Penicillin-Binding Protein 5/6 Acting as a Decoy Target in Pseudomonas aeruginosa Identified by Whole-Cell Receptor Binding and Quantitative Systems Pharmacology

Abstract

The β-lactam antibiotics have been successfully used for decades to combat susceptible Pseudomonas aeruginosa, which has a notoriously difficult to penetrate outer membrane (OM). However, there is a dearth of data on target site penetration and covalent binding of penicillin-binding proteins (PBP) for β-lactams and β-lactamase inhibitors in intact bacteria. We aimed to determine the time course of PBP binding in intact and lysed cells and estimate the target site penetration and PBP access for 15 compounds in P. aeruginosa PAO1. All β-lactams (at 2 × MIC) considerably bound PBPs 1 to 4 in lysed bacteria. However, PBP binding in intact bacteria was substantially attenuated for slow but not for rapid penetrating β-lactams. Imipenem yielded 1.5 ± 0.11 log₁₀ killing at 1h compared to <0.5 log₁₀ killing for all other drugs. Relative to imipenem, the rate of net influx and PBP access was ~ 2-fold slower for doripenem and meropenem, 7.6-fold for avibactam, 14-fold for ceftazidime, 45-fold for cefepime, 50-fold for sulbactam, 72-fold for ertapenem, ~ 249-fold for piperacillin and aztreonam, 358-fold for tazobactam, ~547-fold for carbenicillin and ticarcillin, and 1,019-fold for cefoxitin. At 2 × MIC, the extent of PBP5/6 binding was highly correlated (r² = 0.96) with the rate of net influx and PBP access, suggesting that PBP5/6 acted as a decoy target that should be avoided by slowly penetrating, future β-lactams. This first comprehensive assessment of the time course of PBP binding in intact and lysed P. aeruginosa explained why only imipenem killed rapidly. The developed novel covalent binding assay in intact bacteria accounts for all expressed resistance mechanisms.

Keywords: Penicillin binding proteins (PBP); bacterial permeability; beta-lactams/beta-lactamase inhibitors; intact cells; outer membrane penetration; quantitative and systems pharmacology (QSP).

FIG 1

Normalized protein expression and relative band intensity of the whole cell and isolated membrane PBP binding assays. Normalized PBP expression relative to PBP5/6 in (a) whole cells and (b) isolated membranes. The columns represent the average PBP expression values obtained from at least 3 biological replicates, and the error bars show the standard deviations. One-way ANOVA with post hoc Tukey’s multiple comparison test was run to determine the statistically significant differences over time (*, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001). Bacterial growth (CFU/mL) in the whole cells assay (c). The average viable counts from at least 3 experiments ± standard deviations are shown. The data were normalized to the highest expression PBP, which was always PBP5/6. Therefore, PBP5/6 contained no error bars. (d) Coefficient of variation (CV) of PBP5/6 expression across the 3 replicates.

FIG 2

Difference between whole cells and isolated PBPs fraction unbound. P. aeruginosa PAO1 cultures (whole cells) and PBP-containing membrane preparations (isolated) were incubated for 15, 30, and 60 min in the presence of doripenem (DOR); ertapenem (ETP); imipenem (IPM); meropenem (MEM); cefepime (FEP); cefoxitin (FOX); ceftazidime (CAZ); aztreonam (ATM); carbenicillin (CAR); piperacillin (PIP); ticarcillin (TIC); avibactam (AVI); relebactam (REL); sulbactam (SUL); tazobactam (TZB), and afterwards isolated PBPs labeled with 25 μM Boc-FL. The resulting 0.5 mg/mL of PBP-containing membrane preparations (whole cells) were labeled with 25 μM Boc-FL. The antibiotic concentrations tested were 2 × MIC: DOR = 2 mg/L; ETP = 8 mg/L; IPM = 2 mg/L; MEM = 1 mg/L; FEP = 2 mg/L; FOX = 2,048 mg/L; CAZ = 2 mg/L; ATM = 8 mg/L; CAR = 96 mg/L; PIP = 8 mg/L; TIC = 48 mg/L. The BLIs AVI, REL, SUL, and TZB were used at a fixed concentration of 4 mg/L. Data represent the difference between average fraction of unbound PBPs in the whole-cell and isolated membrane assays from at least three biological replicates; the error bars show the SD of the differences. Unpaired Student’s t tests were applied to determine statistically significant differences (*, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001).

FIG 3

Time-course of PBP binding for PBPs 1a, 1b, 2, 3, 4, and 5/6 in intact (upper rows) and lysed cells (lower rows) for 15 drugs. The markers are the means of the observations, and the lines are the curve-fits from simultaneous modeling of the PBP binding data from QSP modeling. This plot focused (i.e., zoomed in) on the most important PBPs, and the same data with the full y axis range (due to PBP5/6) is shown in the supplement (Fig. S7).

FIG 4

Empirical relationship between the extent of PBP5/6 binding and the number of β-lactam molecules that penetrate the outer membrane and inactivate PBPs per minute at 2 × the MIC (i.e., Rate_{Influx/access}). Please note that the Rate_{Influx/access} accounts for the extracellular drug concentration (as shown in the last column of Table 1). The plot only shows β-lactams, since the BLI were studied at 4 mg/L (and not at 2 × the MIC). The outlier at the bottom left for the lysed cell assay was ertapenem (ETP), which penetrated slowly. The plot highlights that β-lactams which inactivate the highly expressed PBP5/6 need to be rapidly penetrating (i.e., have a high rate of net influx and PBP access) to achieve 2 × MIC. In contrast, slowly penetration β-lactams can be efficacious (i.e., 2 × MIC), as long as they have limited or no PBP5/6 binding.