Rapid beta-lactam-induced lysis requires successful assembly of the cell division machinery

Abstract

Beta-lactam antibiotics inhibit penicillin binding proteins (PBPs) involved in peptidoglycan synthesis. Although inhibition of peptidoglycan biosynthesis is generally thought to induce cell lysis, the pattern and mechanism of cell lysis can vary substantially. Beta-lactams that inhibit FtsI, the only division specific PBP, block cell division and result in growth as filaments. These filaments ultimately lyse through a poorly understood mechanism. Here we find that one such beta-lactam, cephalexin, can, under certain conditions, lead instead to rapid lysis at nascent division sites through a process that requires the complete and ordered assembly of the divisome, the essential machinery involved in cell division. We propose that this assembly process (in which the localization of cell wall hydrolases depends on properly targeted FtsN, which in turn depends on the presence of FtsI) ensures that the biosynthetic machinery to form new septa is in place before the machinery to degrade septated daughter cells is enabled. Beta-lactams that target FtsI subvert this mechanism by inhibiting FtsI without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. One seemingly paradoxical implication of our results is that beta-lactam therapy may be improved by promoting active cell division.

Fig. 1.

Lack of division prevents cephalexin-induced cell lysis. (A) Bacterial cell division proteins localize according to a defined pathway. SulA inhibits the first step in cell division, FtsZ polymerization. (B) OD curves for strain (NWG593) expressing (green) and suppressing (black) sulA from an arabinose-induced promoter. 50 μg/ml cephalexin was added at t = 20 min. (C) Dose–response curves of the sulA induced and wild-type control showing their maximal OD, normalized to its no drug value, as a function of drug dosage. (D) Microscopy images of NWG593 show lysis under cephalexin treatment (50 μg/ml) occurred at mid-cell of sulA-suppressed cells (Left) and no lysis observed for sulA-induced cells (Right). FtsZ-GFP fluorescence images (red) show the disappearance of FtsZ ring in the sulA-induced strain. (Scale bar, 10 μm.) Cells were fixed at 30 min (−SulA) and at 80 min (+SulA) after the addition of cephalexin. For time-lapse movies of wild-type (JOE650) under different cephalexin concentrations, see Movies S1–S4. Note that cephalexin induces filaments, followed by mid cell lysis as in D.

Fig. 2.

FtsN depletion protects against cephalexin killing through a mechanism independent of the drug target localization. (A) OD curves for strain (HSC070) suppressing (purple) and expressing (black) ftsN from an arabinose-induced promoter; 50 μg/ml cephalexin was added at t = 0. (B) The protection of FtsN depletion against cell lysis operates along a wide range of drug dosage, as indicated by its higher normalized OD. (C) Microscopy images of HSC070 show lysis under cephalexin treatment (50 μg/ml) occurred at mid-cell of ftsN-induced (Left) and no lysis observed for ftsN-depleted cells (Right). (Scale bar, 10 μm.) GFP-FtsI fluorescence images (red) show that protection from lysis induced by cephalexin operates without affecting divisome localization of the drug target (FtsI) in the FtsN depletion strains. Cells were fixed at 30 min (+FtsN) and at 60 min (−FtsN) after the addition of cephalexin.

Fig. 3.

Cell wall degradation enzymes are required for fast lysis by cephalexin. (A) OD as a function of time for wild type (JOE309; black) compared to amidase double knockouts (HSC087: ΔamiA ΔamiC (ΔAΔC); orange) in the presence (solid lines) and absence (dotted lines) of cephalexin; 50 μg/ml cephalexin was added at t = 48 min. (B) Dose–response curves showing maximal OD as a function of drug dosage for wild-type and ΔamiA ΔamiC strains. (C) Microscopy images show lysis for wild-type cells (JOE309; black) under cephalexin treatment (50 μg/ml) occurred at midcell and defects in cell separation leading to delayed lysis of ΔamiA ΔamiC strain (HSC087; orange). These images are snapshots taken from Movie S4. For the other 2 amidase double knockouts [HSC086: ΔamiA ΔamiB (ΔAΔB); HSC083: ΔamiB ΔamiC (ΔBΔC)], see Fig. S3.

Fig. 4.

Sensitivity to β-lactams depends on the state of divisome assembly. The bar graph shows the relative change in MIC for mutant versus wild-type strains under treatment with cephalexin (PBP3 selective), ampicillin (PBP1a, PBP1b, and PBP3), or cefsulodin (PBP1a and PBP1b selective). The ratio of MICs for both sulA-induced and ftsN-depleted cells under cephalexin treatment is greater than 35, but sulA induction confers better protection than ftsN depletion.