A broad-spectrum bactericidal lipopeptide with anti-biofilm properties

Abstract

Previous studies of the oligoacyllysyl (OAK) series acyl-lysyl-lysyl-aminoacyl-lysine-amide, suggested their utility towards generating robust linear lipopeptide-like alternatives to antibiotics, although to date, none exhibited potent broad-spectrum bactericidal activity. To follow up on this premise, we produced a new analog (C₁₄KKc₁₂K) and investigated its properties in various media. Mechanistic studies suggest that C₁₄KKc₁₂K uses a non-specific membrane-disruptive mode of action for rapidly reducing viability of Gram-negative bacteria (GNB) similarly to polymyxin B (PMB), a cyclic lipopeptide used as last resort antibiotic. Indeed, C₁₄KKc₁₂K displayed similar affinity for lipopolysaccharides and induced cell permeabilization associated with rapid massive membrane depolarization. Unlike PMB however, C₁₄KKc₁₂K was also bactericidal to Gram-positive bacteria (GPB) at or near the minimal inhibitory concentration (MIC), as assessed against a multispecies panel of >50 strains, displaying MIC₅₀ at 3 and 6 µM, respectively for GPB and GNB. C₁₄KKc₁₂K retained activity in human saliva, reducing the viability of cultivable oral microflora by >99% within two minutes of exposure, albeit at higher concentrations, which, nonetheless, were similar to the commercial gold standard, chlorhexidine. This equipotent bactericidal activity was also observed in pre-formed biofilms of Streptococcus mutans, a major periodontal pathogen. Such compounds therefore, may be useful for eradication of challenging poly-microbial infections.

Figure 1

Molecular structure and organization in aqueous solution. (a) Structure of C₁₄KKc₁₂K (top), PMB (middle; R = 6-methyloctanoyl) and CHX (bottom). (b) Two fold dilutions of three OAKs were prepared in PBS and light scattering was measured after two hours incubation. Symbols: dotted line, C₁₂KKc₁₂K; solid line, C₁₄KKc₁₂K; dashed line, C₁₆KKC₁₂K. Results are from at least two independent experiments. Error bars represent the standard deviation. (c) Representative light scattering experiments showing the signal evolution immediately after adding 10⁵ CFU/ml (top, S. aureus; bottom, E. coli), using the conditions described in panel (b) (OAK at 200 µM). Symbols: triangles, C₁₂KKc₁₂K; inverted triangles, C₁₄KKc₁₂K. The vast majority of data points standard deviations varied by <2%.

Figure 2

Mode of action investigated against E. coli ML-35p as GNB representative. (a) Bactericidal kinetics upon exposure to C₁₄KKc₁₂K 0, 1, 2 and 4 MIC multiples (squares, circles, triangles and inverted triangles, respectively); dashed line, limit of detection (500 CFU/ml); asterisks denote lack of detected CFUs. (b,c) Membrane permeabilization, expressed as percentage of the positive control dermaseptin S4 (1–15) at 6.25 µM. Symbols: triangles, outer membrane; inverted triangles, cytoplasmic membrane. (c) Cytoplasmic membrane permeation to ethidium bromide. The inset shows representative permeation kinetics by the OAK at the MIC (triangles) and the positive control (inverted triangles); Min, minutes; F.U., fluorescence units (excitation: 535 nm, emission: 590 nm). Results are from at least two independent experiments performed in duplicate. Error bars represent the standard deviation.

Figure 3

Dansyl-polymyxin binding assay. Interaction with LPS was assessed by incubation (1.5 hr) of C₁₄KKc₁₂K and polymyxin B with 2 μM pure monodansyl-polymyxin and 3 μg/ml LPS from E. coli (a) or P. aeruginosa (b). Symbols: triangles, C₁₄KKc₁₂K; inverted triangles, Polymyxin B; The Y axis represents fluorescence measurements (excitation: 340 nm, emission: 485 nm). Results are from two independent experiments performed in duplicate. Error bars represent the standard deviation.

Figure 4

Mechanistic studies (GPB). C₁₄KKc₁₂K mode of action was investigated against S. mutans ATCC 35668 as GPB representative. (a) Bactericidal kinetics upon exposure to 0, 1, 2 and 4 MIC multiples (squares, circles, triangles and inverted triangles, respectively); dashed line, limit of detection (500 CFU/ml); asterisks denote lack of detected CFUs. (b,c) Membrane damages instigated by C₁₄KKc₁₂K, expressed as percentage of the positive control dermaseptin S4(1–15) at 6.25 µM. Membrane depolarization (b) assessed by displacement of DiSC₃(5), and membrane permeation (c) assessed by accumulation of EtBr. The insets in (b and c) show representative depolarization and permeation kinetics, of the OAK (triangles) and the positive control (inverted triangles) at 0.78 and 6.25 µM, respectively; Min, minutes; F.U., fluorescence units (excitation: 620 nm, emission: 680 nm in panel b; excitation: 535 nm, emission: 590 nm in panel c). Results are from at least two independent experiments performed in duplicate. Error bars represent the standard deviation.

Figure 5

Potential oral application of C₁₄KKc₁₂K. (a) Bactericidal kinetics of C₁₄KKc₁₂K against S. mutans ATCC 35668 in the supernatant of centrifuged saliva. Symbols: squares, untreated control; triangles, C₁₄KKc₁₂K at 0.1 mM; inverted triangles, C₁₄KKc₁₂K at 1 mM; dashed line, limit of detection (500 CFU/ml); asterisks denote lack of detected CFUs. (b) Bacterial killing in whole saliva after 2 min treatment. Symbols: white bars, untreated control; gray bars, 0.1 mM, black bars, 1 mM; Con, untreated control; OAK, C₁₄KKc₁₂K; CHX, chlorhexidine. (c) Bactericidal kinetics against S. mutans ATCC 35668 upon exposure to chlorhexidine in BHI growth medium. Symbols: squares, untreated control; circles, 20 MIC; triangles, 40 MIC; inverted triangles, 60 MIC; dashed line, limit of detection (500 CFU/ml); asterisks denote lack of detected CFUs. (d) Bactericidal kinetics against S. mutans ATCC 35668 pre-formed biofilm upon exposure to 0.5 mM of C₁₄KKc₁₂K or chlorhexidine. Symbols: squares, untreated control; triangles, C₁₄KKc₁₂K; inverted triangles, chlorhexidine; dashed line, limit of detection (500 CFU/ml); asterisks denote lack of detected CFUs. Results are from at least two independent experiments performed in duplicate. Error bars represent the standard deviation.