Daptomycin Binding Research Articles

Tunable biomimetic bacterial membranes from binary and ternary lipid mixtures and their application in antimicrobial testing

The reconstruction of accurate yet simplified mimetic models of cell membranes is a very challenging goal of synthetic biology. To date, most of the research focuses on the development of eukaryotic cell membranes, while reconstitution of their prokaryotic counterparts has not been fully addressed, and the proposed models do not reflect well the complexity of bacterial cell envelopes. Here, we describe the reconstitution of biomimetic bacterial membranes with an increasing level of complexity, developed from binary and ternary lipid mixtures. Giant unilamellar vesicles composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE); PC and phosphatidylglycerol (PG); PE and PG; PE, PG and cardiolipin (CA) at varying molar ratios were successfully prepared by the electroformation method. Each of the proposed mimetic models focuses on reproducing specific membrane features such as membrane charge, curvature, leaflets asymmetry, or the presence of phase separation. GUVs were characterized in terms of size distribution, surface charge, and lateral organization. Finally, the developed models were tested against the lipopeptide antibiotic daptomycin. The obtained results showed a clear dependency of daptomycin binding efficiency on the amount of negatively charged lipid species present in the membrane. We anticipate that the models proposed here can be applied not only in antimicrobial testing but also serve as platforms for studying fundamental biological processes in bacteria as well as their interaction with physiologically relevant biomolecules.

A Clinically Selected Staphylococcus aureus clpP Mutant Survives Daptomycin Treatment by Reducing Binding of the Antibiotic and Adapting a Rod-Shaped Morphology.

Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations; however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and β-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that inactivation of ClpP reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis, eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10× MIC daptomycin, resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression.

Nafcillin Augmentation of Daptomycin and Cathelicidin LL-37 Killing of Methicillin-resistant Staphylococcus epidermidis: Foundations of Successful Therapy of Endocarditis

Methicillin-resistant Staphylococcus epidermidis (MRSE) endocarditis failing conventional therapy has been successfully treated with nafcillin plus daptomycin in the clinic. In vitro studies showed that nafcillin enhanced daptomycin killing of MRSE in both planktonic cells and biofilm. Nafcillin exposure also sensitized MRSE to killing by human neutrophils and cathelicidin antimicrobial peptide LL-37. Fluorescent microscopy showed increased daptomycin and LL-37 binding to the MRSE bacterial surface upon nafcillin treatment. Ceftaroline also increased MRSE killing by daptomycin in planktonic cultures and biofilms, as well as daptomycin and LL-37 binding on the bacterial surface. Nafcillin, ceftaroline, and possibly other β-lactams, may serve an important role in the therapy of MRSE endocarditis through augmentation of cationic peptide, the innate immune system, and daptomycin killing. Clinical studies will be needed to determine how early these regimens should be deployed to optimize clinical outcome.

DAPTOMYCIN-INDUCED EOSINOPHILIC PNEUMONITIS

DAPTOMYCIN-INDUCED EOSINOPHILIC PNEUMONITIS

Host environment induces daptomycin tolerance in Staphylococcus aureus

Daptomycin is a last-resort antibiotic for the treatment of invasive diseases caused by methicillin-resistant Staphylococcus aureus. However, despite potent activity in vitro,daptomycin fails to resolve up to 30% of cases of staphylococcal bacteraemia, suggesting that the host environment reduces bacterial susceptibility to the antibiotic. Using human serum as a model of bacteraemia, we demonstrated that the host environment induced daptomycin tolerance via activation of the GraRS two-component system. Testing of various antimicrobial peptides present in serum revealed that the human cathelicidin LL-37 was able to bind GraS, induce GraRS signalling and confer daptomycin tolerance. Activation of GraRS by serum led to daptomycin tolerance via changes in the staphylococcal cell envelope. For example, GraRS-dependent increases in positive surface charge and peptidoglycan content occurred in serum, leading to reduced daptomycin binding. Additionally, incubation in serum led to a Cls2-dependent increase in the cardiolipin content of the membrane which contributed to daptomycin tolerance. Finally, inhibition of both peptidoglycan and cardiolipin synthesis together completely abolished acquisition of daptomycin tolerance in serum, demonstrating that these processes fully explain the tolerance phenotype. In summary, host LL-37 activates staphylococcal GraRS signalling, causing changes in the cell surface which confer daptomycin tolerance. This demonstrates how host defences can compromise antibiotic efficacy, and also provides a rationale for combination therapies to prevent the development of daptomycin tolerance and reduce rates of treatment failure.

Phosphate Ions Alter the Binding of Daptomycin to Living Bacterial Cell Surfaces.

Advancements in antibiotic drug design are often hindered by missing information on how these small molecules interact with living cells. The antibiotic, daptomycin, has found clinical success and an emerging resistance, but a comprehensive picture of its mechanism of action has remained elusive. Using a surface-specific spectroscopy technique, second harmonic generation, we are able to quantitatively assess the binding of daptomycin to living cell membranes without the addition of exogenous labels. Our results reveal similar binding affinities for both Gram-positive and Gram-negative bacteria studied, including Escherichia coli. More importantly, we show that the presence of phosphate ions influences the binding of daptomycin to the Gram-positive bacterium Enterococcus faecalis. The role of environmental phosphate has not previously been considered in any proposed mechanism, and its implications are expected to be important in vivo.

Binding Studies Reveal Phospholipid Specificity and Its Role in the Calcium-Dependent Mechanism of Action of Daptomycin.

Multidrug-resistant bacteria pose a serious global health threat as antibiotics are increasingly losing their clinical efficacy. A molecular level understanding of the mechanism of action of antimicrobials plays a key role in developing new agents to combat the threat of antimicrobial resistance. Daptomycin, the only clinically used calcium-dependent lipopeptide antibiotic, selectively disrupts Gram-positive bacterial membranes to illicit its bactericidal effect. In this study, we use isothermal titration calorimetry to further characterize the structural features of the target bacterial phospholipids that drive daptomycin binding. Our studies reveal that daptomycin shows a clear preference for the phosphoglycerol headgroup. Furthermore, unlike other calcium-dependent lipopeptide antibiotics, calcium binding by daptomycin is strongly dependent on the presence of phosphatidylglycerol. These investigations provide new insights into daptomycin’s phospholipid specificity and calcium binding behavior.

Daptomycin exerts bactericidal effect through induction of excessive ROS production and blocking the function of stress response protein Usp2

AbstractDaptomycin, as a lipopeptide antibiotic, exhibits high potency in the treatment of infections caused by clinically relevant drug‐resistant Gram‐positive pathogens, such as methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococci. However, its bactericidal mechanism of action remains controversial. In this study, we report that daptomycin kills bacteria through triggering overproduction of deleterious reactive oxygen species (ROS). This outcome is attributed to daptomycin binding to the universal stress response protein (Usp2) and subsequent blocking its function, triggering stress, and anti‐ROS response. Based on these findings, we conclude that daptomycin causes bacterial cell death by simultaneously triggering ROS production through inflicting cell membrane damages and inhibiting antioxidant defense by blocking Usp2 function. This study depicts molecular mechanisms underlying the bactericidal effect of daptomycin, a combination of triggering ROS production and inhibiting anti‐ROS response.Key points Novel bactericidal mechanism of daptomycin by ROS Identification of first bacterial protein target, Usp2, for daptomycin Deciphering the dual role of Usp2 on daptomycin mediated bacterial killing

Characterization of the Interaction of Daptomycin With Site II on Human Serum Albumin

Daptomycin, a cyclic lipopeptide antibiotic, is clinically used for the treatment of infections caused by Gram-positive bacteria, including the methicillin-resistant Staphylococcus aureus and the vancomycin-resistant Enterococci. While daptomycin shows high plasma protein binding (90–93%), our knowledge of the binding process is not extensive. To address this issue in more detail, we characterized the binding of daptomycin to plasma proteins and the findings indicate that the association constant for the binding of daptomycin to human serum albumin (HSA) is much higher than that for α1-acid glycoprotein, another plasma protein. Daptomycin was also found to bind to a single site on HSA, which was identified as site II. The findings also suggest that the n-decanoyl moiety of daptomycin penetrates into the hydrophobic pocket of site II and that this acyl moiety interacts with Tyr411 at the entrance to site II. Due to this selective interaction with site II, daptomycin binding was significantly inhibited by drugs (ibuprofen or diazepam) and endogenous compounds (uremic toxins or fatty acids) which also strongly bind to site II. In diseased states, such an inhibition in the binding could result in the pharmacokinetics and therapeutic action of daptomycin being substantially altered.

601. TelA and XpaC Are Novel Mediators of Daptomycin Resistance in Enterococcus faecium

BackgroundThe YycFG system is an essential two-component regulatory system involved in cell wall homeostasis associated with the development of daptomycin (DAP) resistance in E. faecium. Importantly, the standard combination of DAP plus β-lactam is ineffective against strains harboring mutations in yycFG. Transcriptional profiling identified a cluster of two genes (xpaC and telA) that is upregulated in the presence of a YycGS333L substitution. xpaC and telA are annotated as 5-bromo-4-chloroindolyl phosphate hydrolysis and tellurite resistance proteins, respectively. Here, we aimed to determine the contribution of xpaC and telA in DAP resistance.MethodsNon-polar in-frame deletions of xpaC/telA and complementation of xpaC were performed in clinical strain E. faecium R446RIF. All mutants were characterized by PFGE and sequencing of the open reading frames to confirm the deletion. DAP MIC determination was performed by Etest on Mueller–Hinton agar. Binding of DAP was evaluated using BODIPY-labeled DAP (BDP-DAP). Cell membrane phospholipid microdomains were visualized using 10-N-nonyl acridine orange. All assays were compared with a DAP-susceptible clinical E. faecium strain S447.ResultsR446RIFΔ telA and R446RIFΔ xpaCtelA did not alter DAP MICs in R446RIF (24–32 μg/mL). However, deletion of xpaC alone (R446RIFΔ xpaC) markedly decreased DAP MIC 8 fold (to 4 μg/mL). R446RIFΔ telA and R446RIFΔ xpaCtelA exhibited similar binding of BDP-DAP compared with parental R446RIF. In contrast, R446RIFΔ xpaC exhibited increased binding of the antibiotic molecule to the cell membrane, similar to that of DAP-susceptible S447. Complementation of xpaC restored DAP MIC to 32–48 µg/mL and decrease binding of DAP. NAO staining of S447, R446RIF, R446RIFΔ telA, R446RIFΔ xpaCtelA, and R446RIFΔ xpaC:: xpaC displayed septal and polar distribution. In stark contrast, R446RIFΔ xpaC showed a redistribution of phospholipid microdomains away from the septa.ConclusionXpaC is a key contributor to DAP binding and phospholipid architecture of E. faecium but only in the presence of an intact TelA. The xpaC and telA gene cluster is a novel mediator of DAP-resistance in E. faecium via theYycFG system and independent of the LiaFSR system Disclosures All authors: No reported disclosures.

A Quantitative Model of Daptomycin Binding to Lipid Bilayers.

Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multidrug resistant infections, including those caused by methicillin-resistant S. aureus strains. Similar to many other antimicrobial peptides, daptomycin binds with preference to anionic membranes such as those typically found in prokaryotes. However, in contrast to most linear α-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions, and its activity in vivo is absolutely Ca2+-dependent. Here, we describe the early events that occur in the binding of daptomycin to lipid bilayers using a quantitative model to analyze both equilibrium and kinetic binding data. The goal of the analysis was to obtain a precise description of the early events that occur in the interaction of daptomycin with lipid and calcium ions at low daptomycin concentrations. In the course of the analysis, we also determined the rate and equilibrium constants for binding of daptomycin to lipid and Ca2+. The model used to describe the experimental data comprises a soluble daptomycin monomer that binds calcium ions in solution with low affinity, a soluble, Ca2+-bound dimer, and a 1:1 daptomycin-lipidCa complex. A strong interaction of daptomycin with Ca2+-complexed lipid, the amount of which depends on the availability of calcium ions in the bulk solution, appears central to its function.

An Exact Model of Daptomycin Binding to Lipid Bilayers: An Update

Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multi-drug resistant infections, including those caused by methicillin-resistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides, daptomycin binds with preference to the anionic cytoplasmic membranes typically found in prokaryotes. However, in contrast to most linear, alpha-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions and its activity is absolutely calcium-dependent. We measured the interaction of daptomycin with anionic lipid membranes using kinetic binding experiments and equilibrium titrations. The data were analyzed using an exact model describing the interactions of daptomycin with the lipid bilayer that includes solution and membrane-bound states, and the influence of calcium ions on daptomycin-lipid interactions. A preliminary version of the model was presented at the Biophysical Society Meeting in 2017. Here, we present a more complete analysis.

Listeria monocytogenes endocarditis: case report, review of the literature, and laboratory evaluation of potential novel antibiotic synergies

Endocarditis is a rare but serious manifestation of Listeria monocytogenes (LM). However, the optimal treatment strategy for LM endocarditis has yet to be established. Current antibiotic strategies for listeriosis include penicillin G or ampicillin (AMP) monotherapy, or AMP + gentamicin combination therapy which is often favored for endocarditis. The primary objective of our investigation was to assess the utility of AMP + ceftriaxone (CRO) and AMP + daptomycin (DAP) against LM, modeling less nephrotoxic antibiotic combinations traditionally used to manage resistant enterococcal endocarditis. Here we report a case of LM endocarditis, review the world literature, and evaluate alternative treatment strategies for listeriosis utilizing in vitro and ex vivo studies. The combination of AMP + CRO and AMP + DAP were each noted to have synergistic activity against a LM endocarditis isolate. Additionally, co-incubation of the isolate with sub-lethal concentrations of antibiotics (AMP, CRO, DAP, AMP + CRO or AMP + DAP) sensitized the bacterium to whole blood killing while pretreatment with CRO and DAP (at 1/4 MIC) sensitized the bacterium to neutrophil killing. However, these effects did not reflect potentiation of antibiotic activity to human cathelicidin peptide LL-37, which is abundant in neutrophils and highly active against LM. Interestingly, AMP pretreatment of the LM endocarditis isolate resulted in increased DAP binding to the bacterium when assessed by fluorescence microscopy. These in vitro and ex vivo studies suggest further investigation of combination therapy using AMP + CRO or AMP + DAP as an alternative treatment for LM infection is warranted.

Daptomycin-induced Eosinophilic Pneumonia and a Review of the Published Literature

A 53-year-old man was admitted to the hospital with a diagnosis of cellulitis and osteomyelitis. Twenty-four days after the initiation of daptomycin and sulbactam/ampicillin, he developed a fever and pulmonary infiltration. Bronchoalveolar lavage revealed a high number of eosinophils, while an intracutaneous test revealed positivity for daptomycin. The patient improved after discontinuing antimicrobial therapy. The plasma daptomycin minimum concentration (Cmin) was elevated (27.4 μg/mL), but plasma protein binding of daptomycin was low (87.8%). Although the pathophysiology of eosinophilic pneumonia remains unclear, antigenic stimulation due to daptomycin accumulation in the alveoli may have caused continuous immune activation.

Plasma Protein Binding Structure-Activity Relationships Related to the N-Terminus of Daptomycin.

Daptomycin is a lipopeptide antibiotic that is highly bound to plasma proteins. To date, the plasma components and structure-activity relationships responsible for the plasma protein binding profile of daptomycin remain uncharacterized. In the present study we have employed a surface plasmon resonance assay together with molecular docking techniques to investigate the plasma protein binding structure-activity relationships related to the N-terminal fatty acyl of daptomycin. Three compounds were investigated: (1) native daptomycin, which displays an N-terminal n-decanoyl fatty acid side chain, and two analogues with modifications to the N-terminal fatty acyl chain; (2) des-acyl daptomycin; and (3) acetyl-daptomycin. The surface plasmon resonance (SPR) data showed that the binding profile of native daptomycin was in the rank order human serum albumin (HSA) ≫ α-1-antitrypsin > low-density lipoprotein ≥ hemoglobin > sex hormone binding globulin > α-1-acid-glycoprotein (AGP) > hemopexin > fibrinogen > α-2-macroglobulin > β2-microglobulin > high-density lipoprotein > fibronectin > haptoglobulin > transferrin > immunoglobulin G. Notably, binding to fatty acid free HSA was greater than binding to nondelipidated HSA. SPR and ultrafiltration studies also indicated that physiological concentrations of calcium increase binding of daptomycin and acetyl-daptomycin to HSA and AGP. A molecular model of the daptomycin-human serum albumin A complex is presented that illustrates the pivotal role of the N-terminal fatty acyl chain of daptomycin for binding to drug site 1 of HSA. In proof-of-concept, the capacity of physiological cocktails of the identified plasma proteins to inhibit the antibacterial activity of daptomycin was assessed with in vitro microbiological assays. We show that HSA, α-1-antitrypsin, low-density lipoprotein, sex hormone binding globulin, α-1-acid-glycoprotein, and hemopexin are responsible for the majority of the sequestering activity in human plasma. The findings are relevant to medicinal chemistry programs focused on the development of next-generation daptomycin lipopeptides. Tailored modifications to the N-terminal fatty acyl domain of the daptomycin molecule should yield novel daptomycin lipopeptides with more ideal plasma protein binding profiles to increase the levels of active (free) drug in plasma and improved in vivo activity.

An Exact Model of Daptomycin Binding to Lipid Bilayers

Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of multi-drug resistant infections, including those caused by methicillin-resistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides, daptomycin binds with preference to the anionic cytoplasmic membranes typically found in prokaryotes. However, in contrast to most linear, alpha-helical peptides, daptomycin binds to lipid bilayers only in the presence of calcium ions and its activity is absolutely calcium-dependent. We measured the interaction of daptomycin with anionic lipid membranes using kinetic binding experiments and equilibrium titrations. The data were analyzed using an exact model describing the interactions of daptomycin with the lipid bilayer that includes solution and membrane-bound states, and the influence of calcium ions on daptomycin-lipid interactions.

Classical β-Lactamase Inhibitors Potentiate the Activity of Daptomycin against Methicillin-Resistant Staphylococcus aureus and Colistin against Acinetobacter baumannii.

ABSTRACTWe asked whether beta-lactamase inhibitors (BLIs) increased the activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA), the peptide antibiotic colistin (COL) against the emerging Gram-negative nosocomial pathogen Acinetobacter baumannii, and the human host defense peptide cathelicidin LL37 against either pathogen. DAP and LL37 kill curves were performed with or without BLIs against MRSA, vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA). COL and LL37 kill curves were performed against A. baumannii. Boron-dipyrromethene (BODIPY)-labeled DAP binding to MRSA grown with the BLI tazobactam (TAZ) was assessed microscopically. The combination of COL plus TAZ was studied in a murine model of A. baumannii pneumonia. TAZ alone lacked in vitro activity against MRSA or A. baumannii. The addition of TAZ to DAP resulted in a 2- to 5-log10 reduction in recoverable MRSA CFU at 24 h compared to the recoverable CFU with DAP alone. TAZ plus COL showed synergy by kill curves for 4 of 5 strains of A. baumannii tested. Growth with 20 mg/liter TAZ resulted in 2- to 2.5-fold increases in the intensity of BODIPY-DAP binding to MRSA and hVISA strains. TAZ significantly increased the killing of MRSA and A. baumannii by LL37 in vitro. TAZ increased the activity of COL in a murine model of A. baumannii pneumonia. Classical BLIs demonstrate synergy with peptide antibiotics. Since BLIs have scant antimicrobial activity on their own and are thus not expected to increase selective pressure toward antibiotic resistance, their use in combination with peptide antibiotics warrants further study.

Comparative Genome Analysis of the Daptomycin-ResistantStreptococcus anginosusStrain J4206 Associated with Breakthrough Bacteremia

Streptococcus anginosus is a member of the normal oral flora that can become a pathogen causing pyogenic infections in humans. The genome of daptomycin-resistant strain J4206, originally isolated from a patient suffering from breakthrough bacteremia and septic shock at the University of Texas Health Science Center at San Antonio, was determined. The circular genome is 2,001,352 bp long with a GC content of 38.62% and contains multiple mobile genetic elements, including the phage-like chromosomal island SanCI that mediates a mutator phenotype, transposons, and integrative conjugative elements. Daptomycin resistance involves multiple alterations in the cell membrane and cell wall, and unique features were identified in J4206 that may contribute to resistance. A cluster of capsular polysaccharide (CPS) genes for choline metabolism and transport are present that may help neutralize cell surface charges, destabilizing daptomycin binding. Further, unique J4206 genes encoding sortases and LPXTG-target proteins that are involved in cell wall modification were present. The J4206 genome is phylogenetically closely related to the recently reported vancomycin-resistant SA1 strain; however, these genomes differ with SNPs in cardiolipin synthetase, histidine kinase yycG, teichoic acid modification genes, and other genes involved in cell surface modification. Transmission electron microscopy showed that the cell walls of both strains J4206 and SA1 were significantly thicker and more electron dense than daptomycin- and vancomycin-sensitive strain J4211. This comparative genomic study has identified unique genes as well as allelic variants in the J4206 genome that are involved in cell surface modification and thus might contribute to the acquisition of daptomycin resistance.

Mutant prevention concentrations of daptomycin for Enterococcus faecium clinical isolates

Owing to the emergence of vancomycin-resistant Enterococcus faecium, treatment of enterococcal infections has become challenging. Although spontaneous in vitro resistance frequencies are low, the emergence of resistance is increasingly reported during daptomycin therapy. The mutant selection window (MSW), comprised between the minimum inhibitory concentration (MIC) and the mutant prevention concentration (MPC), corresponds to the concentration range within which resistant mutants may be selected. Since no data are available for enterococci, the aim of this study was to determine MPCs and MSWs for 12 representative E. faecium clinical isolates. MICs and MPCs were determined by broth microdilution and agar dilution methods, respectively. A basic MSW-derived pharmacodynamic analysis was also performed using mean maximum plasma concentration (Cmax) values obtained with dosages from 4 to 12 mg/kg. MICs and MPCs of daptomycin ranged from 0.5 to 4 mg/L and from 2 to 32 mg/L, respectively, with no correlation between them. The wideness of MSWs ranged from 2× to 32× MIC. Mean plasma Cmax values of daptomycin were calculated from 55 to 174.5 mg/L when using a dosage from 4 to 12 mg/kg. All Cmax values were above the MPCs whatever the dosage. Taking into account the protein binding of daptomycin (ca. 90%), the unbound fraction Cmax was just within the MSW in 67–92% of strains at recommended dosages (4–6 mg/kg) and was above the MPC for the majority of strains only with the highest dosage (12 mg/kg). This study shows that free daptomycin Cmax values usually fell into MSWs when using lower dosages (<10 mg/kg).

Two successive calcium-dependent transitions mediate membrane binding and oligomerization of daptomycin and the related antibiotic A54145

Daptomycin and A54145 are homologous lipopeptide antibiotics that permeabilize the cell membranes of Gram-positive bacteria. Membrane permeabilization depends on the presence of both phosphatidylglycerol (PG) and calcium, and it involves the formation of oligomeric transmembrane pores that consist of approximately 6–8 subunits. We here show that each lipopeptide molecule binds two calcium ions in separable, successive steps. The first calcium ion causes the lipopeptide molecule to bind to the target membrane, and likely to form a loosely associated oligomer. Higher calcium concentrations induce binding of a second ion, which produces the more tightly associated and more deeply membrane-inserted final, functional form of the oligomer. Both calcium-dependent steps are accompanied by fluorescence signals that indicate transition of specific amino acid residues into less polar environments, suggestive of insertion into the target membrane. Our findings agree with the earlier observation that two of the four acidic amino acid residues in the daptomycin molecule are essential for antibacterial activity.