Multifaceted Activity of Fabimycin: Insights from Molecular Dynamics Studies on Bacterial Membrane Models

Membranes—cells’ essential scaffolds—are valid molecular targets for substances with an antimicrobial effect. While certain substances, such as octenidine, have been developed to target membranes for antimicrobial purposes, the recently reported molecule, fabimycin (F2B)—a novel agent targeting drug-resistant Gram-negative bacteria—has not received adequate attention regarding its activity on membranes in the literature. The following study aims to investigate the effects of F2B on different bacterial membrane models, including simple planar bilayers and more complex bilayer systems that mimic the Escherichia coli shell equipped with double inner and outer bilayers. Our results show that F2B exhibited more pronounced interactions with bacterial membrane systems compared to the control PC system. Furthermore, we observed significant changes in local membrane property homeostasis in both the inner and outer membrane models, specifically in the case of lateral diffusion, membrane thickness, and membrane resilience (compressibility, tilt). Finally, our results showed that the effect of F2B differed in a complex system and a single membrane system. Our study provides new insights into the multifaceted activity of F2B, demonstrating its potential to disrupt bacterial membrane homeostasis, indicating that its activity extends the currently known mechanism of FabI enzyme inhibition. This disruption, coupled with the ability of F2B to penetrate the outer membrane layers, sheds new light on the behavior of this antimicrobial molecule. This highlights the importance of the interaction with the membrane, crucial in combating bacterial infections, particularly those caused by drug-resistant strains.


Systems Characteristics
In our study, we constructed various lipid membrane systems with CHARMM GUI, [1][2][3][4] each tailored for specific membrane reflection.The systems included a phosphatidylcholine (PC) bilayer, denoted as a control system and bacterial mimicking such as single inner membrane (sIM), asymmetric single outer membrane (sOM), symmetric outer membrane (LPS) and a complex hybrid dual membrane system with inner and outer membrane included (complex).The sIM system was created using a combination of phosphatidylethanolamine (PYPE 16:0 / 16:1), phosphatidylglycerol (PYPG 16:0 / 16:1), and cardiolipin (PVCL2 16:0,16:0 / 18:1,18:1) lipids. 5sOM was made up of PYPE, PYPG and the E. coli lipopolysaccharide (LPS) composed with LipidA type1, R1 core, and 2 repeating units of the O6-antigen. 6The length of the O6-repeating units was adapted based on the results published by Wu et.al 7,8 and Rzycki et.al . 6The number of LPS molecules and phospholipids was adjusted equally to the total lipid area occupied in each leaflet.Ca 2+ ions were automatically added based on the length of the LPS to neutralize the system.LPS system was created with equally distributed E. coli LPS molecules composed of LipidA type1 and R1 core.

S2
The dual complex system was constructed from the sOM and sIM systems separated by approx.4nm water slab mimicking the periplasm.Some of the systems were enlarged (>200 lipids) to verify whether membrane parameters may depend on the size of the system and the concentration of fabimycin (F2B) (see Figure S1).In all listed systems, F2B molecules were placed in the water phase 15-20 Å from the surface of the bilayer.The detailed specifications of the bilayer systems along with their production time are presented in Table S1.In the snapshots, the charged amine fragment of F2B is highlighted in red.Orange beads indicate the position of phosphorus, while blue beads represent the last carbon atoms in the acyl chain (C16).Due to the dense structure of the LPS in sOM and cOM leaflets, the limited interaction of F2B with lipidA occurs, thus the distribution angle covers the entire orientation range.

Individual parameters of membrane systems
In main section the parameters are represented as change observed due to incorporation of F2B.In this section absolute values of determined parameters are presented.Specifically, membrane thickness and area per lipid are presented in Figure S6; interdigitation and lateral diffusion coefficient are presented in Figure S7; area compressibility and tilt coefficient are presented in Figure S8, membrane curvature are presented in Figure S11; acyl chain order parameter are presented in Figure S10 and S12.Finally, both apparent bending rigidity and bending rigidity are presented in Figure S9.This distinction is caused by the fact that bending rigidity determined using real-space fluctuation method for smaller systems (≤ 200 lipids) returns underestimated values.It is still possible to investigate the change on this parameter, however the absolute values are not related to the experimental ones.To this end term 'bending rigidty' was only used in the case of larger systems.

Figure S5 :
Figure S4: Location of several F2B molecules in the last 100ns of the simulation.The membrane core including the phosphorus atoms have been marked in light blue, while the remaining F2B molecules are marked in the corresponding other colors A) sIM, B) PC, C) sOM, D) Complex, E) LPS systems.

2 ]Figure S6 :Figure S7 :Figure S8 :Figure S9 :
FigureS6: Absolute values of (A) membrane thickness and (B) area per lipid of investigated membrane systems.Since values for outer membranes are presented as average of both leaflets, an inset is added.In the inset distinction between the leaflets is presented.

Figure S10 :Figure S11 :
Figure S10: Order parameters |S cd | for saturated sn − 1 (top) and unsaturated sn − 2 (bottom) acyl chains of sIM (A,D panels), PC (B,E panels), cIM (C,F panels) systems.Low carbon atom numbers correspond to those close to the headgroup.

Figure S12 :
Figure S12: Order parameters |S cd | for subsequent lipidA acyl chains of sOM (A), LPS (B), cOM (C) systems.Low carbon atom numbers correspond to those close to the headgroup.

Table S1 :
Characteristics of simulated membrane systems