Sugar-based bactericides targeting phosphatidylethanolamine-enriched membranes

Abstract

Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.

Fig. 1

Lead series generated by chemical synthesis. Glycones differ in the deoxygenation pattern (2-deoxy, 4-deoxy, 6-deoxy, 2,6-dideoxy and 4,6-dideoxy), d- and l-configuration, anomeric configuration, and atom linkage to the dodecyl chain (details on synthesis are given in Supplementary Methods)

Fig. 2

Tapping^TM mode AFM imaging of B. cereus deposited on mica surface. a, d Depictions of B. cereus before the exposure of compound 1, whereas b, c, and e show the bacteria in the presence of compound 1. a–c 3D representations of topographic images, whereas d and e (scale bars = 750 nm) are detailed topographic (left panels) and phase-contrast (right panels) images of untreated cells and upon interaction with glycoside 1 (8 μg mL⁻¹), respectively. The arrows indicate lesions in the cellular envelope

Fig. 3

Effect of 1 in Gram-positive bacteria/protoplasts and in Gram-negative bacteria/spheroplasts. Phosphatidylethanolamine (PE)—depicted in gray; phosphatidylglycerol (PG)—depicted in light blue; cardiolipin (CL)—depicted in dark blue

Fig. 4

Effect of compound 1 on the stability of pre-formed membrane pores. a Estimated size of pre-formed membrane pores over time without external surface tension. The five replicates of each system, namely pure DMPC (control) and mixtures containing ~ 20% or 50% of compound 1 are presented. A full pore corresponds to a region with ~ 1800 water molecules between leaflets. The gray region represents very small pore sizes and, in many cases, complete closure of the pore. Pore size was estimated using the number of water molecules in the interior of the transmembrane cavity. The data are shown as a floating window average (2 ns) for clarity. A graphical representation of b a large pore and c a small pore is also shown for a 20% molar ratio mixture. DMPC molecules are shown as thin lines while those of compound 1 are shown as thicker sticks. Water molecules and phosphorus atoms are shown as blue and orange spheres, respectively

Fig. 5

Effect of alkyl glycosides on phospholipid acyl chain ordering. a Deuterium order parameters at the sixth methylene group of DMPC, averaged over both sn-1 and sn-2 chains, as a function of the respective glycoside molar fraction for compounds 1, 6, and 7. b Control deuterium order parameters at the seventh methylene group of DMPC in the presence of octyl β-d-glucopyranoside (OG) or dodecyl β-d-glucopyranoside (DG). Experimental data shown have been determined for multilamellar bilayer dispersions containing DPPC and OG at 45 °C. *For x_OG = 0.75, the bilayer structure distorts significantly by the end of the simulation

Fig. 6

Glycoside-membrane interactions and induction of hexagonal phases. a Turbidity increase upon addition of compounds 1 and 13 to LUVs of POPE at 35 °C. Results are given as a quotient between the absorbance of LUVs in the presence of glycoside minus its intrinsic absorbance (A–B) and the absorbance of LUVs in the presence of ethanol (A₀), thus expressing exclusively the changes in turbidity of LUVs caused by their interaction with glycosides. Results are expressed as mean ± SE (n = 3). b Steady-state fluorescence anisotropy of TMA-DPH in binary mixtures of POPE/Soy:PE 3:1 in the presence or absence of compounds 1 and 13 (50 μM). Each dot represents the mean of three replicates for control and compound 1 and five replicates for compound 13, expressed as mean ± SE

Fig. 7

Membrane permeabilizing activity of 1 and 13 towards POPE vs. POPC membranes. a Representative membrane permeability curves of POPE and POPC LUVs in the absence or presence of compound 1 and 13 at 50 µM. The leakage of carboxyfluorescein from LUVs over time, which is a measure of membrane permeability due to the action of 1 and 13, was obtained through the variation of carboxyfluorescein fluorescence intensity at excitation and emission wavelengths of 495 and 535 nm, respectively, according to Equation 1 (see Methods section). The nonlinear fitting of an exponential law (Equation 2, Methods section) allows to quantitatively describe the maximum extent and kinetics of the process. ​In b and c the curve fitting parameters, maximum membrane leakage (L_max) and c leakage time (τ_L) are shown as average ± SD of three independent experiments (Student’s t-test: *p < 0.05; ***p < 0.001)

Fig. 8

Effect of 1 and 13 on the compression isotherm of POPE or POPC. a Compression isotherms of a POPE monolayer before (light blue line) and after (dark blue line) incubation with compound 13 (0.2 µM). b Compression isotherms of a POPE monolayer before (light blue line) and after (purple line) incubation with compound 1 (0.2 µM). c Compression isotherms of a POPC monolayer before (yellow line) and after (green line) incubation with compound 1 (0.2 µM). Changes in surface pressure (Δπ) of preformed POPE monolayers at π ~ 25 mN m⁻¹ induced by compound 1 (purple line) or 13 (dark blue) are also shown (inset in a). The compounds were present at the concentration 0.1 µM after the first addition (5 min) and at concentration 0.2 µM after the second addition (90 min). The maximum values of Δπ are 2.61 ± 0.25 mN m⁻¹ for compound 1 and 0.15 ± 0.06 mN m⁻¹ for compound 13 (n = 3)