Yolanda Cajal

Gram-negative outer and inner membrane models: insertion of cyclic cationic lipopeptides.

Most Gram-negative bacteria are susceptible to polymyxin B (PxB), and development of resistance to this cationic lipopeptide is very rare. PxB mechanism of action involves interaction with both the outer membrane (OM) and the inner membrane (IM) of bacteria. For the design of new antibiotics based on the structure of PxB and with improved therapeutic indexes, it is essential to establish the key features of PxB that are important for activity. We have used an approach based on mimicking the outer layers of the OM and the IM of Gram-negative bacteria using monolayers of lipopolysaccharide (LPS) or anionic 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), respectively, and using a combination of penetration assay, analysis of pressure/area curves, and Brewster angle microscopy to monitor surface morphology changes. Synthetic analogue sp-B maintains the basic structural characteristics of the natural compound and interacts with the OM and the IM in a similar way. Analogue sp-C, with a mutation of the sequence [d-Phe6-Leu7] into [d-Phe6-Dab7], shows that this hydrophobic domain is involved in LPS binding. The significant role of the positive charges is demonstrated with sp-Dap analogue, where l-alpha,gamma-diaminobutyric acid residues Dab1 and Dab8 are replaced by l-alpha,gamma-diaminopropionic acid (Dap), resulting in lower degrees of insertion in both LPS and PG monolayers. The importance of the N-terminal acyl chain is demonstrated with polymyxin B nonapeptide (PxB-np). PxB-np shows lower affinity for LPS compared to PxB, sp-B, or sp-C, but it does not insert into PG monolayers, although it binds superficially to the anionic film. Since PxB microbial killing appears to be mediated by osmotic instability due to OM-IM phospholipid exchange, the ability of the different peptides to induce membrane-membrane lipid exchange has been studied by use of phospholipid unilamellar vesicles. Results indicate that cationic amphipathicity determines peptide activity.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

Salt-triggered intermembrane exchange of phospholipids and hemifusion by myelin basic protein.

Intervesicle phospholipid exchange through molecular contacts induced by the C1 molecular species of myelin basic protein (MBP) are characterized by using methods that amplify the effect of MBP-membrane interaction. The effect of salt concentration (KCl) on the vesicle-vesicle interaction of anionic sonicated covesicles of 30% 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine and 70% 1,2-dimyristoylglycero-sn-3-phosphomethanol (POPC/DMPM) by MBP is dissected by a combination of protocols into individual steps: aggregation of vesicles, apposition and contact formation, and hemifusion. Scattering and resonance energy transfer measurements reveal that, in the absence of KCl, MBP promotes rapid aggregation of the vesicles without lipid mixing. At >40 mM KCl, the extent of aggregation is larger and time-dependent. Fluorescence dequenching due to dilution of labeled phospholipids indicates that on a somewhat slower time scale, hemifusion of vesicles is triggered by salt, with mixing of the outer monolayer lipids but without flip-flop of phospholipids and without mixing or leakage of the aqueous contents. The exchange and hemifusion are seen with anionic vesicles; the effect of the structure of phospholipid, composition of vesicles, and the protein/lipid ratio is primarily on the kinetics of these and other competing processes. Thus, at 0.022 mol % of MBP and less than 100 mM KCl, it is possible to uncouple three sequential steps: (1) aggregation of vesicles by MBP; (2) apposition of bilayers and selective lipid exchange through vesicle-vesicle contacts established by MBP, i.e., anionic and zwitterionic phospholipids exchange, but cationic probes are excluded; and (3) hemifusion and lipid mixing of contacting monolayers of vesicles.

Influence of polymyxins on the structural dynamics of Escherichia coli lipid membranes

Polymyxins are a family of nonribosomic cationic peptide antibiotics highly effective against Gram-negative bacteria. Two members of this family, Polymyxins B and E (PxB, PxE), form molecular vesicle-vesicle contacts and promote a selective exchange of phospholipids at very low concentrations in the membrane, a biophysical phenomenon that can be the basis of their antibiotic mode of action. To get more insight into the interaction of these antibiotics with the lipid membrane, their effect on the structural dynamics of bilayers prepared with lipids extracted from the membrane of Escherichia coli was determined using fluorescently labeled phopholipids. Steady-state anisotropy measurements with probes that localize at different positions in the membrane give information on the effects of polymyxins on the mobility of the phospholipids. Results with PxB, PxE, colymycin M and polymyxin B nonapeptide (PxB-NP), a deacylated derivative with no antibiotic properties, are compared. At low peptide concentrations (<2 mol%) PxB and PxE bind to the membranes superficially, affecting very slightly the ordering of the lipids at the outermost part of the bilayer. Above this concentration, PxB and PxE insert more deeply in the bilayer, increasing lipid order both in the gel and liquid-crystal states and modifying phase transitions. Fluorescence experiments with pyrene labeled phospholipids indicate that the increase in lipid packing is accompanied by an enrichment of phospholipids in the bilayers. In contrast, colymycin M and PxB-NP did not modify lipid packing or phase transition, nor did they induce microdomain formation. The possible significance of these results in the antibiotic mode of action of PxB and PxE is discussed. The combination of spectroscopic techniques described here can be useful as part of a general method of screening for new antibiotics that act on the membrane by the same mechanism as polymyxins.

Tryptophan-containing lipopeptide antibiotics derived from polymyxin B with activity against Gram positive and Gram negative bacteria.

Resistance to all known antibiotics is a growing concern worldwide, and has renewed the interest in antimicrobial peptides, a structurally diverse class of amphipathic molecules that essentially act on the bacterial membrane. Propelled by the antimicrobial potential of this compound class, we have designed three new lipopeptides derived from polymyxin B, sp-34, sp-96 and sp-100, with potent antimicrobial activity against both Gram positive and Gram negative bacteria. The three peptides bind with high affinity to lipopolysaccharide as demonstrated by monolayer penetration and dansyl-displacement. The interaction with the cytoplasmic membrane has been elucidated by biophysical experiments with model membranes of POPG or POPE/POPG (6:4), mimicking the Gram positive and Gram negative bacterial membrane. Trp-based fluorescence experiments including steady-state, quenching, anisotropy and FRET, reveal selectivity for anionic phospholipids and deep insertion into the membrane. All three lipopeptides induce membrane fusion and leakage from anionic vesicles, a process that is favored by the presence of POPE. The molecules bind to zwitterionic POPC vesicles, a model of the eukaryotic membrane, but in a different way, with lower affinity, less penetration into the bilayer and no fusion or permeabilization of the membrane. Results in model membranes are consistent with flow cytometry experiments in Escherichia coli and Staphylococcus aureus using a membrane potential sensitive dye (bis-oxonol) and a nucleic acid dye (propidium iodide), suggesting that the mechanism of action is based on membrane binding and collapse of membrane integrity by depolarization and permeabilization.

Synergistic Antipseudomonal Effects of Synthetic Peptide AMP38 and Carbapenems.

The aim was to explore the antimicrobial activity of a synthetic peptide (AMP38) and its synergy with imipenem against imipenem-resistant Pseudomonas aeruginosa. The main mechanism of imipenem resistance is the loss or alteration of protein OprD. Time-kill and minimal biofilm eradication concentration (MBEC) determinations were carried out by using clinical imipenem-resistant strains. AMP38 was markedly synergistic with imipenem when determined in imipenem-resistant P. aeruginosa. MBEC obtained for the combination of AMP38 and imipenem was of 62.5 μg/mL, whereas the MBEC of each antimicrobial separately was 500 μg/mL. AMP38 should be regarded as a promising antimicrobial to fight MDR P. aeruginosa infections. Moreover, killing effect and antibiofilm activity of AMP38 plus imipenem was much higher than that of colistin plus imipenem.

Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection.

The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage or mixing of aqueous contents in anionic and cationic vesicles at pH 4-6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/PE) results in leakage without lipid mixing, indicating pore formation. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A virus infection of hepatocytes by the endosomal route is discussed.

Fluorescence study on the interaction of a multiple antigenic peptide from hepatitis A virus with lipid vesicles.

The interaction of the multiple antigenic peptide MAP4VP3 with lipid membranes has been studied by spectroscopic techniques. MAP4VP3 is a multimeric peptide that corresponds to four units of the sequence 110-121 of the capsid protein VP3 of hepatitis A virus. In order to evaluate the electrostatic and hydrophobic components on the lipid-peptide interaction, small unilamelar vesicles of different compositions, including zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic dipalmitoylphosphatidylcholine/phatidylinositol (DPPC:PI 9:1), and cationic dipalmitoylphosphatidylcholine/stearylamine (DPPC:SA 9.5:0.5), were used as membrane models. Intrinsic tryptophan fluorescence changes and energy transfer experiments show that MAP4VP3 binds to all three types of vesicles with the same stoichiometry, indicating that the electrostatic component of the interaction is not important for binding of this anionic peptide. Steady-state polarization experiments with vesicles labeled with 1,6-diphenyl-1,3,5-hexatriene or with 1-anilino-8-naphtalene sulphonic acid indicate that MAP4VP3 induces a change in the packing of the bilayers, with a decrease in the fluidity of the lipids and an increase in the temperature of phase transition in all the vesicles. The percentage of lipid exposed to the bulk aqueous phase is around 60% in intact vesicles, and it does not change upon binding of MAP4VP3 to DPPC vesicles, indicating that the peptide does not alter the permeability of the membrane. An increase in the amount of lipid exposed to the aqueous phase in cationic vesicles indicates either lipid flip-flop or disruption of the vesicles. Binding to DPPC vesicles occurs without leakage of entrapped carboxyfluorescein, even at high mol fractions of peptide. However, a time-dependent leakage is seen with cationic DPPC/SA and anionic DPPC/PI vesicles, indicating that the peptide induces membrane destabilization and not lipid flip-flop. Resonance energy transfer experiments show that MAP4VP3 leakage from cationic vesicles is due to membrane fusion, whereas leakage from anionic vesicles is not accompanied by lipid mixing. Results show that MAP4VP3 interacts strongly with the lipid components of the membrane, and although binding is not of electrostatic nature, the bound form of the peptide has different activity depending on the membrane net charge; thus, it is membrane disruptive in cationic and anionic vesicles, whereas no destabilizing effect is seen in DPPC vesicles.

pH-induced destabilization of lipid bilayers by a peptide from the VP3 protein of the capsid of hepatitis A virus†

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.

Spectroscopic studies of the interfacial binding of Humicola lanuginosa lipase

The interaction of Humicola lanuginosa lipase (HLL) with small unilamellar vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) and in the presence of tributyrin (TB) as a substrate was studied by the use of steady-state fluorescence techniques. An inactive mutant with the serine from the catalytic triad changed by alanine (S146A) was used in experiments with TB to avoid interferences from product formation. HLL binds to POPG vesicles in an active or open form for the catalytic turnover, and therefore POPG provides a suitable system for studying the conformational changes involving the movement of the loop of amino acids that covers the active site of the enzyme in solution. Tryptophan (Trp) fluorescence shows that HLL binding to POPG occurs with a change in the environment of Trp residue(s) and that there is only one type of bound form, even in the presence of TB. Accessibility to aqueous quenchers indicates shielding of Trp in the membrane. Fluorescence anisotropy of the enzyme increases on binding to the vesicles, indicating restricted rotational freedom for the Trp due to penetration in the bilayer. Resonance energy transfer experiments using an interfacial membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene p-toluenesulfonate (TMA-DPH), and an internal membrane probe, 1,6-diphenylhexa-1,3,5-triene (DPH), indicate that HLL does not penetrate very deeply in the hydrophobic core of the membrane, but preferentially stays close to the lipid interface. Addition of substrate (TB) does not result in any additional changes in the spectroscopic properties of HLL. It is suggested that the observed changes are due to the ‘opening of the lid’ on binding to POPG vesicles, leaving the active site accessible for the substrate to bind.