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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) (α(12)-5α(10)). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC(90) of 6.2 μM. The OAK's 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 α(12)-5α(10) 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 OAK's 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.
Antimicrobial Agents and Chemotherapy 07/2012; 56(9):4827-32. · 4.84 Impact Factor
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ABSTRACT: 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, C(12)K-7α(8), 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.
The FASEB Journal 06/2011; 25(10):3336-43. · 5.71 Impact Factor
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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, C(16(ω7))-KK-C(12)-K(amide), 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 OAK's capacity to breach the cell membrane permeability barrier, whereas the death of the related strain resulted from the OAK's 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 OAK's 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.
Antimicrobial Agents and Chemotherapy 11/2010; 55(2):688-95. · 4.84 Impact Factor
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ABSTRACT: 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 C(12(omega7))K-beta(12)) 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 C(12(omega7))K-beta(12) 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.
The FASEB Journal 06/2010; 24(6):1904-13. · 5.71 Impact Factor
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ABSTRACT: 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 OAK's ability to significantly affect bacterial viability upon single-dose systemic treatment (2 mg/kg).
Chemistry & biology 12/2009; 16(12):1250-8. · 6.52 Impact Factor
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ABSTRACT: 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.
Biophysical Journal 10/2009; 97(8):2250-7. · 3.65 Impact Factor
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ABSTRACT: We investigated the potency, selectivity, and mode of action of the oligo-acyl-lysine (OAK) NC(12)-2 beta(12), 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 NC(12)-2 beta(12) exerted potent activity against gram-positive bacteria while exhibiting negligible hemolysis up to at least 100 times the MIC. Interestingly, NC(12)-2 beta(12) 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 [K(app)], 3.7 x 10(6) 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 NC(12)-2 beta(12) 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.
Antimicrobial Agents and Chemotherapy 07/2009; 53(8):3422-9. · 4.84 Impact Factor
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ABSTRACT: We investigated both the structural and functional consequences of modifying the hydrophobic, lipopeptide-mimetic oligo-acyl-lysine (OAK) N(alpha)-hexadecanoyl-l-lysyl-l-lysyl-aminododecanoyl-l-lysyl-amide (c(16)KKc(12)K) to its unsaturated analog hexadecenoyl-KKc(12)K [c(16(omega7))KKc(12)K]. Despite similar tendencies for self-assembly in solution (critical aggregation concentrations, approximately 10 muM), the analogous OAKs displayed dissimilar antibacterial properties (e.g., bactericidal kinetics taking minutes versus hours). Diverse experimental evidence provided insight into these discrepancies: whereas c(16(omega7))KKc(12)K created wiry interconnected nanofiber networks, c(16)KKc(12)K 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 c(16(omega7))KKc(12)K 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.
Antimicrobial Agents and Chemotherapy 11/2008; 52(12):4308-14. · 4.84 Impact Factor
