cb9b00977_si_001.pdf (9.49 MB)
Harnessing β‑Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis
journal contribution
posted on 2020-03-20, 19:13 authored by Shabnam Sharifzadeh, Felix Dempwolff, Daniel B. Kearns, Erin E. CarlsonSelective 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.