The lantibiotic mersacidin inhibits peptidoglycan synthesis by targeting lipid II

Abstract

The lantibiotic mersacidin exerts its bactericidal action by inhibition of peptidoglycan biosynthesis. It interferes with the membrane-associated transglycosylation reaction; during this step the ultimate monomeric peptidoglycan precursor, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (lipid II) is converted into polymeric nascent peptidoglycan. In the present study we demonstrate that the molecular basis of this inhibition is the interaction of mersacidin with lipid II. The adsorption of [14C]mersacidin to growing cells, as well as to isolated membranes capable of in vitro peptidoglycan synthesis, was strictly dependent on the availability of lipid II, and antibiotic inhibitors of lipid II formation strongly interfered with this binding. Direct evidence for the interaction was provided by studies with isolated lipid II. [14C]mersacidin associated tightly with [14C]lipid II micelles; the complex was stable even in the presence of 1% sodium dodecyl sulfate. Furthermore, the addition of isolated lipid II to the culture broth efficiently antagonized the bactericidal activity of mersacidin. In contrast to the glycopeptide antibiotics, complex formation does not involve the C-terminal D-alanyl-D-alanine moiety of the lipid intermediate. Thus, the interaction of mersacidin with lipid II apparently occurs via a binding site which is not targeted by any antibiotic currently in use.

FIG. 1

Binding of [¹⁴C]mersacidin to M. luteus ATCC 4698. At time zero an exponentially growing culture was treated with [¹⁴C]mersacidin (7 μg/ml; 70 times the MIC), and binding (•) was determined by filtration of culture aliquots. ▴, A₆₀₀.

FIG. 2

Chase experiment with [¹⁴C]mersacidin. M. luteus ATCC 4698 was grown in the presence of [¹⁴C]mersacidin (1 μg/ml). At the time indicated by the arrow, the culture was divided into two aliquots, one of which was chased with a 900-fold excess of unlabeled mersacidin (▴), while the other served as a control (•).

FIG. 3

Cycle of the lipid carrier in peptidoglycan biosynthesis. The target reactions of the antibiotics employed in this study, all of which interfere with one of the membrane-associated stages, are depicted. Moenomycin and penicillin interact directly with the respective enzymes (6, 39), while for vancomycin and bacitracin, complex formation with the peptidoglycan precursors has been established (27, 37). For ramoplanin an interaction with lipid I has been discussed (35).

FIG. 4

Binding of [¹⁴C]mersacidin to isolated membranes of B. megaterium KM. Adsorption to either a wall fragment-containing membrane preparation capable of synthesizing polymeric peptidoglycan (A) or a protoplast membrane preparation which forms lipid II but no peptidoglycan (B) was measured. The binding capacity of membranes performing peptidoglycan synthesis in vitro in the presence of soluble UDP-linked peptidoglycan precursors (+ substrates) was compared to that of membranes in the absence of these precursors (− substrates). The effects of an incubation of the membranes with the combination of [¹⁴C]mersacidin (100 μg/ml) and 300 μg/ml of either actagardine (acta), vancomycin (vanc), moenomycin (moen), bacitracin (baci), or ramoplanin (ramo) are shown. The amount of mersacidin bound is given as a percentage of the binding capacity of control membranes (cont) in the presence of substrates. The 100% value corresponds to an adsorption of 3.8 ng of [¹⁴C]mersacidin per μg of membrane protein for the wall membrane preparation and 4.7 ng/μg of protein for the protoplast membrane preparation. n.d., not determined.

FIG. 5

(A and B) Interaction of mersacidin with isolated lipid II. Various amounts of [¹⁴C]lipid II and either [¹⁴C]mersacidin or unlabeled mersacidin were incubated and subjected to PAGE under nondenaturing conditions (A) or in the presence of 1% SDS in the sample buffer and 0.1% SDS in the running buffer (B). Lane 1, 0.4 nmol of [¹⁴C]mersacidin and 0.4 nmol of [¹⁴C]lipid II; lane 2, 0.8 nmol of [¹⁴C]mersacidin and 0.4 nmol of [¹⁴C]lipid II; lane 3, 0.4 nmol of [¹⁴C]mersacidin and 0.8 nmol of [¹⁴C]lipid II; lane 4, 4 nmol of unlabeled mersacidin and 0.4 nmol of [¹⁴C]lipid II; lane 5, 0.4 nmol of [¹⁴C]mersacidin; lane 6, 0.4 nmol of [¹⁴C]lipid II. The lipid II bands mark the upper boundary of the separating gel. Most of the stacking gel was removed prior to autoradiography. (C and D) SDS-PAGE of M. luteus ATCC 4698 after adsorption of [¹⁴C]mersacidin to the cells. (C) Autoradiogram; (D) Coomassie stain of gels run in parallel. Lanes 7 and 12, 5 mg of cells (wet weight); lanes 8 and 13, 2.5 mg of cells; lanes 9 and 14, 1 mg of cells; lane 10, 0.25 nmol of [¹⁴C]mersacidin; lane 11, ¹⁴C-methylated Rainbow molecular mass marker (Amersham-Buchler).

FIG. 6

Structure of the peptidoglycan precursor lipid II as synthesized in vitro by M. luteus membranes.