Penicillin-Binding Protein Occupancy Dataset for 18 β-Lactams and 4 β-Lactamase Inhibitors in Neisseria gonorrhoeae

Abstract

The lack of effective first-line antibiotic treatments against Neisseria gonorrhoeae, and the worldwide dissemination of resistant strains, are the main drivers of a worsening global health crisis. β-lactam antibiotics have been the backbone of therapeutic armamentarium against gonococci. However, we are lacking critical insights to design rationally optimized therapies. In the present work, we generated the first PBP-binding data set on 18 currently available and clinically relevant β-lactams and 4 β-lactamase inhibitors in two N. gonorrhoeae ATCC type collection strains, 19424 and 49226 (PBP2 type XXII and A39T change in mtrR). PBP binding (IC₅₀) was determined via the Bocillin FL binding assay in isolated membrane preparations. Three clusters of differential PBP IC₅₀s were identified and were mostly consistent across both strains, but with quantitative differences. Carbapenems were coselective for PBP2 and PBP3 (0.01 to 0.03 mg/L). Third- and fourth-generation cephalosporins cefixime, cefotaxime, ceftazidime, cefepime, and ceftriaxone showed the lowest IC₅₀ values for PBP2 (0.01 mg/L), whereas cefoxitin, ceftaroline, and ceftolozane required higher concentrations (0.04 to >2 mg/L). Aztreonam was selective for PBP2 in both strains (0.03 to 0.07 mg/L); amdinocillin bound this PBP at higher concentrations (1.33 to 2.94 mg/L). Penicillins specifically targeted PBP2 in strain ATCC 19424 (0.02 to 0.19 mg/L) and showed limited inhibition in strain ATCC 49226 (0.01 to >2 mg/L). Preferential PBP2 binding was observed by β-lactam-based β-lactamase inhibitors sulbactam and tazobactam (1.07 to 6.02 mg/L); meanwhile, diazabicyclooctane inhibitors relebactam and avibactam were selective for PBP3 (1.27 to 5.40 mg/L). This data set will set the bar for future studies that will help the rational use and translational development of antibiotics against multidrug-resistant (MDR) N. gonorrhoeae. IMPORTANCE The manuscript represents the first N. gonorrhoeae PBP-binding data set for 22 chemically different drugs in two type strains with different genetic background. We have identified three clusters of drugs according to their PBP binding IC₅₀s and highlighted the binding differences across the two strains studied. With the currently available genomic information and the PBP-binding data, we have been able to correlate the target attainment differences and the mutations that affect the drug uptake with the MIC changes. The results of the current work will allow us to develop molecular tools of great practical use for the study and the design of new rationally designed therapies capable of combating the growing MDR gonococci threat.

Keywords: N. gonorrhoeae; gonococcus; penicillin-binding proteins (PBP); β-lactam resistance; β-lactams.

FIG 1

Penicillin-binding protein profiles of N. gonorrhoeae strains ATCC 19424 and ATCC 49226. PBPs were labeled with Bocillin FL, separated by SDS-PAGE, and quantified via the ImageQuantTL program. ^aApparent molecular mass relative to Precision Plus Protein Dual Color Standards (range 10 to 250 kDa) (Bio-Rad Laboratories, Hercules, CA). (a) Comparison of the PBP profiles of the two studied N. gonorrhoeae strains. (b) Membrane preparation loaded with and without Bocillin FL labelling. ^#Two autofluorescence bands were present in both strains with and without Bocillin FL labelling (not bound by any of the drugs tested); they were excluded from any further analysis. *The band below PBP3 is a potential proteolytic band and was excluded from further analysis.

FIG 2

Binding patterns of β-lactams in N. gonorrhoeae PBPs from strain ATCC 19424. DOR, doripenem; ETP, ertapenem; IPM, imipenem; MEM, meropenem; FEP, cefepime; CFM, cefixime; CTX, cefotaxime; FOX, cefoxitin; CPT, ceftaroline; CAZ, ceftazidime; TOL, ceftolozane; CRO, ceftriaxone; ATM, aztreonam; MEC, amdinocillin (amdinocillin); CAR, carbenicillin; PenG, penicillin G; PIP, piperacillin; TIC, ticarcillin; AVI, avibactam; REL, relebactam; SUL, sulbactam; TZ, tazobactam. The membrane preparations were incubated with the indicated β-lactams for 30 min before Bocillin FL labeling. Labeled PBPs were separated by SDS-PAGE and detected using a fluorimager. The range of concentrations tested was 0.015 to 2 mg/L. *MEC and BLIs studied ranged from 2 to 256 mg/L.

FIG 3

Binding patterns of β-lactams for N. gonorrhoeae PBPs from strain ATCC 49226. The membrane preparations were incubated with the indicated β-lactams for 30 min before Bocillin FL labeling. The range of concentrations tested was 0.015 to 2 mg/L. Please see Fig. 2 for abbreviations. *MEC and BLIs studied ranged from 2 to 256 mg/L.

FIG 4

Agglomerative hierarchical clustering for logarithmic IC₅₀ data of the tested 22 drugs with penicillin-binding proteins in isolated membranes of N. gonorrhoeae strains (a) ATCC 19424 and (b) ATCC 49226 using the XLSTAT program. C1 to C3 represent clusters 1 to 3. Please see Fig. 2 for abbreviations.