The lipopeptide antibiotic Friulimicin B inhibits cell wall biosynthesis through complex formation with bactoprenol phosphate

Abstract

Friulimicin B is a naturally occurring cyclic lipopeptide, produced by the actinomycete Actinoplanes friuliensis, with excellent activity against gram-positive pathogens, including multidrug-resistant strains. It consists of a macrocyclic decapeptide core and a lipid tail, interlinked by an exocyclic amino acid. Friulimicin is water soluble and amphiphilic, with an overall negative charge. Amphiphilicity is enhanced in the presence of Ca(2+), which is also indispensable for antimicrobial activity. Friulimicin shares these physicochemical properties with daptomycin, which is suggested to kill gram-positive bacteria through the formation of pores in the cytoplasmic membrane. In spite of the fact that friulimicin shares features of structure and potency with daptomycin, we found that friulimicin has a unique mode of action and severely affects the cell envelope of gram-positive bacteria, acting via a defined target. We found friulimicin to interrupt the cell wall precursor cycle through the formation of a Ca(2+)-dependent complex with the bactoprenol phosphate carrier C(55)-P, which is not targeted by any other antibiotic in use. Since C(55)-P also serves as a carrier in teichoic acid biosynthesis and capsule formation, it is likely that friulimicin blocks multiple pathways that are essential for a functional gram-positive cell envelope.

FIG. 1.

Chemical structure of the lipopeptide antibiotic friulimicin B. The 10-membered cyclopeptide ring is linked by an exocylic Asn to a branched fatty acid side chain. The positions of the amino acid residues are indicated by numbers next to the amino acid abbreviations. Asn, asparagine; Dab, diaminobutyric acid; Pip, pipecolinic acid; Me-Asp, methylaspartic acid; Asp, aspartic acid; Gly, glycine; Val, valine; Pro, proline.

FIG. 2.

Impact of friulimicin B on the incorporation of [³H]glucosamine into macromolecules in B. subtilis 168. Error bars show standard deviations. ○, untreated controls; ▪, friulimicin (10× MIC)-treated cells.

FIG. 3.

Intracellular accumulation of the soluble cell wall precursor UDP-MurNAc-pp in S. simulans 22. (A) Results for untreated (dashed line) and vancomycin-treated (solid line) cells are shown. (B) Results for friulimicin B-treated (solid line) and daptomycin-treated (dashed line) cells are shown. The experiment was performed with 10× the MIC of each antibiotic for 30 min. Treated cells were extracted with boiling water, and the intracellular nucleotide pool was analyzed by reversed-phase high-performance liquid chromatography. UDP-MurNAc-pp was identified by mass spectrometry.

FIG. 4.

Impact of friulimicin B on the membrane potential of B. subtilis 168. The potential was calculated from the distribution of the lipophilic cation TPP⁺ inside and outside the cells. The experiment was started by the addition of TPP⁺ to a growing culture; after 10 min of incubation, the culture was divided into two, and one part was run as control (▪) while the second was treated with friulimicin (10× MIC) (○). To further control the depolarization assay, the pore-forming lantibiotic nisin (□) was used. Arrows indicate the time points of antibiotic addition. Mean membrane potential values were calculated from the results of four independent experiments. Error bars show standard deviations.

FIG. 5.

Impact of friulimicin B on the integrity of the cytoplasmic membrane of S. simulans 22 cells. Peptides were added after 30 s, and potassium release was monitored with a potassium-sensitive electrode. Potassium leakage was expressed relative to the total amount of potassium released after the addition of 1 μM of the pore-forming lantibiotic nisin (100%; ▴). The experiment was further controlled with the non-pore-forming lantibiotic mersacidin (1 μM; ⋄). ▪, untreated cells; ○, friulimicin-treated cells (10× MIC).

FIG. 6.

Impacts of friulimicin B and daptomycin on the membrane-associated steps of cell wall biosynthesis. The peptide was added at increasing molar ratios of 0.5 to 2 with respect to the amount of the substrate C₅₅-P. Reaction products synthesized in the absence of friulimicin were taken as the 100% level. Analysis was performed as described in Materials and Methods. (A, B) Impacts of friulimicin on the overall in vitro lipid II synthesis catalyzed by membrane preparations of M. luteus. Reaction products were excised following TLC (A), and the amount of [¹⁴C]GlcNAc incorporated was quantified (B). (C, D) Inhibition of the MraY-catalyzed reaction by friulimicin. The conversion of [³H]C₅₅-P to lipid I using purified MraY-His₆ was analyzed in the presence of friulimicin by using TLC (C), and quantification was carried out by analysis of radioactivity incorporated (D). The specific MraY inhibitor tunicamycin was used as a control. Error bars show standard deviations. FRI, friulimicin; DAP, daptomycin; +, present.

FIG. 7.

Effects of friulimicin B on the reactions catalyzed by MurG, FemX, and PBP2. Friulimicin B (striped bars) or nisin (dotted bars) was added to the reaction mixture in a molar ratio of 1:1 or 2:1, respectively, with respect to the concentration of lipid intermediate substrate. Filled bars, untreated control.

FIG. 8.

Model for the mode of action of friulimicin B. We postulate friulimicin B to form a stoichiometric complex with bactoprenol phosphate (C₅₅-P). Abduction of the central carbohydrate carrier interrupts precursor cycling and blocks all biosynthetic pathways which make use of C₅₅-P, such as peptidoglycan (right side), wall teichoic acid (left side), and polysaccharide capsule biosyntheses. The simultaneous interference with these pathways obstructs the formation of a functional cell envelope in gram-positive bacteria. FRI, friulimicin B.