NMR gets the needle: Bacterial target pinpointed

Needle pinned

Getting the needle
Credit: Christian Goosmann
Diane Schad, Rashmi Gupta
and Michael Kolbe

The structure of a "needle" used by various bacteria, among them strains that cause bubonic plague, enterohaemorrhagic colitis, typhoid fever, Shigella dysentery and cholera, to get into host cells has been obtained at atomic resolution using NMR and other techniques. The research could ultimately lead to the development of novel drug to combat these and other diseases.

Although all very different diseases, plague, bacterial dysentery and cholera have one important factor in common, the bacterial pathogen involved exploits a needle-like protein structure as an injection apparatus to preclude detection of their host cell by the body's immune system. Now, researchers at the Max Planck Institute for Biophysical Chemistry in Goettingen, Germany, working with colleagues at the MPI for Infection Biology in Berlin and the University of Washington in Seattle, USA, have obtained an atomic-resolution of the structure that could help in the development of pharmaceutical interventions to block this aspect of the bacterial infection process and so allow the immune system to do its job unimpeded.

The membrane of the cholera bacterium, Vibrio cholera, and that of several other pathogens has hundreds of microscopic hollow needles. These needles coupled with a base embedded in the membrane form the pathogen's type III secretion system. The payload of this system are molecules that interfere with essential metabolic processes in the host cells and thwart the immune response, making infection lethal as the pathogens can spread without hindrance. Inevitably, the pathogens have evolved to cope with conventional antibiotics and there is an urgent need to find novel drugs that work via very different mechanisms to cope with the re-emergence of these diseases particularly in the developing world.

Conventional methods, including the usually very powerful X-ray crystallography, have been unable to generate a model structure for the bacterial needles. The 60-80 nanometre long structures have proved intractable to standard crystallisation techniques and so Adam Lange and Stefan Becker at the MPI for Biophysical Chemistry together with physicist, biologist and chemist colleagues have taken an entirely different tack.

Anti-infective collaboration

The team worked with David Baker at Washington and Michael Kolbe at the MPI for Infection Biology to combine the production of the needle from Salmonella typhimurium T3SS in the laboratory with solid-state NMR spectroscopy, cryo-electron microscopy, and computer modelling. The result was new data that allowed the researchers to tease apart the needles atom by atom to produce a structure in the angstrom range.

"We have made big steps forward concerning sample production as well as solid-state NMR spectroscopy," explains Lange. "Finally, we were also able to use one of the most powerful solid-state NMR spectrometers in Christian Griesinger's [20 Tesla, 850 MHz] NMR-based Structural Biology Department at our Institute."

Lange adds that the team was surprised to see how the needles are constructed. As expected, the needles from the different pathogens are strikingly similar but only on the inside. The exterior of the needles from each bacterium are astonishingly variable, the team says. This, the researchers suggest, is probably down to bacterial evolution that has allowed the pathogens to avoid immune recognition. Changes on the surface of the needle make it difficult for the host's immune system to recognize the pathogen.

With MPI colleagues Christian Griesinger and Arturo Zychlinsky, the team has focused on bacterial syringes for many years. In 2010, working with the Federal Institute for Materials Research and Testing they demonstrated how the microscopic syringes are assembled. The new structure determination adds to the growing body of knowledge surrounding these pathogens and offers hope of finding a way to block needle assembly at an early stage using novel anti-infective agents, precluding the need for antibiotics, which are usually given once infection has taken place and symptoms are apparent. "Thanks to our new technique, we can produce large amounts of needles in the lab. Our aim is now to develop a high-throughput method. This will allow us to search for new agents that prevent the formation of the needle," explains Becker.

"In contrast to established models of the needle in which the amino terminus of the protein subunit was assumed to be a-helical and positioned inside the needle, our model reveals an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen," the team concludes.

"Atomic Model of the Type III Secretion System Needle", Nature, 2012; DOI: 10.1038/nature11079