New nanonets target, trap, and kill specific bacteria

The nanonets could be used instead of antibiotics once fully developed.

The microscopic nets consist of antimicrobial peptides (AMPs) that form a mesh when they detect certain chemicals in the bacterial cell membrane. Once the AMPs have attached themselves to the bacteria via these chemical sites, they attract other peptides, self-organizing to project long, interwoven tendrils to surround the bacteria – trapping and killing them in infected mice.

In a major breakthrough, National University of Singapore (NUS) researchers have developed prolongated bacteria busters as a way to treat antibiotic-resistant bacteria. A burgeoning public health crisis, the United Nations recently projected that these superbugs will kill 10 million people by 2050-causing more deaths than cancer and posing an imminent threat to humanity. 

As misuse and overprescribing of antimicrobials are the main drivers in developing drug-resistant pathogens, it stands to reason that using more antibiotics to treat them will be highly detrimental to society.

Therefore, major organizations have prioritized the development of alternative therapies to fight the illnesses caused by these stubborn strains of bacteria that are getting harder to shake.

Professor Rachel Ee from NUS, who co-led the team with Professor Rajamani Lakshminarayanan, says: “Faced with the rapid emergence of antimicrobial resistance globally, there is an urgent demand for innovative strategies to ‘outwit’ the microbes’ ability to evolutionarily adapt through mutations.”

In her team’s whitepaper published in Advanced Functional Materials, a new nanonet platform is described that self-assembles to ensnare and kill specific bacteria in mice without using antimicrobials. The system comprises synthetic AMPs, which cluster to form a sticky web once they come into contact with either lipopolysaccharide or lipoteichoic acid in bacterial cell membranes–an action that also disrupts these structures, killing the microbes.

In their whitepaper, the team state, considering all data, “the findings in this work hold potential to advance the development of synthetic nanonets as anti-infective biomaterials that can help tackle the antibiotic resistance crisis.” Professor Ee added that the next challenge “is to optimize the design for clinical application in humans.”

 Proposed mechanistic pathways of bacteria-induced formation of peptide nanonets. Credit: Advanced Functional Materials (2022).

Nanonet chemistry

In nature, trap-and-kill is a common immune reaction employed in sites such as the small intestine, urinary tract, and blood vessels in response to the presence of a foreign body. Once the immune system recognizes these trespassers, it deploys native peptides, which form nanonets incapacitating the bacteria: causing them to clump together, making it easier for white blood cells to kill them.

In past research, scientists have explored the development of synthetic nanonets to address widespread antibiotic resistance. However, according to Professor Ee, these AMP nanonets failed as they could only form short, disjointed strands unable to encompass or disable bacteria.

To overcome this, the team used ‘click chemistry,’ changing one or two amino acids in the peptide sequences for a preferred function: “By modifying the chemical compositions of the AMPs previously used, our team’s peptides could self-assemble into extensive, cross-linked nanonets, which are more suitable for physically entrapping and immobilising bacteria cells,” says Prof Ee in an interview with The Strait Times.

“In addition, most of the synthetic peptide nanonets that have been developed so far can only trap bacteria, whereas the modifications we made allowed our nanonets to both trap and kill them,” she adds.

The team made these changes to the short peptide sequence by adding a special group of peptides shaped like a loop called a ‘β-hairpin turn’ to the end of the backbone amides. As the β-hairpin isn’t part of the backbone, researchers can rearrange them to recognize and react to certain chemicals in bacteria or stick to other peptides while the backbone remains unreactive and unchanged.

Once the group had tweaked the AMP’s hairpin, they tackled E.coli infections in mice. The bacteria, which causes food poisoning, presents a challenge as it is known to resist even the most potent last-resort antibiotics.

Trap-and-kill E.coli

First, the team established the action of their peptide arrangement against gram-positive and negative bacteria in glassware. They then moved on to toxicity tests in a mouse model of peritonitis – an infection of the abdominal cavity lining that the E.coli had caused. They began slowly, only giving the animals one AMP injection, running tests after 2 hours to ensure the creature’s systems weren’t overloaded.

Blood tests post-injection showed that the AMPs had an almost immediate effect, lowering the bacteria load and peritoneal fluid in the animal’s abdomens. However, the group observed issues with the treatment reaching and treating distant infected organs.

Once the researchers completed low-dosage toxicity tests, they evaluated the efficacy of a multiple-injection regimen over 24 hours, noting that the E. coli-infected mice showed no signs of discomfort or distress during this time. After 24 hours, tests showed negligible changes to the animal’s body weight, with urine tests indicating healthy renal functions and no damage to any other vital murine organs. Overall, the team says their nanonets displayed excellent in vivo efficacy.

Commenting on the promising results, Prof Ee highlighted that even if the bacteria become resistant to the killing functionality of the nanonets, their immobilizing effect alone can already render them vulnerable to the immune system’s antimicrobial defenses: “This means that the bacteria cannot just mutate and alter their proteins to evade detection by the nets, which is one of the ways germs become resistant to antibiotics.”

Regarding the initial limitations of their platform, in their whitepaper, the group writes that further improvement to the formulation is required to achieve greater antimicrobial efficacy at distant organs; however, overall, the findings are promising.

To end, the scientists hope health organizations worldwide will use their nanoscale bacterial trawler to wage war on superbugs. In the future, they plan to reformulate the AMPs into a hydrogel that can be injected directly into the infected part of the body before beginning human trials.

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