table shows means ± SD, significance is indicated by *=p < 0.05, ns = non-significant.
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Figure 5—figure supplement 1—source data 1
Original files of the full raw unedited immunoblots used to prepare Figure 5—figure supplement 1B.
‘Left’ and ‘Right’ in the file names refer to the part of the immunoblot to the left or to the right of the vertical black line shown in the final figure, respectively.
- https://cdn.elifesciences.org/articles/91082/elife-91082-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Uncropped immunoblots used to prepare Figure 5—figure supplement 1B.
The figure included in the paper is shown on the left and relevant bands used for each part of the figure are marked with red boxes on the uncropped immunoblots on the right. ‘Left’ and ‘Right’ in the file names refer to the part of the immunoblot to the left or to the right of the vertical black line shown in the final figure, respectively.
- https://cdn.elifesciences.org/articles/91082/elife-91082-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Complementation of dsbA restores the β-lactam MIC values for E. coli MC1000 dsbA expressing β-lactamases.
Re-insertion of dsbA at the attTn7 site of the chromosome restores representative β-lactam MIC values for E. coli MC1000 dsbA harboring (A) pDM1-blaPOM-1 (imipenem MIC), and (B) pDM1-blaSMB-1 (ceftazidime MIC). Graphs show MIC values (µg/mL) and are representative of two biological experiments, each conducted as a single technical repeat.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Escherichia coli) | DH5α | Hanahan, 1985 | F– endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZ∆M15 ∆(lacZYA-argF)U169 hsdR17(rK–mK+) λ– | - |
| Genetic reagent (E. coli) | DH5αλpir | Martínez-García and de Lorenzo, 2011 | λpir | - |
| Genetic reagent (E. coli) | CC118λpir | Herrero et al., 1990 | araD Δ(ara, leu) ΔlacZ74 phoA20 galK thi-1 rspE rpoB argE recA1 λpir | - |
| Genetic reagent (E. coli) | HB101 | Boyer and Roulland-Dussoix, 1969 | supE44 hsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 | - |
| Genetic reagent (E. coli) | MC1000 | Casadaban and Cohen, 1980 | araD139 ∆(ara, leu)7697 ∆lacX74 galU galK strA | - |
| Genetic reagent (E. coli) | MC1000 dsbA | Kadokura et al., 2004 | dsbA::aphA, KanR | - |
| Genetic reagent (E. coli) | MC1000 dsbA attTn7::Ptac-dsbA | Furniss et al., 2022 | dsbA::aphA attTn7::dsbA, KanR | Can be obtained from the Mavridou lab |
| Strain, strain background (Pseudomonas aeruginosa) | PAO1 | Holloway, 1969 | wild-type prototroph | - |
| Strain, strain background (P. aeruginosa) | PA14 | Rahme et al., 1995 | wild-type prototroph | - |
| Genetic reagent (P. aeruginosa) | PA14 attTn7::accC | This study | attTn7::accC, GentR | Can be obtained from the Mavridou lab |
| Strain, strain background (P. aeruginosa) | G4R7 | Dortet et al., 2018 | blaAIM-1 | Human clinical strain |
| Genetic reagent (P. aeruginosa) | G4R7 dsbA1 | This study | dsbA1 blaAIM-1 | Can be obtained from the Mavridou lab |
| Strain, strain background (P. aeruginosa) | G6R7 | Dortet et al., 2018 | blaAIM-1 | Human clinical strain |
| Genetic reagent (P. aeruginosa) | G6R7 dsbA1 | This study | dsbA1 blaAIM-1 | Can be obtained from the Mavridou lab |
| Strain, strain background (P. aeruginosa) | CDC #769 | CDC AR Isolate Bank | blaGES-19blaGES-26 | Human clinical strain |
| Genetic reagent (P. aeruginosa) | CDC #769 dsbA1 | This study | dsbA1 blaGES-19blaGES-20 | Can be obtained from the Mavridou lab |
| Strain, strain background (Stenotrophomonas maltophilia) | AMM | Emeraud et al., 2019 | blaL2-1blaL1-1 | Human clinical strain |
| Genetic reagent (S. maltophilia) | AMM dsbA dsbL | This study | dsbA dsbL blaL2-1blaL1-1 | Can be obtained from the Mavridou lab |
| Genetic reagent (S. maltophilia) | AMM attTn7::accC msfgfp | This study | blaL2-1blaL1-1 attTn7::accC msfgfp, GentR | Can be obtained from the Mavridou lab |
| Genetic reagent (S. maltophilia) | AMM dsbA dsbL attTn7::accC msfgfp | This study | dsbA dsbL blaL2-1blaL1-1attTn7::accC msfgfp, GentR | Can be obtained from the Mavridou lab |
| Genetic reagent (S. maltophilia) | AMM dsbA dsbL attTn7::accC msfgfp dsbA1 | This study | dsbA dsbL blaL2-1blaL1-1attTn7::accC msfgfp dsbA1, GentR | Can be obtained from the Mavridou lab |
| Strain, strain background (S. maltophilia) | GUE | Emeraud et al., 2019 | blaL2-1blaL1-1 | Human clinical strain |
| Genetic reagent (S. maltophilia) | GUE dsbA dsbL | This study | dsbA dsbL blaL2-1blaL1-1 | Can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1 (plasmid) | Mavridou lab stock | GenBank MN128719 | pDM1 vector, p15A ori, Ptac promoter, MCS, TetR |
| Recombinant DNA reagent | pDM1-blaL2-1 (plasmid) | Furniss et al., 2022 | - | blaL2-1 cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaLUT-1 (plasmid) | This study | - | blaLUT-1 cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaCARB-2 (plasmid) | This study | - | blaCARB-2 cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaBPS-1m (plasmid) | This study | - | blaBPS-1m cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaBPS-6 (plasmid) | This study | - | blaBPS-6 cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaAIM-1 (plasmid) | This study | - | blaAIM-1 cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaPOM-1 (plasmid) | This study | - | blaPOM-1 cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaSMB-1 (plasmid) | This study | - | blaSMB-1 cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaOXA-50 (plasmid) | This study | - | blaOXA-50 cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaL2-1-StrepII (plasmid) | Furniss et al., 2022 | - | blaL2-1 encoding L2-1 with a C-terminal StrepII tag cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaBEL-1-StrepII (plasmid) | This study | - | blaBEL-1 encoding BEL-1 with a C-terminal StrepII tag cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaCARB-2-StrepII (plasmid) | This study | - | blaCARB-2 encoding CARB-2 with a C-terminal StrepII tag cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaBPS-1m -StrepII (plasmid) | This study | - | blaBPS-1m encoding BPS-1m with a C-terminal StrepII tag cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaAIM-1-StrepII (plasmid) | This study | - | blaAIM-1 encoding AIM-1 with a C-terminal StrepII tag cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaPOM-1-StrepII (plasmid) | This study | - | blaPOM-1 encoding POM-1 with a C-terminal StrepII tag cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaSMB-1-StrepII (plasmid) | This study | - | blaSMB-1 encoding SMB-1 with a C-terminal StrepII tag cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pDM1-blaOXA-50-StrepII (plasmid) | This study | - | blaOXA-50 encoding OXA-50 with a C-terminal StrepII tag cloned into pDM1, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pKNG101 (plasmid) | Kaniga et al., 1991 | - | Gene replacement suicide vector, oriR6K, oriTRK2, sacB, StrR |
| Recombinant DNA reagent | pKNG102 (plasmid) | Bernal lab stock | - | Gene replacement suicide vector, oriR6K, oriTRK2, sacB, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pKNG101-dsbA1 (plasmid) | Furniss et al., 2022 | - | PCR fragment containing the regions upstream and downstream P. aeruginosa dsbA1 cloned in pKNG101; when inserted into the chromosome, the strain is a merodiploid for dsbA1 mutant, StrR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pKNG102-dsbA1-769 (plasmid) | This study | - | PCR fragment containing the regions upstream and downstream P. aeruginosa CDC #769 (Supplementary file 2) dsbA1 cloned in pKNG102; when inserted into the chromosome, the strain is a merodiploid for dsbA1 mutant, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pKD4 (plasmid) | Datsenko and Wanner, 2000 | - | Conditional oriRγ ori, (template for the aphA cassette), AmpR |
| Recombinant DNA reagent | pCB112 (plasmid) | Paradis-Bleau et al., 2014 | - | Inducible lacZ expression under the control of the Plac promoter, pBR322 ori, CamR |
| Recombinant DNA reagent | pKNG101 (plasmid) | Kaniga et al., 1991 | - | Gene replacement suicide vector, oriR6K, oriTRK2, sacB, (template for the strAB cassette), StrR |
| Recombinant DNA reagent | pKNG101-dsbA1 (plasmid) | This study | - | PCR fragment containing the regions upstream and downstream P. aeruginosa dsbA1 cloned in pKNG101; when inserted into the chromosome the strain is a merodiploid for dsbA1 mutant, StrR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pKNG102-dsbA1-773 | This study | - | PCR fragment containing the regions upstream and downstream P. aeruginosa CDC #773 (Supplementary file 2) dsbA1 cloned in pKNG102; when inserted into the chromosome, the strain is a merodiploid for dsbA1 mutant, TetR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pKNG101-dsbA dsbL-AMM (plasmid) | This study | - | PCR fragment containing the regions upstream and downstream S. maltophilia AMM dsbA and dsbL genes cloned in pKNG101; when inserted into the chromosome, the strain is a merodiploid for dsbA dsbL mutant, StrR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pKNG101-dsbA dsbL-GUE (plasmid) | This study | - | PCR fragment containing the regions upstream and downstream S. maltophilia GUE dsbA and dsbL genes cloned in pKNG101; when inserted into the chromosome, the strain is a merodiploid for dsbA dsbL mutant, StrR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pRK600 (plasmid) | Kessler et al., 1992 | - | Helper plasmid, ColE1 ori, mobRK2, traRK2, CamR |
| Recombinant DNA reagent | pTn7-M (plasmid) | Zobel et al., 2015 | - | Mini-Tn7 delivery transposon vector containing the Tn7 flanking regions and a GentR marker, R6K ori, KanR, GentR |
| Recombinant DNA reagent | pBG42 (plasmid) | Zobel et al., 2015 | - | Mini-Tn7 delivery transposon vector containing the Tn7 flanking regions, a GentR marker and msfgfp, R6K ori, KanR, GentR |
| Recombinant DNA reagent | pBG42-PAO1dsbA1 (plasmid) | This study | - | dsbA1 encoding DsbA1 from P. aeruginosa PAO1 cloned into pBG42, KanR, GentR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pTNS2 (plasmid) | Choi et al., 2005 | - | Helper plasmid, R6K ori; encodes the TnsABC +D specific transposition pathway, AmpR |
| Recombinant DNA reagent | pMK-RQ carb-2 (plasmid) | This study | - | GeneArt cloning vector containing carb-2, ColE1 ori, (template for carb-2), KanR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pMK-RQ bps-1m (plasmid) | This study | - | GeneArt cloning vector containing bps-1m, ColE1 ori, (template for bps-1m), KanR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pMK-RQ bps-6 (plasmid) | This study | - | GeneArt cloning vector containing bps-6, ColE1 ori, (template for bps-6), KanR; can be obtained from the Mavridou lab |
| Recombinant DNA reagent | pMK-RQ smb-1 (plasmid) | This study | - | GeneArt cloning vector containing smb-1, ColE1 ori, (template for smb-1), KanR; can be obtained from the Mavridou lab |
| Chemical compound, drug | Ampicillin | Melford | A40040-10.0 | - |
| Chemical compound, drug | Imipenem | Cambridge Bioscience | CAY16039-100 mg | - |
| Chemical compound, drug | Kanamycin | Gibco | 11815032 | - |
| Chemical compound, drug | Gentamicin | VWR | A1492.0025 | - |
| Chemical compound, drug | Streptomycin | ACROS Organics | AC612240500 | - |
| Chemical compound, drug | Tetracycline | Duchefa Biochemie | T0150.0025 | - |
| Chemical compound, drug | Colistin sulphate | Sigma | C4461-1G | - |
| Chemical compound, drug | Tazobactam | Sigma | T2820-10MG | - |
| Chemical compound, drug | Isopropyl β-D-1-thiogalactopyranoside (IPTG) | Melford | I56000-25.0 | - |
| Chemical compound, drug | KOD Hotstart DNA Polymerase | Sigma | 71086–3 | - |
| Chemical compound, drug | Nitrocefin | Abcam | ab145625-25mg | - |
| Chemical compound, drug | 4,5-dibromo-2-(2-chlorobenzyl)pyridazin-3(2 H)-one | Bioduro-Sundia | - | Custom synthesis |
| Commercial assay or kit | NEBuilder HiFi DNA Assembly | New England Biolabs | E5520S | |
| Commercial assay or kit | BugBuster Mastermix | Sigma | 71456–3 | - |
| Commercial assay or kit | SigmaFast BCIP/NBT tablets | Sigma | B5655-25TAB | - |
| Commercial assay or kit | ETEST - Amoxicillin | Biomerieux | 412242 | - |
| Commercial assay or kit | ETEST - Cefuroxime | Biomerieux | 412304 | - |
| Commercial assay or kit | ETEST - Ceftazidime | Biomerieux | 412292 | - |
| Commercial assay or kit | ETEST - Imipenem | Biomerieux | 412373 | - |
| Commercial assay or kit | ETEST - Aztreonam | Biomerieux | 412258 | - |
| Commercial assay or kit | ETEST - Gentamicin | Biomerieux | 412367 | - |
| Antibody | Strep-Tactin-AP conjugate (mouse monoclonal) | Iba Lifesciences | NC0485490 | (1:3,000) in 3 w/v % BSA/TBS-T |
| Antibody | anti-DnaK 8E2/2 (mouse monoclonal) | Enzo Life Sciences | ADI-SPA-880-D | (1:10,000) in 5% w/v skimmed milk/TBS-T |
| Antibody | anti-mouse IgG-AP conjugate (goat polyclonal) | Sigma | A3688-.25ML | (1:6,000) in 5% w/v skimmed milk/TBS-T |
| Software, algorithm | Prism | GraphPad | - | version 8.0.2 |
| Software, algorithm | USEARCH | Edgar, 2010 | - | version 7.0 |
| Software, algorithm | MUSCLE | Edgar, 2004 | - | - |
| Software, algorithm | FastTree | Price et al., 2010 | - | version 2.1.7 |
| Software, algorithm | HMMER | Finn et al., 2015 | - | version 3.1b2 |
Additional files
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Supplementary file 1
Analysis of the cysteine content and phylogeny of all identified β-lactamases.
7741 unique β-lactamase protein sequences were clustered with a 90% identity threshold and the centroid of each cluster was used as a phylogenetic cluster identifier for each sequence (‘Phylogenetic cluster (90% ID)’ column). All sequences were searched for the presence of cysteine residues (‘Total number of cysteines’ and ‘Positions of all cysteines’ columns). Proteins with two or more cysteines after the first 30 amino acids of their primary sequence (cells shaded in gray in the ‘Number of cysteines after position 30’ column) are potential substrates of the DSB system for organisms where oxidative protein olding is carried out by DsbA and provided that translocation of the β-lactamase outside the cytoplasm is performed by the Sec system. The first 30 amino acids of each sequence were excluded to avoid considering cysteines that are part of the signal sequence mediating the translocation of these enzymes outside the cytoplasm. Cells shaded in gray in the ‘Reported in pathogens’ column mark β-lactamases that are found in pathogens or organisms capable of causing opportunistic infections. The Ambler class of each enzyme is indicated in the ‘Ambler class column’ and each class (A, B1, B2, B3, C, and D) is highlighted in a different color.
- https://cdn.elifesciences.org/articles/91082/elife-91082-supp1-v1.xlsx
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Supplementary file 2
Data used to generate Figure 1, Figure 5—figure supplement 1, Figure 4E, Figure 4—figure supplement 1B.
(A) MIC values (µg/mL) used to generate Figure 1 are in rows 2–7 (strains serving as negative controls; E. coli MC1000 strains harboring pDM1 (vector alone), pDM1-blaL2-1 or pDM1-blaLUT-1 (cysteine-containing β-lactamases which lack disulfide bonds)) and rows 9–20. MIC values (µg/mL) used to generate Figure 5—figure supplement 1 are in rows 22–25. The aminoglycoside antibiotic gentamicin serves as a negative control for all strains. Cells marked with a dash (-) represent strain-antibiotic combinations that were not tested. (B) P. aeruginosa PA14 colony forming unit (CFU) counts used to generate Figure 4E. (C) P. aeruginosa PA14, S. maltophilia AMM, and S. maltophilia AMM dsbA dsbL CFU counts used to generate Figure 4—figure supplement 1B. For all tabs, three biological experiments are shown; for (B) and (C), each biological replicate was conducted in technical triplicate and mean CFU values are shown.
- https://cdn.elifesciences.org/articles/91082/elife-91082-supp2-v1.xlsx
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Supplementary file 3
Full immunoblots and SDS-PAGE analysis of the immunoblot samples for total protein content.
(Pages 1–6). Full immunoblots for Figure 2A, Figure 5—figure supplement 1B. On the left of each page, the relevant figure panel is shown and the lanes in question are marked with red outline. On the right of each page, the full immunoblot is displayed with the corresponding area also marked with red outline. (Pages 7–9) SDS PAGE analysis of the immunoblot samples for total protein content. In each page, the immunoblot in question is indicated (by “Figure 2A” or “Figure 5—figure supplement 1B”) and lanes are marked accordingly to identify the immunoblot lane that they correspond to (see white labels at the bottom of the gel).
- https://cdn.elifesciences.org/articles/91082/elife-91082-supp3-v1.pdf
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Supplementary file 4
Analysis of Stenotrophomonas spp. for the presence of MCR proteins.
Hidden Markov Models built from validated sequences of MCR-like and EptA-like proteins were used for the identification of MCR-like analogs in a total of 106 complete genomes of the Stenotrophomonas genus downloaded from the NCBI repository. (A) Most genomes that were investigated (‘Stenotrophomonas maltophilia genome’ column), encoded one or two MCR-like proteins (‘Number of MCR analogues column’). (B) The 146 MCR-like sequences (‘Protein ID column’) that were identified (only hits with evalues <1e-10 were considered; ‘Evalue’ column) belong to the same phylogenetic group as validated MCR-5 or MCR-8 proteins (‘Phylogenetic group’ column).
- https://cdn.elifesciences.org/articles/91082/elife-91082-supp4-v1.xlsx
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Supplementary file 5
Quality control information on 4,5-dibromo-2-(2-chlorobenzyl)pyridazin-3(2 H)-one.
1H-NMR and LCMS spectra of 4,5-dibromo-2-(2-chlorobenzyl)pyridazin-3(2 H)-one (compound 36) demonstrating the correctness and purity of the synthesized compound by Bioduro-Sundia.
- https://cdn.elifesciences.org/articles/91082/elife-91082-supp5-v1.pdf
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Supplementary file 6
Supplemary file including supplementary Tables 1 - 5 and supplementary reference citations.
- https://cdn.elifesciences.org/articles/91082/elife-91082-supp6-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/91082/elife-91082-mdarchecklist1-v1.pdf
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Antibiotic potentiation and inhibition of cross-resistance in pathogens associated with cystic fibrosis
eLife 12:RP91082.
https://doi.org/10.7554/eLife.91082.3
