19-Aug-2022
The continuous rise in drug-resistant bacteria is a significant concern for global health, made even more critical as few new antibiotics have been added to the market in recent years. That is, until teixobactin was discovered as a potential new antibiotic, which displayed broad activity against several drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA).
This study, from Shukla et al. (2022), aimed to understand the mechanism by which teixobactin attacked bacterial cells, in order to get a clearer picture of the potential role it could play as a clinical antibiotic, with the key technology being solid-state NMR (ssNMR)
Markus Weingarth, Associate Professor at Utrecht University, said, “In 2019, my colleague Prof. Moreno Lelli from CERM/Florence, and I obtained an Instruct-ERIC Research award (JRA award) that was endowed with €20,000. Our project was entitled 'Solid-State NMR to monitor native drug - receptor interactions in cellular surfaces'. This was a project that I incited because we had a major bottleneck in our understanding of the mode of action of teixobactin.”
It was through the use of ssNMR and several other techniques, that Markus and the team investigated the binding mechanism of teixobactin to the bacterial membrane and discovered how it displays such potent antibiotic efficiency. These techniques included HS-AFM, confocal microscopy and molecular dynamic simulations.
Markus: “Teixobactin, using backbone amino-protons, targets the pyrophosphate group of the cell-wall precursor molecules Lipid II. But it was not known how exactly this worked. To understand this precisely, it was necessary to determine proton - phosphorous distances with solid-state NMR.”
The C-terminus of teixobactin contains the amino acid, enduracididine (End10). The team found that this bound to lipid II in the sugar moiety (specifically the MurNAc-PPi site). This is also then anchored by the N-terminus of teixobactin.
Lipid II is a peptidoglycan precursor, and is essential for bacterial cell wall and membrane function and synthesis. It is not found in eukaryotic cells. The use of ssNMR at CERM, Florence, through Instruct funding, helped the team to make the discovery. The binding of End10 to MurNAc-PPi of lipid II is shown in Figure 1.
Figure 1. The backbone amino-protons of teixobactin, the End10 sidechain and the N terminus of an adjacent teixobactin coordinate the lipid II PPi group. In addition, End10 interacts with the MurNAc sugar via hydrogen bonds. Blue spheres represent backbone nitrogens; numbers indicate the residue numbers.
Markus: “Measuring H-P distances in solids required a special piece of hardware (a 31P-capable ssNMR probehead that enables fast sample spinning up to 60 kHz MAS) that we did not have in Utrecht, but that was available in Florence. With the JRA award, we could kick off our collaboration, and these H-P distances were a key contribution to precisely resolve the teixobactin - Lipid II interface.”
The result was that as teixobactin bound to lipid II, it formed β-sheets. This created an irreversible supramolecular complex as more teixobactin molecules bound, forming more β-sheets. This complex inhibited the function of peptidoglycan (limiting cell membrane synthesis), but more importantly thinned the membrane itself through hydrophobic interactions within the complex, allowing ion leaking and a drop in membrane potential.
This explains the two-pronged method by which teixobactin attacks the cell envelope, but it has several other properties that make it such an exciting antibiotic prospect.
Lipid II is made of two components: the sugar moiety (bound to by enduracididine in teixobactin), and a pentapeptide (bound to by other antibiotics such as vancomycin, and ignored by teixobactin). The pentapeptide is liable to variation and, therefore, drug-resistance. By contrast, the sugar component (specifically MurNAc-PPi) is invariable, and present in several cell wall precursors (lipid I, lipid II, and lipid III).
Furthermore, as mentioned, lipid II is not found in eukaryote cells. The supramolecular structure (which destabilises the membrane) formed by teixobactin in contact with the cell can only take place by binding to lipid II. As eukaryote (human) cells do not express lipid II, they will remain unaffected by teixobactin, providing another “elegant” feature that makes it such an effective antibiotic.
This ground-breaking study should provide the foundation for further teixobactin development, and hopefully will lead to the generation of a novel, non-resistant antibiotic – something which is sorely needed in the medicinal world. The access to ssNMR at CERM through Instruct allowed the team to carry out their research into this potentially crucial antibiotic.
