Neutrons help reveal details of the binding of a human pathogen to our cells
Pseudomonas aeruginosa is a human opportunistic pathogen that causes severe infections in immunocompromised patients. P. aeruginosa and other pathogenic bacteria use several virulence factors to promote their infectivity, including sugar-binding proteins.
Teams of researchers at CERMAV (CNRS) and the Institut Laue-Langevin in Grenoble have used neutron protein crystallography and the production of deuterated protein and sugar to study how Pseudomonas aeruginosa binds to host cells. The results of the study have been published in Nature Communications.
Antibiotic resistance is a rapidly increasing global threat to human health. The World Health Organization has identified P. aeruginosa as a critical target among the antibiotic-resistant bacteria against which efficient new drugs need to be developed. Researchers are now working on new compounds to fight multi-drug resistant bacterial infections, targeting strategies which do not directly kill the microbes. Anti-adhesive therapies are one such strategy, as they work by preventing microbes from adhering to human cells, which is one of the mechanisms reinforcing drug resistance.
Essentially all human cells are covered with carbohydrate molecules (sugars) bound to the proteins and lipids embedded in cell membranes. These carbohydrates are involved in the communication between the cells and their environment. They often act as binding targets for lectins, which are proteins capable of recognising and binding to sugars via highly coordinated networks of hydrogen bonds.
Amongst its arsenal of virulence factors Pseudomonas aeruginosa produces several sugar-binding proteins. One of these is LecB, involved in biofilm stabilization and lung colonization. It is present on the outer membrane of the bacteria and binds fucose, a small sugar present on the host-cell surface, in a calcium-dependent manner. The interaction between LecB and fucose is unusually strong; this study was conducted to provide the structural details required to better understand the interaction and the role of the two closely located calcium ions.
Neutron crystallography was used to locate the hydrogen atoms playing a crucial role in the binding. Neutrons are ideal for this as they reveal the location of the hydrogen atoms directly without causing radiation damage to the sample; the data can be collected at ambient temperature, close to physiological conditions. To gain as much information as possible from the neutron diffraction experiments, both the protein and the sugar were produced in deuterated forms with all hydrogene atoms replaced by deuterium atoms.
The room temperature neutron structure of the complex was collected on the LADI-III beamline at ILL. This provided an outstanding level of detail which would not have been possible using X-ray crystallography alone. A complete hydrogen-bond network could be observed between the protein and its ligand. A low-barrier hydrogen bond was also identified ‑ a special type of strong hydrogen bond which plays a role in the unusually high affinity of LecB with fucose. LecB has become a target for medical research as great efforts are being made to develop LecB inhibitors to block the binding process and prevent biofilm formation. “Glycomimetic” compounds, for example, are part of this strategy, but their development requires more detailed structural data. The results described here show that neutrons have an important role to play in this field.
Re.: “Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding", by Lukas Gajdos et al. , Nature Communications (2022).
The article can be accessed at https://doi.org/10.1038/s41467-021-27871-8.
ILL instrument : Quasi-Laue diffractometer LADI-III