Far reaching changes to invasion protein

Pathogens and the organisms they infect are engaged in a constant evolutionary contest: The hosts build up cellular and molecular defence lines, while pathogens continuously refine their invasion methods. To bind precisely to the corresponding structures on their host cells, pathogens often carry distinct modules on their surface. Changes to those docking modules enable them to infect new host species. This can lead to disastrous consequences when animal pathogens become human pathogens: The Plague, Spanish Influenza, and AIDS developed like this. Today, the whole world closely monitors new pathogens when they arise for signs of cross-species contamination potential.

Structural biologists, headed by Prof. Dirk Heinz at the Helmholtz Centre for Infection Research in Braunschweig are studying the molecular interaction between the docking proteins of pathogens and their hosts. For the first time, they succeeded in emulating the change process of a pathogen that normally takes place spontaneously in nature, and thus changing the host spectrum of the pathogen.“ We wanted to produce lab mice which can be infected with Listeria monocytogenes, the bacterium responsible for the disease listeriosis. Usually, mice are immune to this disease,” says Dr. Wolf-Dieter Schubert, head of the research group „Molecular Host-Pathogen Interactions”. “Because we use mice for research, this will be necessary to develop and test new drugs against listeriosis.”

The pathogen enters the human body via contaminated food and leads to severe – often fatal – illnesses in immunocompromised people. With the help of its invasion protein internalin A, the pathogen binds the protein E-cadherin on the surface of human intestinal wall. This binding initiates the invasion of the bacterium. In mice, this process does not work since internalin A cannot bind to murine E-cadherin. The structural biologists from Braunschweig wanted to change that by altering the binding region of internalin A in such a way that it can also bind the mouse receptor protein.

Their studies took them into the microscopic world of molecules and atoms, where measurements are made in Ångstrom – one ten millionth of a millimetre. This high resolution is necessary to determine the spatial arrangement of atoms of the amino acids, i.e. those building blocks that provide a protein with both structure and function. The question was: Which amino acids are necessary for the binding of the docking modules and which block this interaction? Powerful particle accelerators found at the DESY or BESSY synchrotrons in the Helmholtz Association and capable of a targeted high performance X-ray structural analysis can give the necessary answers.

At first, Heinz and his co-workers analyzed the crystal structures of the single internalin A molecule and of its complex bound to human E-cadherin. These analyses gave a clear picture of the interaction between the binding modules. “Consequently, we could extrapolate changes to optimise the binding – without having to revert to using computer simulations or randomly generated mutants,” says Schubert. A substitution of only two out of more than 500 amino acids in the docking module of the internalin should be sufficient.

In the next step, the researchers generated an internalin A variant in which those two amino acids have been replaced by others. This modified protein now binds to the murine E-cadherin almost as well as the native protein binds to the human E-cadherin. Finally, in the key biological experiment, the researchers applied the genetically changed Listeria bacteria carrying the modified internalin A on their surface to mice. Remarkably, the pathogen now was able to infect the epithelial cells of the small bowel of the mouse. It is the assumption that they would spread similarly from there, as they do in humans. Currently, Listeria with modified internalin are used for the development of new therapies against listeriosis. The structural biologists from Braunschweig, together with cooperation partners, are following new approaches that have arisen from their work on Listeria.

Their findings can also lead to the development of so-called bacterial ferries: targeted stimulation of the immune system within an organism using antigens – effectively ‘vaccination with a pill’. “In Listeria, small changes in a single invasion protein were sufficient to broaden the potential host spectrum,” says Heinz. “Such small changes like this also take place in nature and can lead to dangerous zoonoses such as SARS or swine flu.”