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. No changes in MIC values 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 (Table S1), 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 File S2A (rows 2-7 and 9-20).

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 or a Strep-Tactin-HRP 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. (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 minutes. n=3, table shows means ±SD, significance is indicated by * = p < 0.05, ns = non-significant.

The biogenesis of numerous cysteine-containing β-lactamase enzymes is dependent on disulfide bond formation.

Cysteine-containing β-lactamase enzymes, which are translocated to the periplasm through the Sec system, rely on DsbA-mediated disulfide bond formation for their stability and folding. We have previously shown that disulfide bond formation is essential for the activity of clinically important broad-spectrum enzymes, usually carried on plasmids [15]. Data presented here (Figures 1 and 2) demonstrate that oxidative protein folding is also critical for several species-specific β-lactamases from P. aeruginosa and Burkholderia spp. After translocation to the periplasm and DsbA-assisted folding, these enzymes are active and can hydrolyze β-lactam antibiotics, rendering bacteria resistant. 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. After each round of oxidative protein folding, DsbA is regenerated by the quinone (Q)-containing protein DsbB, which in turn transfers the reducing equivalents to the respiratory chain [46].

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 hours post infection, while only 52.5% of larvae infected with P. aeruginosa G6R7 and treated with piperacillin (red curve) survive 28 hours post infection. Treatment of larvae infected with P. aeruginosa G6R7 dsbA1 with piperacillin (pink curve) results in 77.5% survival, 28 hours 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 after application of each treatment 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; n=40; p=0.3173 (non-significance) (P. aeruginosa versus P. aeruginosa dsbA1), p<0.0001 (significance) (P. aeruginosa vs P. aeruginosa treated with piperacillin), p<0.0001 (significance) (P. aeruginosa dsbA1 versus P. aeruginosa treated with piperacillin), p=0.0147 (significance) (P. aeruginosa treated with piperacillin versus 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 hours post infection. Treatment of larvae infected with P. aeruginosa CDC #773 dsbA1 with ceftazidime (pink curve) results in 96.7% survival, 24 hours 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 after application of each treatment 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; n=30; p<0.0001 (significance) (P. aeruginosa versus P. aeruginosa dsbA1), p>0.9999 (non-significance) (P. aeruginosa vs P. aeruginosa treated with ceftazidime), p<0.0001 (significance) (P. aeruginosa dsbA1 versus P. aeruginosa treated with ceftazidime), p<0.0001 (significance) (P. aeruginosa treated with ceftazidime versus P. aeruginosa dsbA1 treated with ceftazidime). No changes in MIC values are observed for the aminoglycoside antibiotic gentamicin upon deletion of dsbA1 from a representative clinical strain of P. aeruginosa (P. aeruginosa G6R7), confirming that disruption of disulfide bond formation does not compromise the general ability of this organism to resist antibiotic stress (Fig. S2A). 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.

(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 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 drastic decrease of its ceftazidime and colistin MIC values. No changes in MIC values are observed for the aminoglycoside antibiotic gentamicin upon deletion of dsbA and dsbL from a representative clinical strain of S. maltophilia (S. maltophilia AMM), confirming that disruption of disulfide bond formation does not compromise the general ability of this organism to resist antibiotic stress (Fig. S2B). 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 Figure 5A and [15]). The graph shows P. aeruginosa PA14 colony forming unit counts (CFUs) 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 File S2B.

Inhibition of oxidative protein folding prevents antibiotic resistance and inter-species interactions in CF-associated pathogens.

(Left) Abrogation of DsbA activity results in incapacitation of antibiotic resistance proteins and allows treatment of recalcitrant pathogens with existing antibiotics (the schematic depicts inhibition of β-lactam hydrolysis as an example). (Right) In multispecies bacterial communities, protection of antibiotic susceptible strains by species that can degrade antibiotics is prevalent. Targeting disulfide bond formation impairs beneficial interactions that are reliant on the activity of DsbA-dependent resistance proteins (for example β-lactamase enzymes), thus allowing eradication of all species.