Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins inBacillus subtilis

Abstract

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b or PBP4. Almost all penicillin scaffolds were co-selective for all or combinations of PBP2a, 2b and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin), 2b (cefotaxime) or 2a, 2b and 4 (cephalothin).Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid) and various co-selective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

A regulatory pathway that selectively up-regulates elongasome function in the absence of class A PBPs

“…We tested 4 β-lactams (cefuroxime, oxacillin, ampicillin and penicillin G) for their inhibition profiles against ΔrasP and ΔponA strains. Oxacillin and cefuroxime (CEF) were previously suggested to preferentially inhibit aPBPs ( Sassine et al, 2017 ; Sharifzadeh et al, 2020 ), whereas penicillin G preferentially inhibits bPBPs ( Sassine et al, 2017 ). Consistently, oxacillin and CEF had highest activity against ∆ rasP , whereas penicillin G and ampicillin had the highest activity against ∆ ponA, which encodes the major aPBP, PBP1 ( Figure 2B ).…”

Mutations inrpoBthat confer rifampicin resistance can alter levels of peptidoglycan precursors and affect β-lactam susceptibility

“…We next tested the sensitivity of the three clinically relevant RIF resistance mutants towards additional β-lactams and other antibiotics that target the cell wall (Figure 3B). All β-lactams inhibit the formation of PG layer by targeting different PBPs with varying affinities (35). Three additional β-lactams (oxacillin, ampicillin and penicillin) were similar to CEF with S487L increasing resistance, and H482Y conferring sensitivity.…”

“…MurA initiates synthesis of the second sugar required for PG synthesis, UDP-MurNAc. We included ponA , which encodes PBP1, the primary aPBP involved in PG synthesis during vegetative growth and a major target of CEF inhibition (23, 35). PG synthesis can also be supported by import of amino sugars such as GlcNAc present in the growth medium.…”

Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

“…Indeed, previous work by us and others to identify PBP-selective inhibitors has yielded β-lactam-based ABPs. 21,22,65,66 There is currently only one report in the literature describing the use of a similar assay in a non-hypersusceptible organism, the PA01 strain of Pseudomonas aeruginosa, that investigated the Bocillin-FL labeling profiles with dual-combination treatments. 67 We have also observed Bocillin-FL labeling of the entire PBP complement in P. aeruginosa (Figure S4), and it is unclear why these results were obtained without the need of cell permeabilization.…”

“…To address these questions, we have taken a chemical biology approach to develop PBP-isoform selective chemical probes that have enabled us to study the activity and spatial-temporal regulations of individual PBPs in different species. 10,17,20,21 To identify PBP-selective molecules, we previously developed a live-cell screening assay for PBP inhibitors in Streptococcus pneumoniae, 13 Bacillus subtilis, 22 and hypersusceptible Escherichia coli. 12 Historically, similar assays have been performed using the membrane fraction of bacterial cell lysates with either radiolabeled penicillins 23−27 or Bocillin-FL (a fluorescent penicillin V analogue that labels most PBPs).…”