Hadar Sarig

Impact of self-assembly properties on antibacterial activity of short acyl-lysine oligomers.

ABSTRACT We investigated both the structural and functional consequences of modifying the hydrophobic, lipopeptide-mimetic oligo-acyl-lysine (OAK) Nα-hexadecanoyl-l-lysyl-l-lysyl-aminododecanoyl-l-lysyl-amide (c16KKc12K) to its unsaturated analog hexadecenoyl-KKc12K [c16(ω7)KKc12K]. Despite similar tendencies for self-assembly in solution (critical aggregation concentrations, ∼10 μM), the analogous OAKs displayed dissimilar antibacterial properties (e.g., bactericidal kinetics taking minutes versus hours). Diverse experimental evidence provided insight into these discrepancies: whereas c16(ω7)KKc12K created wiry interconnected nanofiber networks, c16KKc12K formed both wider and stiffer fibers which displayed distinct binding properties to phospholipid membranes. Unsaturation also shifted their gel-to-liquid transition temperatures and altered their light-scattering properties, suggesting the disassembly of c16(ω7)KKc12K in the presence of bacteria. Collectively, the data indicated that the higher efficiency in interfering with bacterial viability emanated from a wobbly packing imposed by a single double bond. This suggests that similar strategies might improve hydrophobic OAKs and related lipopeptide antibiotics.

A miniature mimic of host defense peptides with systemic antibacterial efficacy

Oligomers of acylated lysines (OAKs) are synthetic mimics of host defense peptides (HDPs) with promising antimicrobial properties. Here we challenged the OAK concept for its ability to generate both systemically efficient and economically viable lead compounds for fighting multidrug‐resistant bacteria. We describe the design and characterization of a miniature OAK composed of only 3 lysyls and 2 acyls (designated C12(ω7)K‐β12) that preferentially targets gram‐positive species by a bacteriostatic mode of action. To gain insight into the mechanism of action, we examined the interaction of OAK with various potential targets, including phospholipid bilayers, using surface plasmon resonance, and Langmuir monolayers, using insertion assays, epifluorescence microscopy, and grazing incidence X‐ray diffraction, in a complementary manner. Collectively, the data support the notion that C12(ω7)K β12 damages the plasma‐membrane architecture similarly to HDPs, that is, following a near‐classic 2‐step interaction including high‐affinity electrostatic adhesion and a subsequent shallow insertion that was limited to the phospholipid head group region. Notably, preliminary acute toxicity and efficacy studies performed with mouse models of infection have consolidated the potential of OAK for treating bacterial infections, including systemic treatments of methicillin‐resistant Staphylococcus aureus. Such simple yet robust chemicals might be useful for various antibacterial applications while circumventing potential adverse effects associated with cytolytic compounds.—Sarig, H., Livne, L., Held‐Kuznetsov, V., Zaknoon, F., Ivankin, A., Gidalevitz, D., Mor, A. A miniature mimic of host defense peptides with systemic antibacterial efficacy. FASEB J. 24, 1904–1913 (2010). www.fasebj.org

Cell-Wall Interactions and the Selective Bacteriostatic Activity of a Miniature Oligo-Acyl-Lysyl

The oligo-acyl-lysyl, C(12(omega 7))K-beta(12), is comprised of only three Lys residues. Despite its small size, it exhibits potent bacteriostatic activity against Gram-positive bacteria, but it is approximately 10-fold less potent against Gram-negative bacteria. We followed the interactions of C(12(omega 7))K-beta(12) from its initial contact with the bacterial surface across the cell wall down to the cytoplasmic membrane. Binding to anionic lipids, as well as to negatively charged LPS and LTA, occurs with very high affinity. The C(12(omega 7))K-beta(12) does not cross the outer membrane of Gram-negative bacteria; rather, it achieves its action by depositing on the LPS layer, promoting surface adhesion and blocking passage of solutes. In Gram-positive bacteria, the thick peptidoglycan layer containing LTA allows passage of C(12(omega 7))K-beta(12) and promotes its accumulation in the small periplasm. From that location it is then driven to the membrane by strong electrostatic interactions. Despite its high potency against Gram-positive bacteria, this agent is not capable of efficiently breaking down the permeability barrier of the cytoplasmic membrane or of reaching an intracellular target, as suggested by the fact that it does not interact with DNA.

Sensitization of gram-negative bacteria by targeting the membrane potential

Toward generating new tools for fighting multidrug‐resistant (MDR) bacteria, we assessed the ability of a membrane‐active peptide to sensitize gram‐negative bacteria to various antibiotics. The mechanism for affecting inner and/or outer membrane functions was assessed by complementary biophysical methods (SPR, DSC, ITC). The implication of efflux pumps was examined using Acr‐AB mutants, as tested with representative antibiotics, host defense peptides, and synthetic mimics. The ability to affect disease course systemically was compared for a single therapy and combination therapy, using the mouse thigh‐infection model. The data show that potent antibiotic action can be provoked in vitro and in vivo, by a treatment combining two antibacterial compounds whose individual inefficiency against gram‐negative bacteria stems from their efflux. Thus, at subminimal inhibitory concentrations, the lipopeptide‐like sequence, Nα(ω7)dodecenoyl‐lysyl‐[lysyl‐aminodode‐canoyl‐lysyl]‐amide (designated C12(ω7)K‐β12), has, nonetheless, rapidly achieved a transient membrane depolarization, which deprived bacteria of the proton‐motive force required for active efflux. Consequently, bacteria became significantly sensitive to intracellular targeting antibiotics. Collectively, these findings suggest a potentially useful approach for expanding the antibiotics sensitivity spectrum of MDR gram‐negative bacteria to include efflux substrates.—Goldberg, K., Sarig, H., Zaknoon, F., Epand, R. F., Epand, R. M., Mor, A., Sensitization of gram‐negative bacteria by targeting the membrane potential. FASEB J. 27, 3818–3826 (2013). www.fasebj.org

Design and Characterization of a Broad -Spectrum Bactericidal Acyl-lysyl Oligomer

Previously characterized chemical mimics of host defense peptides belonging to the oligo-acyl-lysyl (OAK) family have so far failed to demonstrate broad-spectrum antibacterial potency combined with selectivity toward host cells. Here, we investigated OAK sequences and characterized a promising representative, designated C(12)K-3beta(10), with broad-spectrum activity (MIC(90) = 6.2 microM) and low hemotoxicity (LC(50) > 100 microM). Whereas C(12)K-3beta(10) exerted an essentially bactericidal effect, E. coli bacteria were killed faster than S. aureus (minutes versus hours). Mechanistic studies addressing this difference revealed that unlike E. coli, S. aureus bacteria undergo a transient rapid bactericidal stage that over time converts to a bacteriostatic effect. This behavior was dictated by interactions with cell wall-specific components. Preliminary efficacy studies in mice using the thigh infection model demonstrated the OAKs ability to significantly affect bacterial viability upon single-dose systemic treatment (2 mg/kg).

Functional studies of cochleate assemblies of an oligo-acyl-lysyl with lipid mixtures for combating bacterial multidrug resistance

The cationic antimicrobial oligo‐acyl‐lysyls (OAKs) interact with lipid mixtures mimicking the composition of bacterial cytoplasmic membranes. We have reported the ability of one such OAK, C12K‐7α8, to cluster anionic lipids and to promote a structural change with lipid bilayers to form rolled cylindrical structures or cochleates, without requiring divalent cations for their assembly. These assemblies can be exploited for drug delivery, permitting their synergistic use with antibiotics in systemic therapy to increase efficacy and reduce toxicity. Our previous studies of the biophysical properties of these systems led us to select mixtures with the goal of optimizing their potential for enhancing effectiveness in combating bacterial multidrug resistance. Here, we further investigate the properties of such mixtures that result in enhanced in vivo activity. The role of erythromycin in the assembly of cochleates with OAK in the gel and the liquid crystalline states were assessed, as well as the encapsulation efficiency of the systems chosen. In addition, we found that erythromycin did not undermine the ability of OAKs to induce fusion of vesicles, fusion being an essential component of cochleate formation. The in vivo activity of the new assemblies tested resulted in higher survival rates of animals infected with multidrug resistant bacteria.—Sarig, H., Ohana, D., Epand, R. F., Mor, A., Epand, R. M. Functional studies of cochleate assemblies of an oligo‐acyl‐lysyl with lipid mixtures for combating bacterial multidrug resistance. FASEB J. 25, 3336–3343 (2011). www.fasebj.org

Antibacterial Properties and Mode of Action of a Short Acyl-Lysyl Oligomer

ABSTRACT We investigated the potency, selectivity, and mode of action of the oligo-acyl-lysine (OAK) NC12-2β12, which was recently suggested to represent the shortest OAK sequence that retains nonhemolytic antibacterial properties. A growth inhibition assay against a panel of 48 bacterial strains confirmed that NC12-2β12 exerted potent activity against gram-positive bacteria while exhibiting negligible hemolysis up to at least 100 times the MIC. Interestingly, NC12-2β12 demonstrated a bacteriostatic mode of action, unlike previously described OAKs that were bactericidal and essentially active against gram-negative bacteria only. The results of various experiments with binding to model phospholipid membranes correlated well with those of the cytotoxicity experiments and provided a plausible explanation for the observed activity profile. Thus, surface plasmon resonance experiments performed with model bilayers revealed high binding affinity to a membrane composition that mimicked the plasma membrane of staphylococci (global affinity constant [Kapp], 3.7 × 106 M−1) and significantly lower affinities to mimics of Escherichia coli or red blood cell cytoplasmic membranes. Additional insertion isotherms and epifluorescence microscopy experiments performed with model Langmuir monolayers mimicking the outer leaflet of plasma membranes demonstrated the preferential insertion of NC12-2β12 into highly anionic membranes. Finally, we provide mechanistic studies in support of the view that the bacteriostatic effect resulted from a relatively slow process of plasma membrane permeabilization involving discrete leakage of small solutes, such as intracellular ATP. Collectively, the data point to short OAKs as a potential source for new antibacterial compounds that can selectively affect the growth of gram-positive bacteria while circumventing potential adverse effects linked to lytic compounds.

Simultaneous breakdown of multiple antibiotic resistance mechanisms in S. aureus

In previous studies, the oligo‐acyl‐lysyl (OAK) C12(ω7)K‐β12 added to cultures of gram‐positive bacteria exerted a bacteriostatic activity that was associated with membrane depolarization, even at high concentrations. Here, we report that multidrug‐resistant Staphylococcus aureus strains, unlike other gram‐positive species, have reverted to the sensitive phenotype when exposed to subminimal inhibitory concentrations (sub‐MICs) of the OAK, thereby increasing antibiotics potency by up to 3 orders of magnitude. Such chemosensitization was achieved using either cytoplasm or cell‐wall targeting antibiotics. Moreover, eventual emergence of resistance to antibiotics was significantly delayed. Using the mouse peritonitis‐sepsis model, we show that on single‐dose administration of oxacillin and OAK combinations, death induced by a lethal staphylococcal infection was prevented in a synergistic manner, thereby supporting the likelihood for synergism to persist under in vivo conditions. Toward illuminating the molecular basis for these observations, we present data arguing that sub‐MIC OAK interactions with the plasma membrane can inhibit proton‐dependent signal transduction responsible for expression and export of resistance factors, as demonstrated for β‐lactamase and PBP2a. Collectively, the data reveal a potentially useful approach for overcoming antibiotic resistance and for preventing resistance from emerging as readily as when bacteria are exposed to an antibiotic alone.—Kaneti, G., Sarig, H., Marjieh, I., Fadia, Z., Mor, A., Simultaneous breakdown of multiple antibiotic resistance mechanisms in S. aureus. FASEB J. 27, 4834‐4843 (2013). www.fasebj.org

Mechanisms Mediating Bactericidal Properties and Conditions That Enhance the Potency of a Broad-Spectrum Oligo-Acyl-Lysyl

ABSTRACT Previous studies have established the potential of the oligo-acyl-lysyl (OAK) concept in generating simple chemical mimics of host defense peptides (HDPs) with improved antimicrobial properties. We investigated the antibacterial properties of such an OAK, C16(ω7)-KK-C12-Kamide, to obtain a better understanding of the complex mode(s) of action of cationic antibacterial peptides. The average MIC, determined against a multispecies panel of 50 strains, was 6 ± 5 μg/ml. However, although the OAK exerted an essentially dose-dependent bactericidal effect (time-kill curves typically exhibited 99% death within 2 h), marked differences in the killing rates occurred among inter- and intraspecies strains. Mechanistic comparison between equally sensitive and related strains revealed death of one strain to stem from the OAKs capacity to breach the cell membrane permeability barrier, whereas the death of the related strain resulted from the OAKs direct interference with DNA functions in vivo, without detectable membrane damage. These findings therefore support the notion that the antibacterial mechanism of action of a single HDP can vary among inter- and intraspecies strains. In addition, we present data illustrating the differential effects of environmental conditions (pH, ionic strength and temperature), on the OAKs antibacterial properties, and ultimately demonstrate potency enhancement (by orders of magnitude) through selection of optimal incubation conditions. Such attributes might be useful in a variety of antibacterial applications.

Antibacterial Properties of an Oligo-Acyl-Lysyl Hexamer Targeting Gram-Negative Species

ABSTRACT Toward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl]5 (α12-5α10). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC90 of 6.2 μM. The OAKs bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α12-5α10 from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAKs effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.