Antimicrobial Lipopeptides Research Articles

An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State

The cyclic antimicrobial lipopeptide daptomycin (DAP) triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence-specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, non-susceptibility to DAP by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated E. faecium LiaR protein at 3.2Å resolution and, in combination with solution studies, show that the activation of LiaR induces the formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to DAP by the upregulation of the LiaFSR regulon. A comparison of the E. faecalis LiaR, E. faecium LiaR, and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with DAP resistance suggests that physicochemical properties such as oligomerization state and DNA specificity, although tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.

Co-immobilization of Palm and DNase I for the development of an effective anti-infective coating for catheter surfaces.

Biomaterial-associated infections, in particular, catheter-associated infections (CAI) are a major problem in clinical practice due to their ability to resist antimicrobial treatment and the host immune system. This study aimed to co-immobilize the antimicrobial lipopeptide Palm and the enzyme DNase I to introduce both antimicrobial and anti-adhesive functionalities to polydimethylsiloxane (PDMS) material, using dopamine chemistry. Surface characterization confirmed the immobilization of both compounds and no leaching of Palm from the surfaces for up to 5days. Co-immobilization of both agents resulted in a bifunctional coating with excellent surface antimicrobial and anti-biofilm properties against both Staphylococcus aureus and Pseudomonas aeruginosa. The modified surfaces demonstrated superior biocompatibility. To better discriminate co-adhesion of both species on modified surfaces, PNA FISH (Fluorescence in situ hybridization using peptide nucleic acid probes) was employed, and results showed that P. aeruginosa was the dominant organism, with S. aureus adhering afterwards on P. aeruginosa agglomerates. Furthermore, Palm immobilization exhibited no propensity to develop bacterial resistance, as opposite to the immobilization of an antibiotic. The overall results highlighted that co-immobilization of Palm and DNase I holds great potential to be applied in the development of catheters. Catheter-associated infections (CAI) are the most common hospital-acquired infections worldwide. Several coating strategies have been proposed to fight these infections but most of them present some important limitations, including the emergence of resistant bacteria and toxicity concerns. The present work describes a two-step polydopamine-based surface modification strategy to successfully co-immobilize an antimicrobial peptide (Palm) and an enzyme targeting an important component of biofilm matrix (DNase I). This immobilization approach imparted polydimethylsiloxane surfaces with both anti-adhesive and antimicrobial properties against the adhesion of relevant bacteria as single and dual-species, with excellent stability and biocompatible and anti-biofilm properties, holding, therefore, great potential in the development of catheters able to prevent CAI.

Functional Role of Bacteria from Invasive Phragmites australis in Promotion of Host Growth.

We hypothesize that bacterial endophytes may enhance the competitiveness and invasiveness of Phragmites australis. To evaluate this hypothesis, endophytic bacteria were isolated from P. australis. The majority of the shoot meristem isolates represent species from phyla Firmicutes, Proteobacteria, and Actinobacteria. We chose one species from each phylum to characterize further and to conduct growth promotion experiments in Phragmites. Bacteria tested include Bacillus amyloliquefaciens A9a, Achromobacter spanius B1, and Microbacterium oxydans B2. Isolates were characterized for known growth promotional traits, including indole acetic acid (IAA) production, secretion of hydrolytic enzymes, phosphate solubilization, and antibiosis activity. Potentially defensive antimicrobial lipopeptides were assayed for through application of co-culturing experiments and mass spectrometer analysis. B. amyloliquefaciens A9a and M. oxydans B2 produced IAA. B. amyloliquefaciens A9a secreted antifungal lipopeptides. Capability to promote growth of P. australis under low nitrogen conditions was evaluated in greenhouse experiments. All three isolates were found to increase the growth of P. australis under low soil nitrogen conditions and showed increased absorption of isotopic nitrogen into plants. This suggests that the Phragmites microbes we evaluated most likely promote growth of Phragmites by enhanced scavenging of nitrogenous compounds from the rhizosphere and transfer to host roots. Collectively, our results support the hypothesis that endophytic bacteria play a role in enhancing growth of P. australis in natural populations. Gaining a better understanding of the precise contributions and mechanisms of endophytes in enabling P. australis to develop high densities rapidly could lead to new symbiosis-based strategies for management and control of the host.

Modeling the effects of different conditions on the inhibitory activity of antimicrobial lipopeptide (AMPNT-6) against Staphylococcus aureus growth and enterotoxin production in shrimp meat

Several bacteria, including Staphylococcus aureus (S. aureus), have been identified as food-borne pathogenic bacteria that cause many cases of food poisoning. This has prompted researchers to identify novel antimicrobial compounds. NT-6 antimicrobial lipopeptide (AMPNT-6) secreted by Bacillus subtilis can inhibit S. aureus growth and enterotoxin B production. The effects of temperature, sodium chloride, pH and sodium metabisulfite on the activity of AMPNT-6 applied to control S. aureus growth and enterotoxin B production in shrimp were investigated by using the response surface method. Design-Expert software was chosen to conduct data analysis and develop the models to demonstrate whether AMPNT-6 is able to control the growth of S. aureus and toxin production in shrimp exposed to different environmental conditions. The results showed that temperature, sodium chloride concentration, pH and sodium metabisulfite concentration had no significant effects on the antimicrobial activity of AMPNT-6 and there were no significant interactions between them. Quadratic polynomial mathematical models were established, and the quadratic regression equations of predicted value LgN (logarithm of bacterial colony number) and Y (enterotoxin B) to the independent variables x1 (temperature), x2 (sodium chloride concentration), x3 (pH), x4 (sodium metabisulfite concentration) and x5 (AMPNT-6 concentration) were constructed as: $${\text{LgN}} = 6.93 + 0.29x_{1} - 0.11x_{3} - 0.46x_{5} - 0.35x_{1}^{2} - 0.30x_{3} x_{4} \left( {R^{2 } = 0.9049} \right);$$ $$Y = 22.83 + 4.33x_{1} - 6.02x_{5} - 5.28x_{1}^{2} - 4.68x_{1} x_{4} - 3.80x_{3} x_{4} \left( {R^{2 } = 0.8874} \right).$$ AMPNT-6 showed stable antimicrobial activity that was hardly affected by external factors such as temperature, salinity, pH and sodium metabisulfite. Observed experimental values were comparable with the predicted values. It is concluded that models that showed good fitness can be used to predict the inhibitory effects of AMPNT-6 on S. aureus growth and enterotoxin B production in shrimp exposed to different external variables.

Isolation and Structural Elucidation of Brevibacillin, an Antimicrobial Lipopeptide from Brevibacillus laterosporus That Combats Drug-Resistant Gram-Positive Bacteria.

A new environmental bacterial strain exhibited strong antimicrobial characteristics against methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of Enterococcus faecalis and Lactobacillus plantarum, and other Gram-positive bacteria. The producer strain, designated OSY-I1, was determined to be Brevibacillus laterosporusvia morphological, biochemical, and genetic analyses. The antimicrobial agent was extracted from cells of OSY-I1 with isopropanol, purified by high-performance liquid chromatography, and structurally analyzed using mass spectrometry (MS) and nuclear magnetic resonance (NMR). The MS and NMR results, taken together, uncovered a linear lipopeptide consisting of 13 amino acids and an N-terminal C6 fatty acid (FA) chain, 2-hydroxy-3-methylpentanoic acid. The lipopeptide (FA-Dhb-Leu-Orn-Ile-Ile-Val-Lys-Val-Val-Lys-Tyr-Leu-valinol, where Dhb is α,β-didehydrobutyric acid and valinol is 2-amino-3-methyl-1-butanol) has a molecular mass of 1,583.0794 Da and contains three modified amino acid residues: α,β-didehydrobutyric acid, ornithine, and valinol. The compound, designated brevibacillin, was determined to be a member of a cationic lipopeptide antibiotic family. In addition to its potency against drug-resistant bacteria, brevibacillin also exhibited low MICs (1 to 8 μg/ml) against selected foodborne pathogenic and spoilage bacteria, such as Listeria monocytogenes,Bacillus cereus, and Alicyclobacillus acidoterrestris Purified brevibacillin showed no sign of degradation when it was held at 80 °C for 60 min, and it retained at least 50% of its antimicrobial activity when it was held for 22 h under acidic or alkaline conditions. On the basis of these findings, brevibacillin is a potent antimicrobial lipopeptide which is potentially useful to combat drug-resistant bacterial pathogens and foodborne pathogenic and spoilage bacteria.

From Pigs to Patients: Transmissible, Single Gene-mediated Resistance to Colistin

The last decade has seen a stark increase in the number of multidrug resistant Gram-negative bacterial pathogens. Of particular importance are highly carbapenem resistant Escherichia coli and Klebsiella pneumoniae, as well as fluoroquinolone resistant Pseudomonas aeruginosa and Acinetobacter baumanii [1]. Owing to the lack of new drugs entering the marketplace, physicians are increasingly revisiting older drugs that had fallen out of common use. One such drug is colistin and it has increasingly become the drug of last resort for these infections [2]. Colistin is a polymyxin type of antimicrobial lipopeptide that is produced by Paenobacillus species. The polymxyins were discovered 65 years ago but have limited clinical use due to their neurotoxic and nephrotoxic side effects. Despite this toxicity in humans, colistin is used extensively in agriculture as growth promoters and estimates of global colistin usage in agriculture is >12,000 tonnes per annum. A recent report in the Lancet Infectious Diseases describes the first example of a transmissible plasmid that encodes colistin resistance by the mcr-1 gene. Colistin resistance was first detected in Escherichia coli isolates from pigs, but was ultimately found in meats for sale, and later in Chinese patients [3].

Characterization of antimicrobial lipopeptides produced by Bacillus sp. LM7 isolated from chungkookjang, a Korean traditional fermented soybean food

A wild-type microorganism exhibiting antimicrobial activities was isolated from the Korean traditional fermented soybean food Chungkookjang and identified as Bacillus sp. LM7. During its stationary growth phase, the microorganism secreted an antimicrobial substance, which we partially purified using a simple two-step procedure involving ammonium sulfate precipitation and heat treatment. The partially purified antimicrobial substance, Anti-LM7, was stable over a broad pH range (4.0–9.0) and at temperatures up to 80°C for 30min, and was resistant to most proteolytic enzymes and maintained its activity in 30% (v/v) organic solvents. Anti-LM7 inhibited the growth of a broad range of Gram-positive bacteria, including Bacillus cereus and Listeria monocytogenes, but it did not inhibit lactic acid bacteria such as Lactobacillus plantarum and Lactococcus lactis subsp. Lactis. Moreover, unlike commercially available nisin and polymyxin B, Anti-LM7 inhibited certain fungal strains. Lastly, liquid chromatography-mass spectrometry analysis of Anti-LM7 revealed that it contained eight lipopeptides belonging to two families: four bacillomycin D and four surfactin analogs. These Bacillus sp. LM7-produced heterogeneous lipopeptides exhibiting extremely high stability and a broad antimicrobial spectrum are likely to be closely related to the antimicrobial activity of Chungkookjang, and their identification presents an opportunity for application of the peptides in environmental bioremediation, pharmaceutical, cosmetic, and food industries.

Modeling: Antimicrobial Lipopeptides Form Micelles along Target Membranes

Computer simulations suggest that antimicrobial lipopepides (AMLPs) clump into micelles, rather than act individually when they target microbial cells, according to biophysicist Alan Grossfield at the University of Rochester Medical Center (URMC) in Rochester, N.Y., and his collaborators. This insight about the AMLP binding mechanism could help to improve the design and function of this emerging class of synthetic antibacterial compounds, he says. Details appeared August 2015 in Biophysical Journal (doi:10.1016/j.bpj.2015.07.011).

Total Synthesis and Stereochemical Assignment of the Antimicrobial Lipopeptide Cerexin A1

The isolation and total synthesis of the antimicrobial lipopeptide cerexin A1 is reported. This synthesis includes the preparation of orthogonally protected γ-hydroxylysine, utilizing a nitrile Reformatsky-type reaction as a key step to yield both diastereomers more efficiently than previously reported methods. The configuration of the β-hydroxyl in the lipid tail was determined by the use of a modified Ohrui-Akasaka approach. Furthermore, new cerexin analogues from Bacillus mycoides ATCC 21929 were isolated and characterized, revealing an ε-amino succinylation of a hydroxylysine residue that is unusual in a nonribosomal peptide synthetase product.

Thermodynamics of Micelle Formation and Membrane Fusion Modulate Antimicrobial Lipopeptide Activity

Antimicrobial lipopeptides (AMLPs) are antimicrobial drug candidates that preferentially target microbial membranes. One class of AMLPs, composed of cationic tetrapeptides attached to an acyl chain, have minimal inhibitory concentrations in the micromolar range against a range of bacteria and fungi. Previously, we used coarse-grained molecular dynamics simulations and free energy methods to study the thermodynamics of their interaction with membranes in their monomeric state. Here, we extended the study to the biologically relevant micellar state, using, to our knowledge, a novel reaction coordinate based on hydrophobic contacts. Using umbrella sampling along this reaction coordinate, we identified the critical transition states when micelles insert into membranes. The results indicate that the binding of these AMLP micelles to membranes is thermodynamically favorable, but in contrast to the monomeric case, there are significant free energy barriers. The height of these free energy barriers depends on the membrane composition, suggesting that the AMLPs’ ability to selectively target bacterial membranes may be as much kinetic as thermodynamic. This mechanism highlights the importance of considering oligomeric state in solution as criterion when optimizing peptides or lipopeptides as antibiotic leads.

Better Together: Lipopeptide Micelle Formation Enhances Antimicrobial Selectivity

Antimicrobial resistance stands as a growing threat to public health (1). Antibiotics, designed to specifically target bacterial cells, have inadvertently accelerated the development of resistant microorganisms, making any infection potentially dangerous. Antimicrobial lipopeptides (AMLPs—part lipid, part peptide) have recently gained increasing interest (2), because they combine two attractive features: just like antimicrobial peptides, their mode of action is very generic and thus evolutionarily conserved; but unlike antimicrobial peptides, they involve much smaller molecular-weight peptides and are thus more in line with both practical and economical considerations of drug development.

Effect of acyl chain length on therapeutic activity and mode of action of the CX-KYR-NH2 antimicrobial lipopeptide

Peptide lipidation has proven to be an inexpensive and effective strategy for designing next-generation peptide-based drug compounds. In this study, the effect of the acyl chain length of ultrashort LiPs (CX-KYR-NH2; X=10, 12, 14 and 16) on their bacterial killing and membrane disruption kinetics was investigated. The geometric mean of the minimum inhibitory concentration (MIC) values for 4 pathogenic bacterial strains was 25μM, with a selectivity index of 10.24 for C14-KYR-NH2. LiPs at all concentrations exhibited no cytotoxicity towards human erythrocytes, but towards Vero cells at 80μM. All the LiPs adopted secondary structure in a membrane mimicking environment. C14-KYR-NH2 aggregated above 256μM, while C16-KYR-NH2 did above 80μM. All LiPs showed outer membrane permeabilization within 3min after treatment, yet the extent and kinetics of inner membrane penetration and depolarization were dependent on the acyl chain length. Cell death subsequently occurred within 10min, and killing activity appeared to correlate most with depolarization activity but not with outer or inner membrane permeability. AFM imaging of cells treated with C14-KYR-NH2 revealed rupture of the cell surface and cytosolic leakage depending on the length of incubation. This study highlights and follows the progression of events that occur during the membrane disintegration process over time, and determines the optimal amphipathicity of ultrashort LiPs with 12–14 carbon atoms for this membrane disrupting activity. The fast acting bactericidal properties of ultrashort LiPs with optimal chain lengths make them promising candidates for drug lead compounds.

Isolation and characterization of an antimicrobial lipopeptide produced by Paenibacillus ehimensis MA2012.

In this study, a novel lipopeptide antibiotic was isolated from the culture supernatant of Paenibacillus ehimensis strain MA2012. After analyses by mass spectrometry (MS), nuclear magnetic resonance (NMR), and high resolution mass spectrometry (HR-MS/MS) the compound was identified to be polypeptin C consisting of 3-hydroxy-4-methyl-hexanoic acid moiety and nine amino acids as peptide body. It has the same molecular mass (1115 Da) with that of polypeptin A and B but the amino acid positions differ. A relatively low concentration (125 ppm) of polypeptin C lowered the surface tension of water from 72.2 to 36.4 mN/m. It showed antimicrobial activity against several plant pathogenic bacteria and fungi. When the polypeptin C was applied to the ripe pepper fruits previously inoculated with conidia of Colletotrichum gloeosporioides, the hyphal growth on the fruit was significantly suppressed. Moreover, the hyphal morphology of C. gloeosporioides was greatly affected by the purified compound. All these data suggest the great potential of P. ehimensis MA2012 to control plant fungal and bacterial diseases.

Thermodynamics Govern the Mechanism of Antimicrobial Lipopeptides: Insights from Coarse-Grained Molecular Dynamics Simulations

Antimicrobial lipopeptides (AMLPs) are a series of acylated cationic peptides with broad-spectrum antimicrobial activity and low hemolytic activity. We used microsecond-scale coarse-grained molecular dynamics simulations with the MARTINI force field to understand AMLPs’ modes of action. Using rigorous free energy calculations with a novel reaction coordinate, we quantified formation of lipopeptide micelles in solution, as well as the the affinity of those micelles for different membrane compositions. The results yield both kinetic and thermodynamics arguments for the lipopeptides selectivity for bacterial over mammalian membranes. Our results indicate that the acyl chain of C16-KGGK, one of the AMLPs, is mainly responsible for its affinity to membrane while the peptide portion determines the selectivity towards different membrane lipid compositions. The micelle results suggest both kinetic and thermodynamics arguments for the lipopeptides selectivity for bacterial over mammalian membranes. Our results provide biophysical insights into the mechanism of lipopeptides’ antimicrobial action.

Bacillus-related Spore Formers: Attractive Agents for Plant Growth Promotion.

Bacillus-related Spore Formers: Attractive Agents for Plant Growth Promotion.

Impact of Antimicrobial Lipopeptides from Bacillus sp. on Suppression of Fusarium Yellows of Tatsoi.

Iturin A and surfactin are antimicrobial lipopeptides produced by antagonistic Bacillus spp. We herein demonstrated that both lipopeptides amended the soil-mediated suppression of the soil-borne disease, Fusarium yellows of tatsoi (Brassica rapa var. rosularis). Significant disease suppression was conferred by the amendments of purified iturin A or surfactin to soil. However, an excess amount of iturin A or surfactin to soil resulted in the loss of disease suppression activity.

Green surfactant of marine origin exerting a cytotoxic effect on cancer cell lines

The present work reveals the efficacy of a marine antimicrobial lipopeptide biosurfactant in blocking proliferation of breast cancer and colon cancer cell lines, without displaying any significant antioxidant activity.

Membrane interaction of a new synthetic antimicrobial lipopeptide sp-85 with broad spectrum activity

Antimicrobial peptides offer a new class of therapeutic agents to which bacteria may not be able to develop genetic resistance, since their main activity is in the lipid component of the bacterial cell membrane. We have developed a series of synthetic cationic cyclic lipopeptides based on natural polymyxin, and in this work we explore the interaction of sp-85, an analog that contains a C12 fatty acid at the N-terminus and two residues of arginine. This analog has been selected from its broad spectrum antibacterial activity in the micromolar range, and it has a disruptive action on the cytoplasmic membrane of bacteria, as demonstrated by TEM. In order to obtain information on the interaction of this analog with membrane lipids, we have obtained thermodynamic parameters from mixed monolayers prepared with POPG and POPE/POPG (molar ratio 6:4), as models of Gram positive and Gram negative bacteria, respectively. Langmuir–Blodgett films have been extracted on glass plates and observed by confocal microscopy, and images are consistent with a strong destabilizing effect on the membrane organization induced by sp-85. The effect of sp-85 on the membrane is confirmed with unilamelar lipid vesicles of the same composition, where biophysical experiments based on fluorescence are indicative of membrane fusion and permeabilization starting at very low concentrations of peptide and only if anionic lipids are present. Overall, results described here provide strong evidence that the mode of action of sp-85 is the alteration of the bacterial membrane permeability barrier.

Differential proteomics analysis of Bacillus amyloliquefaciens and its genome-shuffled mutant for improving surfactin production.

Genome shuffling technology was used as a novel whole-genome engineering approach to rapidly improve the antimicrobial lipopeptide yield of Bacillus amyloliquefaciens. Comparative proteomic analysis of the parental ES-2-4 and genome-shuffled FMB38 strains was conducted to examine the differentially expressed proteins. The proteome was separated by 2-DE (two dimensional electrophoresis) and analyzed by MS (mass spectrum). In the shuffled strain FMB38, 51 differentially expressed protein spots with higher than two-fold spot density were detected by gel image comparison. Forty-six protein spots were detectable by silver staining and further MS analysis. The results demonstrated that among the 46 protein spots expressed particularly induced in the genome-shuffled mutant, 15 were related to metabolism, five to DNA replication, recombination and repair, six to translation and post-translational modifications, one to cell secretion and signal transduction mechanisms, three to surfactin synthesis, two to energy production and conversion, and 14 to others. All these indicated that the metabolic capability of the mutant was improved by the genome shuffling. The study will enable future detailed investigation of gene expression and function linked with surfactin synthesis. The results of proteome analysis may provide information for metabolic engineering of Bacillus amyloliquefaciens for overproduction of surfactin.

Biofilm eradication kinetics of the ultrashort lipopeptide C12 -OOWW-NH2 utilizing a modified MBEC Assay(™).

In this study, we report the antimicrobial planktonic and biofilm kill kinetics of ultrashort cationic lipopeptides previously demonstrated by our group to have a minimum biofilm eradication concentration (MBEC) in the microgram per mL (μg/mL) range against clinically relevant biofilm-forming micro-organisms. We compare the rate of kill for the most potent of these lipopeptides, dodecanoic (lauric) acid-conjugated C12 -Orn-Orn-Trp-Trp-NH2 against the tetrapeptide amide H-Orn-Orn-Trp-Trp-NH2 motif and the amphibian peptide Maximin-4 via a modification of the MBEC Assay(™) for Physiology & Genetics (P&G). Improved antimicrobial activity is achieved upon N-terminal lipidation of the tetrapeptide amide. Increased antimicrobial potency was demonstrated against both planktonic and biofilm forms of Gram-positive micro-organisms. We hypothesize rapid kill to be achieved by targeting of microbial membranes. Complete kill against established 24-h Gram-positive biofilms occurred within 4h of exposure to C12 -OOWW-NH2 at MBEC values [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 15.63μg/mL] close to the values for the planktonic minimum inhibitory concentration (MIC) [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 1.95μg/mL]. Such rapid kill, especially against sessile biofilm forms, is indicative of a reduction in the likelihood of resistant strains developing with the potential for quicker resolution of pathogenic infection. Ultrashort antimicrobial lipopeptides have high potential as antimicrobial therapy.