Approximately five million deaths in 2019 were associated with antibiotic resistant bacteria according to a 2022 Lancet  report (1). To fight resistance, scientists are seeking new ways to make these superbugs easier to kill. One strategy is to target the bacterial cell membrane, which plays an important role in developing antibiotic resistance due to its direct interaction with drugs. 

In a recent study published in Nature, structural biologist Filippo Mancia and his team at Columbia University analyzed the structure of an enzyme ligase called WaaL that transfers O-antigen sugars to the bacterial lipid core during the formation of lipopolysaccharide (LPS) (2). In gram-negative bacteria, lipopolysaccharides (LPS) are made out of a lipid and sugar chain.   This macromolecule resides in the bacterial membrane and is one of its greatest defenses against antibiotics. 

“The membrane protein is the first thing that a drug sees. It is responsible for how the cell responds to extracellular stimuli,” said Mancia. 

While scientists knew that LPS protects gram-negative bacteria from foreign invaders, how the molecule forms remained a mystery. Understanding the steps that produce LPS could help researchers develop drugs that interrupt this process, making bacterial cells more vulnerable to attack.

To investigate the structure of WaaL, Mancia’s team turned to cryogenic electron microscopy (CryoEM) and antigen-binding fragment (Fab) technology. Unlike methods that measure X-ray diffractions, CryoEM does not require crystallizing the protein, which can be challenging. Instead, they froze the proteins and analyzed them close to their native conditions. Still, “WaaL is a very complex protein and has several transmembrane helices that go through the membrane, which makes it much harder to work with,” said Stephen Trent, a molecular microbiologist at the University of Georgia and coauthor of the study.

To find a stable form of WaaL that would be easier to study, the researchers screened 200 unique bacterial sources. They identified Cupriavidus metallidurans, a gram-negative bacterium that has adapted to survive environments with high concentrations of heavy metals and isolated its WaaL to image with CryoEM. 

By combining their high-resolution CryoEM images with molecular dynamics simulations and biochemical assays, the researchers deciphered the steps leading to sugar transfer. During this enzymatic reaction, the lipid carrier undecaprenyl pyrophosphate (UndPP) carries the O-antigen sugars and forms a complex structure with WaaL. The lipid A olligosaccharide then binds to a separate binding site on the WaaL enzyme where the O-antigen is subsequently transferred from the UndPP. Finally, the UndPP detaches from the mature, fully formed LPS consisting of O-antigen-linked-lipid A oligosaccharide.  

This finding revealed a critical role for WaaL in catalyzing the final step of LPS synthesis and identified the enzyme as a promising drug target for weakening the bacterial cell membrane.

According to Trent, this knowledge has important implications for drug development. The LPS is “like the armor that protects the cell,” he said. “Anything you can do to disrupt that armor makes them more susceptible.” 

On gram-negative bacteria, LPS is critical for resistance to antibiotics that attack their outer structures such as beta-lactams like penicillin. A WaaL inhibitor could prevent a bacterial cell from making the correct LPS structure and allow antibiotics or the immune system to attack it more easily, Trent explained. 

The researchers’ use of CryoEM to investigate the formation of LPS is “very clever,” said Yaoqin Hong, a molecular microbiologist at Queensland University of Technology who was not involved in the study. Hong would be interested to learn if the effects of inhibiting WaaL are reversible. “There are definitely ways to extend from this work to understand how [bacterial cell walls] are regulated,” he said. 

References 

1. Murray C.J.L., Ikuta, K.S., Sharara, F. et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet  390, 629-655 (2022). https://doi.org/10.1016/S0140-6736(21)02724-0
2. Ashraf, K.U., Nygaard, R., Vickery, O.N. et al. Structural basis of lipopolysaccharide maturation by the O-antigen ligase. Nature  604, 371–376 (2022). https://doi.org/10.1038/s41586-022-04555-x