Project

Discipline

silver; silver efflux pump; biomineralization; silver resistance; Antimicrobial activity; peptides; electron transfer

Lead
Silber - und wie es Bakterien tötet
Lay summary
Von Silber weiss man schon seit Jahrtausenden, dass es gesundheitsförderlich ist - allein schon, dass man eine Silbermünze in einen Brunnen wirft, um sich Glück und Gesundheit zu wünschen. Heute weiss man, dass Silber antimikrobiell wirkt und Bakterien abtöten kann - man weiss aber nicht genau, wie das auf molekularer Ebene passiert. Zudem gibt es auch Silber-resistente Bakterien, die gelernt haben, mit Silber in der Umwelt umzugehen. Wir möchten in diesem Projekt neue Silber-Verbindungen herstellen, die auch gegen resistente Bakterien wirken. Zu diesem Zweck untersuchen wir auch die Resistenzmechanismen von Bakterien gegenüber Silber. Ein solcher Mechanismus findet sich in machen Bakterien, die gezielt Silberionen aus ihrem Inneren nach aussen pumpen können. Man nennt dies eine sogenannte Silber-Efflux-Pumpe. Diese besteht aus verschiedenen Molekülen, die man zwar identifiziert hat, von denen man aber die Rolle nicht genau kennt. Diese Rolle wollen wir genau untersuchen, denn wenn wir verstehen, wie genau die Pumpe funktioniert, kann man die Pumpe auch "austricksen". Im ersten Teil wollen wir also den Mechanismus der Pumpe genau verstehen und womöglich auch beeinflussen lernen. Ein zweiter Resistenzmechanismus liegt darin, dass manche Bakterien Silberionen ausserhalb ihrer Zelle "unlöslich" machen, indem sie die Silberionen zu Silber-Nanopartikeln umwandelt. Dazu müssen Elektronen aus der Bakterienzelle nach aussen transportiert und auf die Silberionen übertragen werden. Wie genau das passiert, ist nicht bekannt und nur sehr schwierig zu untersuchen. Wir haben hier eine Methode entwickelt, wie man diesen Elektronentransport verfolgen kann. Wenn wir verstehen, wie dieser Transport funktioniert, kann man ihn auch beeinflussen. Im Idealfall lernen wir also, wie Bakterien mit dem Silber umgehen, wie es wirkt und wie man die resistenten Bakterien dennoch abtöten kann.

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Last update: 04.04.2018

Lead
Silver - knowing how it kills bacteria
Lay summary
Silver is long known for having beneficial effects on health. Just imagine the tradition of throwing a silver coin into a well, wishing good health. Today, we know that silver can be antimicrobial while being non-toxic for humans, but the details of how silver acts is not known to the molecular level. We want to learn more about the exact mechanism of action of silver in order to better design silver-containing drugs, and to combat even resistant bacteria. Bacterial resistance against silver can occur for example by pumping the silver out of the bacterial cell, using what is called a silver efflux pump. We study this pump to learn in detail its functioning. Knowing itsfunction means that we will also be able to block this efflux pump andhence trick out one kind of silver resistance mechanism. So, we will investigate how the molecules that build the efflux pump can bind silver ions, and how these are then transported out of the bacterial cell. If we understand how this works, we can develop for instance drugs that are able to block the silver pump, so that the silver ions cannot be transported out of the cell. If they stay inside the bacteria, they will end up killing the bacterium. A second mechanism of bacterial resistance is the precipitation of silver outside of the bacterial cell. Soluble silver ions are thus transformed into less soluble metallic silver particles. Not much is known on how bacteria can transform silver ions to silver nanoparticles. This means that bacteria need to produce electrons, which are dumped onto the silver ions to obtain silver atoms. These electrons are produced inside of the bacterial cell and have to be transported through the bacterial cell membrane to the outside, where the metal ions "attack". This transport process is difficult to observe, but if we can learn how this occurs, we will be able to inhibit this mechanism. We have thus developed a method on how to "see" bacteria producing and transporting electrons. Our observations will help us to combat the resistance mechanism. Ideally, we will understand these mechanisms by which bacteria deal with silver ions and develop compounds that allow to kill resistant bacteria.

Direct link to Lay Summary

Last update: 04.04.2018

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Silver is known for its antimicrobial properties since ancient times and was used in medical applications until the 1940s, when antibiotics were discovered. Forgotten until the upraise of antibiotic-resistant bacteria, it has since seen a revival in research. With the use of silver e.g. in wound dressings, silver-resistant bacteria emerged. Resistance may occur e.g. via expression of an efflux pump, while other bacteria are able to reduce ionic silver to silver nanoparticles (AgNPs).This research project copes in part A with the design of improved silver-based antimicrobial coordination compounds by combining the action of silver with another antibacterial metal ion, e.g. Cu2+ and Zn2+. To achieve this goal, we will use biocompatible ligands that possess two different binding sites. Dual action compounds are expected to be more efficient, also against Ag-resistant bacteria. Compounds will be characterized in solution and the solid state by NMR titration studies, single crystal structure analysis, UV-Vis, IR, elemental analysis and tested for antimicrobial and cytotoxic properties. To follow the compounds in cells, newly developed luminescent ligands allow monitoring via confocal fluorescence microscopy.It is at the same time important to understand the silver-resistance mechanisms of bacteria. In part B, we will continue to investigate the silver efflux pump Sil of some Gram-negative bacteria, in particular the unique periplasmic SilE protein whose role is still not fully understood (Ag+ binding with SilE and how Ag+ is exported). Another mechanism by which bacteria can reduce the toxicity of silver ions is by generating AgNPs outside of the cell (biomineralization), either at the membrane using cytochromes, or at the pili via electron transport through proteins (part C). For both the efflux pump and the biomineralization processes, we will start with model peptides to approach the full protein bottom-up. We have shown this technique successfully in a first study on short peptide excerpts from SilE, using NMR-titrations and competition fluorescence titrations for the determination of the Ag+ binding constants. Larger model peptides will lead us to a final understanding of the proteins themselves, hence of the mechanisms. Studies of the biomineralization at different concentrations will help to establish the electron transfer rates through the membrane to AgNPs. Understanding the antimicrobial and the silver resistance mechanisms is important for the future development of active compounds.