Tackling the toughest infections; researchers reveal the mechanism of action of synthetic antimicrobials

AMR is a growing global problem. The UN has set a target to reduce deaths associated with bacterial antimicrobial resistance by 10% compared to a 2019 baseline, when 4.95 million people globally died due to AMR[1]. Infections caused by Gram-negative bacteria are especially problematic; they possess a second cell membrane which limits the effectiveness of many antimicrobials, allowing them to acquire resistance to multiple drugs.

The team of researchers examined the effects of two synthetic nanoengineered antimicrobial polymers, or SNAPs, on cells of the Gram-negative bacteria Pseudomonas aeruginosa LESB58, which causes lung disease in cystic fibrosis patients. They used neutron reflectometry alongside scanning electron microscopy, transmission electron microscopy, atomic force microscopy and fluorescence assays to investigate the effects of two different SNAP molecules on bacterial cells. They aimed to elucidating their mechanisms of action and understand how differences in molecular architecture, specifically how chemical moieties are arranged along the polymer chain, influence these mechanisms.

Designing new medicines

To tackle AMR, researchers are developing new medicines that are effective against drug-resistant bacteria. SNAPs are one of the promising options for developing new antimicrobials, inspired by short antimicrobial peptides that disrupt bacterial membranes, leading to cell death.

Both of the SNAPs used in the research show antimicrobial activity, although there is some variation in their effects, implying that they rely on different mechanisms. To maximise the effectiveness of SNAPs, it is essential to understand at the molecular level how differences in the chemical structure impact the mechanism of action. The microscopy studies highlighted those differences. When the researchers exposed bacterial cells to the shorter di-block SNAP, consisting of a hydrophobic chain attached to a positively charged chain, they saw more damaged cells and more cells split open and leaking their contents, suggesting their outer membranes were disrupted. The longer tri-block SNAP, consisting of a central positively charged chain flanked on either side by hydrophobic chains, appeared to damage the cell envelope and cause bacterial cell death without causing the cells to burst. The microscopy results suggested it disrupted the membrane by creating nanoscale pores.

The neutron reflectometry experiments, conducted on the OFFSPEC beamline at ISIS, confirmed the findings. By exploiting the unique isotopic sensitivity of neutrons, the researchers obtained precise molecular-level information on how each polymer impacted the membrane structure. Using a model bacterial membrane designed to mimic the outer membrane of the Pseudomonas aeruginosa cells, the researchers found that the shorter di-block SNAP greatly reduced coverage of the membrane from around 88% to around 25%. The membrane consists of a lipid bilayer; the outermost layer containing lipopolysaccharides and the inner layer made of glycerophospholipids. The SNAP almost completely destroyed that asymmetry. In contrast, the longer tri-block SNAP did not greatly reduce membrane coverage but did increase the roughness of the bilayer, the thickness of specific regions, and crucially the amount of water which was able to penetrate through the membrane, indicating that it had formed pores.

The results demonstrate that it is possible to design the next generation of SNAPs to employ different polymer architectures that act through specific mechanisms to target and destroy bacterial cells, including those that are typically more difficult to treat.

The full paper can be found here: https://doi.org/10.1021/acs.biomac.5c01175

[1] World leaders commit to decisive action on antimicrobial resistance