Bacteria of the Yersinia genus elicit intestinal wall inflammation and serious diarrhoeal disease as well as other afflictions in humans. This genus includes the plague pathogen. Scientists from the Helmholtz Centre for Infection Research (HZI) and their colleagues from the Ruhr-Universität Bochum and the University of Leipzig recently discovered various molecular switches in Yersinia pseudotuberculosis that change their three-dimensional structures when they become exposed to a temperature of 37 °C. Only then the bacteria can spread in the host and cause disease. The newly identified structures present targets for future medications that block the temperature-dependent change to render the bacteria harmless. The scientists published their results in the renowned professional journal "Proceedings of the National Academy of Sciences (PNAS)".
The role of thermometer is assumed in the bacteria by certain RNA molecules, which are copies of the genetic information stored in the DNA. The bacteria utilise these copies to produce all the proteins they need for survival. At lower temperatures, these special RNA thermometers fold into a complex structure such that their protein production information is not available for reading. At higher temperatures, these structures unfold and the RNA becomes readable again. Various intestinal bacteria, such as the diarrhoeal pathogen Yersinia pseudotuberculosis or the cholera pathogen Vibrio cholerae, use this trick to recognise their host: Once they enter a human body, the ambient temperature quickly rises to 37 °C. This is exactly the temperature at which the RNA thermometers unfold and make their information accessible.
It has been known for a number of years that one of these molecular thermometers in Yersinia regulates a gene that is involved in the pathogenic programme of these bacteria. In collaboration with the research group of Franz Narberhaus of the Ruhr-Universität Bochum, Petra Dersch, who directs the Molecular Infection Biology department at the HZI, and her postdoc Aaron Nuss wanted to find out if there are additional thermometers in Yersinia that have an impact on the infection process. For this purpose, the team grew bacterial cultures at 25 °C and at 37 °C and isolated their entire RNA. Then they cut the RNA with two enzymes. One of these enzymes only cuts folded, double-stranded RNA regions, whereas the other cuts only unfolded single-strand RNA. All RNA molecules generated by this procedure were then sequenced.
"The technique of high throughput sequencing allowed us to sequence the entire RNA of an organism at once and to elucidate the structure of more than 1,750 RNA structures that are present in the bacterial cell," says Aaron Nuss, and doctoral student Francesco Righetti from Bochum adds: "Just a few years ago this would have been a very laborious and tedious effort." Important contributions to the computer-based analysis were also made by the bioinformatics specialists working with Peter Stadler at the University of Leipzig. Based on these results, the researchers were able to identify RNA molecules, in which binding sites for the protein synthesis machinery are hidden in double-stranded regions at 25 °C, which means that they are blocked, but become unfolded and accessible at 37 °C. These molecules act as RNA thermometers that are regulated by the temperature. The recently discovered thermometers control a range of very different functions in the bacteria including 16 functions in infection processes. "This means that we found a whole range of possible targets in Yersinia which might be used to inactivate the bacteria," says Petra Dersch. "One conceivable strategy is to construct an agent that blocks the unfolding of the RNA thermometers." However, the design itself might be the least problem, since the agent would also have to be delivered to the infecting bacteria inside a body and then needs to be taken up by the bacteria - a challenge that has not been solved yet.
The new method developed through the cooperation of the research groups opens many other opportunities since it can be applied universally: "Our method works in all organisms including bacteria, plants, animals and human cells," says Franz Narberhaus. Moreover, the method can be applied not only to the investigation of temperature influences on RNA structure, but also to very different ambient conditions, such as nutrient deficiency or pollution load, Dersch adds.
Original publication:
Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis: Francesco Righetti, Aaron Mischa Nuss, Christian Twittenhoff, Sascha Beele, Kristina Urban, Sebastian Will, Stephan H. Bernhart, Peter F. Stadler, Petra Dersch, and Franz Narberhaus. PNAS, 2016, DOI: 10.1073/pnas.1523004113
