Screen for Inhibitors of the Coupled Transglycosylase-Transpeptidase of Peptidoglycan Biosynthesis in Escherichia coli

Abstract

Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of Escherichia coli have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an E. coli ponB::Spc r strain. Using this assay, we show that PBP1a is ϳ10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC 50 ) of moenomycin, a TG inhibitor, is ϳ10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC 50 in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the E. coli ponB::Spc r strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother. 48:30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.

Microplate Assay for Inhibitors of the Transpeptidase Activity of PBP1b of Escherichia coli

“…Ampicillin, too, inhibited the TP with a similar IC 50 . Cephaloridine and cefsulodin also inhibited the TP assay (>90% inhibition at 100 µM), but cephalexin and cephradine, β-lactam antibiotics with greater affinity for PBP1a, 26,27 did not inhibit the TP (<20% inhibition at 100 µM). This was expected in the background of our previous results, that under similar conditions in wild-type E. coli membranes, cephalexin and cephradine do not inhibit peptidoglycan synthesis.…”

“…This is in contrast to cases of inhibition of the TG when radioactivity at the origin of the paper chromatogram decreased. 24,26 However, penicillin (300 µM) caused complete inhibition of TP activity as seen in the WGA-SPA + N-laurylsarcosine counts; it is expected that penicillin, a TP inhibitor, would completely inhibit the crosslinking reaction. The same profile was obtained with moenomycin, a TG inhibitor: complete inhibition of the cross-linking reaction (i.e., WGA-SPA + N-laurylsarcosine), confirming it as TP inhibitor.…”

“…3D), a concentration similar to that at which it inhibits the TG or the entire pathway of peptidoglycan synthesis. 1,26 Nisin, an inhibitor of MurG, which was recently reported to inhibit the TG, 26 also inhibited the TP, with an IC 50 between 1 and 17 µg/ml (Fig. 3E).…”

“…We have shown earlier that this can be used as a source of PBP1b and that it shows activity similar to purified PBP1b. 26 For testing the effect of step 1 incubation time on the TP assay, the step 1 incubation time was varied while monitoring the quantity of cross-linked peptidoglycan formed in a 15-min incubation time for step 2 (Fig. 2B).…”

“…26 Using the protocol described above, membranes were made from E. coli AMA1004, pBS96, that is, wild-type E. coli bearing a plasmid with the gene encoding PBP1b (ponB) under the control of its own promoter (B. Spratt, personal communication). The membranes were incubated for 60 min on ice with 1% CHAPS, 1 M NaCl, 100 mM Tris-HCl, pH 7.5, at a concentration of 5 mg/ml protein, followed by centrifugation at 150,000g for 15 min.…”