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. 2017 Sep 6;7(1):10697.
doi: 10.1038/s41598-017-10543-3.

Targeting Bacterial Cardiolipin Enriched Microdomains: An Antimicrobial Strategy Used by Amphiphilic Aminoglycoside Antibiotics

Micheline El Khoury  1 , Jitendriya Swain  1 , Guillaume Sautrey  1   2 , Louis Zimmermann  3 , Patrick Van Der Smissen  4 , Jean-Luc Décout  3 , Marie-Paule Mingeot-Leclercq  5
Affiliations

Targeting Bacterial Cardiolipin Enriched Microdomains: An Antimicrobial Strategy Used by Amphiphilic Aminoglycoside Antibiotics

Micheline El Khoury et al. Sci Rep. .

Abstract

Some bacterial proteins involved in cell division and oxidative phosphorylation are tightly bound to cardiolipin. Cardiolipin is a non-bilayer anionic phospholipid found in bacterial inner membrane. It forms lipid microdomains located at the cell poles and division plane. Mechanisms by which microdomains are affected by membrane-acting antibiotics and the impact of these alterations on membrane properties and protein functions remain unclear. In this study, we demonstrated cardiolipin relocation and clustering as a result of exposure to a cardiolipin-acting amphiphilic aminoglycoside antibiotic, the 3',6-dinonyl neamine. Changes in the biophysical properties of the bacterial membrane of P. aeruginosa, including decreased fluidity and increased permeability, were observed. Cardiolipin-interacting proteins and functions regulated by cardiolipin were impacted by the amphiphilic aminoglycoside as we demonstrated an inhibition of respiratory chain and changes in bacterial shape. The latter effect was characterized by the loss of bacterial rod shape through a decrease in length and increase in curvature. It resulted from the effect on MreB, a cardiolipin dependent cytoskeleton protein as well as a direct effect of 3',6-dinonyl neamine on cardiolipin. These results shed light on how targeting cardiolipin microdomains may be of great interest for developing new antibacterial therapies.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
diNn targets P. aeruginosa microdomains of cardiolipin leading to their redistribution. Bacteria were incubated with 5 µM of diNn for 10 min at 37 °C. (a) Fluorescence images of TF-CL in control cells or (b) treated cells; arrows indicate CL microdomains as revealed by enriched areas with TF-CL. (c) Demographs representing axial signal profiles of TF-CL in control P. aeruginosa cells and (d) treated cells. (e) Fluorescence images of P. aeruginosa CL microdomains as revealed by NAO in control and (f) treated cells; arrows indicate CL microdomains. Nucleoids were visualized using 100 µM DAPI. Scale bars in a, b, e, f correspond to 2 µm. (N = 3)
Figure 2
Figure 2
diNn induces modifications of lipid domains shape, enhancement of lipid phase separation and PE and CL segregation. Imaging of GUVs composed of PE/PG/CL in the absence (a) and in the presence (b) of diNn. Top: visualization through epifluorescence microscopy of lipid domain shape using the red TR-PE. Middle: visualization through confocal microscopy of Le/Lc phase separation using TR-PE and NBD-PE (green), respectively. Bottom: visualization through confocal microscopy of PE and CL localization using TR-PE and TF-CL (green), respectively. Arrows indicate a region in control GUVs without CL with a decreased curvature. White scale bars correspond to 10 µm. (N = 3).
Figure 3
Figure 3
diNn interacts with CL through polar and electrostatic interactions. (a) Assembly of diNn with CL (left). The Neamine derivative in mauve is represented in real volume, nitrogen atoms are in blue. Lipids are in a skeleton representation. (b) Calculated energies of the interaction between the diNn molecule and cardiolipin tested experimentally. Ephi corresponds to polar and electrostatic interactions; Epho corresponds to Van der Waals and hydrophobic interactions. Calculated mean interfacial area (Å2) of the lipid monolayer in the presence and in the absence of diNn.
Figure 4
Figure 4
diNn decreases bacterial membrane fluidity/ hydration. Bacterial cells were incubated for 10 mins at 37 °C in the presence of diNn before analysis. (a) General polarization (GP) of Laurdan, (b) TMA-DPH and (c) DPH anisotropy in P. aeruginosa non treated (NT) cells or treated (T) with different concentrations of diNn. Valinomycin was used at 50 µM as a positive control. *p < 0.05 (N = 3, error bar represent SEM). Valinomycin was used at 50 µM as a positive control. *p < 0.05 (N = 3, error bar represent SEM).
Figure 5
Figure 5
diNn induces outer and inner membrane permeabilization in addition to a decrease of bacterial cell length. Time lapse studies were conducted for 5 hours at 37 °C in CaMHB agarose pad supplemented with NPN, PI, and diNn when needed. Fluorescence and wide field images were analyzed in order to evaluate outer and inner membrane permeabilization and length of the cells. An increase in fluorescence indicates membrane permeabilization. Kinetics of the (a) outer and (b) inner bacterial membrane permeabilization in the presence of increasing concentrations of diNn. (c) Slopes of the sigmoidal curves fitting of outer and inner bacterial membrane permeabilization. The rate of permeabilization process is inversely proportional to the slope. According to one way ANOVA’s test and Tukeys comparison test, outer membrane permeabilization at 5 µM was significantly different (p < 0.001) than permeabilization at 1 µM and 3 µM whereas no statistically significant difference was observed in inner membrane permeabilization between the three concentrations (N = 3, values are mean ± SEM). (d) Outer (green) and inner (red) membrane permeabilization and bacterial length (blue) evaluation as a function of time and in the presence of diNn at 5 µM (N = 3).
Figure 6
Figure 6
diNn alters bacterial length and curvature of P. aeruginosa cells in a time dependent manner. Time lapse studies were conducted for 5 hours at 37 °C in CaMHB agarose pad supplemented with the neamine derivative (a,b). Wide field images were analyzed and the (a) length and (b) width distribution of the cells were calculated. P aeruginosa cells were incubated in the presence (T) or absence (NT) of diNn at its MIC; the cells were imaged and analyzed as a function of time (c,d). (c) Distribution of the bacterial cells width, (d) length, and (e) curvature up to 5 hours of incubation; insert shows wide field microscopy images of non-treated (left) or treated (right) bacterial cells after 5 hours of incubation. (N ≥ 3)
Figure 7
Figure 7
diNn induces defects on P. aeruginosa cells morphology. P. aeruginosa were incubated in the presence of diNn at 1 and 5 µM for 1 hour before sample preparation. (a) Scanning electron microscopy of control and (b,c) cells previously treated with diNn at 1 and 5 µM, respectively. Scale bars correspond to 2 µm. Damage in the cell membrane can be seen when compared with the control sample.
Figure 8
Figure 8
diNn inhibits the regeneration of rod-shaped cells from L-spheroplasts. The non-treated (NT) spheroplasts which divided (red arrows) regenerated their rod shape after 3 divisions. In the presence of diNn, spheroplasts lost totally their shape and their division potential after 2 cycles. Representative images of two independent experiments are shown.
Figure 9
Figure 9
diNn inhibits the respiratory chain in P. aeruginosa cells leading a dose-dependently decrease of the bacterial growth rate, CFU count, and cell death. Effect of diNn after 10 min of incubation on (a) CTC redox chain, (b) intracellular ATPi, (c) intracellular pHi, (d) bacterial growth rate, (e) CFU, and (f) dead cells counts. Results in b, d and c are expressed in percentage when compared to the control condition. (N ≥ 3, Values are mean ± SEM). *P < 0.05; in comparison to untreated control cells.
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