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 coselective 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) and 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 coselective 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 β-lactam 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.
Bibliographical noteFunding Information:
We thank O. Kocaoglu for performing initial titration studies. This work was supported by the National Institutes of Health (R01 GM128439-01A1 to E.E.C. and R35 GM131783 to D.B.K.), a Sloan Research Fellow Award (E.E.C.), a University of Minnesota Interdisciplinary Doctoral Fellowship (S.S.), and the University of Minnesota Department of Chemistry. The authors thank Y. Zhao and P. Villalta for assistance with the operation mass spectrometers. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, supported in part by the Cancer Center Support Grant NIH CA077598.
© 2020 American Chemical Society.
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't