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Ever since daptomycin was introduced to the clinic, daptomycin-resistant isolates have been reported. In most cases, the resistant isolates harbor point mutations in MprF, which produces and flips the positively charged phospholipid LysPG. This has led to the assumption that the resistance mechanism relies on the overproduction of LysPG, given that increased LysPG production may lead to increased electrostatic repulsion of positively charged antimicrobial compounds, including daptomycin. Here we show that the resistance mechanism is highly specific and relies on a different process that involves a functional MprF flippase, suggesting that the resistance-conferring mutations may enable the flippase to accommodate daptomycin or an unknown component that is crucial for its activity. Our report provides a new perspective on the mechanism of resistance to a major antibiotic., Daptomycin, a calcium-dependent lipopeptide antibiotic whose full mode of action is still not entirely understood, has become a standard-of-care agent for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Daptomycin-resistant (DAP-R) S. aureus mutants emerge during therapy, featuring isolates which in most cases possess point mutations in the mprF gene. MprF is a bifunctional bacterial resistance protein that synthesizes the positively charged lipid lysyl-phosphatidylglycerol (LysPG) and translocates it subsequently from the inner membrane leaflet to the outer membrane leaflet. This process leads to increased positive S. aureus surface charge and reduces susceptibility to cationic antimicrobial peptides and cationic antibiotics. We characterized the most commonly reported MprF mutations in DAP-R S. aureus strains in a defined genetic background and found that only certain mutations, including the frequently reported T345A single nucleotide polymorphism (SNP), can reproducibly cause daptomycin resistance. Surprisingly, T345A did not alter LysPG synthesis, LysPG translocation, or the S. aureus cell surface charge. MprF-mediated DAP-R relied on a functional flippase domain and was restricted to daptomycin and a related cyclic lipopeptide antibiotic, friulimicin B, suggesting that the mutations modulate specific interactions with these two antibiotics. Notably, the T345A mutation led to weakened intramolecular domain interactions of MprF, suggesting that daptomycin and friulimicin resistance-conferring mutations may alter the substrate range of the MprF flippase to directly translocate these lipopeptide antibiotics or other membrane components with crucial roles in the activity of these antimicrobials. Our study points to a new mechanism used by S. aureus to resist calcium-dependent lipopeptide antibiotics and increases our understanding of the bacterial phospholipid flippase MprF.

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.

The related lipo(depsi)peptide antibiotics daptomycin and friulimicin B show great potential in the treatment of multiply resistant gram-positive pathogens. Applying genome-wide in-depth expression profiling, we compared the respective stress responses of Bacillus subtilis . Both antibiotics target envelope integrity, based on the strong induction of extracytoplasmic function σ factor-dependent gene expression. The cell envelope stress-sensing two-component system LiaRS is exclusively and strongly induced by daptomycin, indicative of different mechanisms of action in the two compounds.

Four novel lipopeptide antibiotics, friulimicins A, B, C, and D, were isolated from cultures of Actinoplanes friuliensis HAG 010964 after fermentation in different nutrient media. The new compounds were separated by ion-exchange chromatography from the acidic lipopeptides of the amphomycin type also present in the culture fluid, compounds A-1437 A, B, E, and G. The principal constituent friulimicin B, C59H94N14O19, was structurally characterized by mass spectrometric investigations of its hydrolysis and partial degradation products and by sequencing of the cyclic acyl peptide. The NMR data of friulimycin B and the amphomycin constituent A-1437 B were completely assigned by a variety of 2-D experiments, and confirmed the structures determined by mass spectrometry. All 8 lipopeptides possess an identical peptide macrocycle as their central element, linked via a diaminobutyric acid N-terminal either to an acylated asparagine residue or, in the case of the amphomycin series, to an acylated aspartic acid residue. The structures of the amphomycins have now been revised to take account of the peptide framework described herein and the determined cis-configuration of the exocyclic double bond. As a consequence of their higher isoelectric points, the new compounds friulimicin A, B, C, and D have different properties than the amphomycins.