Bacteria under fire
RUB researchers in search of new antibiotics
by Tabea Steinhauer
February 3, 2014
In nature, antibiotic agents are produced for example by fungi and bacteria. In addition, certain antibiotics are now also manufactured synthetically. Humans use them to prevent or to cure infections by destroying harmful bacteria. However, bacteria are highly adaptable and quickly develop defence mechanisms against drugs. “It is possible that, in ten years’ time, none of the currently approved antibiotics will be effective, because bacteria will have become resistant against them,” says Julia Bandow. Despite these bleak prognoses, pharmaceutical companies have reduced their efforts in antibiotic research to a large extent, one of the reasons being that the profits are relatively small due to comparably short treatment times.
Consequently, other research institutes are called upon to do their bit. Julia Bandow dedicated herself to antibiotic research as a PhD student and is continuing this quest today. Together with five partners, she launched the project “Innovative Antibiotics from NRW” (InA) in 2009. Bandow’s team looks for yet undiscovered naturally occurring antibacterial agents that have not yet been exploited in pharmaceutical products. Due to the elusive character of such agents, the researchers at the same time attempt to manufacture antibiotics in the lab. An advantage of synthetic drugs may be that bacteria develop a resistance against them more slowly, because they have not been exposed to these agents before.
Proteome analysis: the microbiologists study the cellular protein composition and synthesis rates with 2D gels. First, they treat bacteria with an antibiotic and then they feed the bacteria radioactive precursors of proteins; these precursors are incorporated into all proteins newly synthesised by the bacteria. Subsequently, the researchers isolate the proteins and separate them, among other factors, according to size. Each dot in the autoradiograph above represents one particular protein species; the larger the dot, the more of a particular protein is newly synthesised. Green: proteins that bacteria generate without antibiotic treatment. Yellow: proteins that bacteria generate with and without antibiotic treatment. Red: proteins that bacteria generate only after antibiotic treatment; pictured here: proteome profile of Bacillus subtilis after treatment with cerulenin, an antibiotic that inhibits fatty acid synthesis. The proteins generated at higher rates after treatment (red, labelled) are crucial for the synthesis of the fatty acids of the cell membrane. © Adapted with permission from ACS Chemical Biology „Wenzel, Patra, Senges, Ott, Stepanek, Pinto, Prochnow, Vuong, Langklotz, Metzler-Nolte, Bandow: Analysis of the Mechanism of Action of Potent Antibacterial Hetero-tri-organometallic Compounds: A Structurally New Class of Antibiotics”. Copyright (2013) American Chemical Society
Certain synthetic substances that are studied by the researchers with regard to their antibiotic properties have been supplied by the RUB Department for Inorganic Chemistry I, headed by Prof Dr Nils Metzler-Nolte. The chemists also test them for cytotoxicity – investigating whether they also affect human cells, in addition to bacteria. If the compounds turn out to be non-toxic for human cells, the antibiotic agents are sent back to the microbiologists. “We specialise in finding out how these chemical substances prevent bacterial growth,” explains Bandow. For an antibiotic to be approved as a drug, it is necessary to understand the mechanism by which the agent interferes with the vital processes of a bacterium. In order to decode the mechanisms of action, Bandow’s team compares the protein make-up of untreated bacteria with the protein make-up of bacteria that have been exposed to the antibiotic substance (fig. 1). Antibiotics interfere with certain cellular processes, for example inhibiting enzymes important for the synthesis of the cell membrane. Bacteria attempt to counteract this blockade by producing more of the membrane-producing proteins. When, following antibiotic treatment, researchers observe an increase in production of proteins belonging to a particular biosynthetic pathway, these changes typically indicate that the tested antibiotic interferes with that particular process.
The InA Consortium has tested over 2,500 substances. The microbiologists found that 60 show promising antibiotic activity, 25 of which were not toxic to human cells. Julia Bandow’s team is now investigating the mechanisms of action of these potential antibiotics. One of the most promising substances contains three residues that are made up of a hydrocarbon fraction and a metal atom. These so-called organometallic residues are connected via a peptide nucleic acid (PNA) backbone; the PNA backbone is a molecule similar to DNA with peptide bonds (fig. 2). As it does not occur in this form in nature, bacteria find it difficult to degrade it; the researchers hope this might delay the development of resistance.
Structure of a potential new antibiotic. The PNA backbone (red) is bonded with three organometallic residues which contain an iron complex (Fe), a manganese complex (Mn) resp. a rhenium complex (Re). The negatively charged ion hexafluorophosphate (PF6-) is the counterion of the positively charged PNA molecule. © RUBIN
The organometallic PNA molecule attacks bacteria via several routes at the same time (fig. 3). “The PNA targets the bacterial membrane. Thus, it interferes with the diffusion barrier separating the cell’s interior from its exterior,” says Bandow. The cell’s energy supply breaks down. In addition, the PNA inhibits the synthesis of the cell wall, i.e. the layer of the bacterium that encloses the cell and its membrane. “Thus, the stability of the cell is compromised,” says the biologist.
Deciphering the mode of action: the microbiologists subjected the PNA substance with an iron complex (FcPNA) and without an iron complex (RcPNA) to several tests (a to d) and compared it with the antibiotic nisin that is utilised as a preservative in food products. The control cells were not treated. a) The green fluorescent protein MinD is typically localised at the cell poles (control). Following the antibiotic treatment, the usual ion distribution at the cell membrane (proton gradient) breaks down. As a result, MinD spreads across the cell. b) Among the substances tested, nisin is the only one to cause holes in the cell membrane, which allow the red fluorescent dye to enter the cells. The cells treated with PNA are not penetrated by the dye, because the membrane remains intact. c) Light-microscopic images of cells fixed with methanol/acetic acid. All antibiotics tested here damage the cell wall, causing the membrane to extrude through the holes in the cell wall. d) The PNA with the iron complex triggers oxidative stress in bacterial cells indicated by another red fluorescent dye. © Adapted with permission from ACS Chemical Biology „Wenzel, Patra, Senges, Ott, Stepanek, Pinto, Prochnow, Vuong, Langklotz, Metzler-Nolte, Bandow: Analysis of the Mechanism of Action of Potent Antibacterial Hetero-tri-organometallic Compounds: A Structurally New Class of Antibiotics”. Copyright (2013) American Chemical Society
In order to boost the antibiotic’s efficacy, the researchers inserted a ferrous organometallic residue into the PNA molecule. The iron complex results in the generation of reactive oxygen species that damage the bacterial DNA and proteins; a condition referred to as oxidative stress. Even without this additional component, the PNA may destroy bacteria. However, the iron-containing complex enhances the antibiotic by adding an additional mechanism of action. As a result, it becomes even more difficult for bacteria to become resistant. “It is less likely that a cell can defend itself against several lines of attack than if you shoot with just one bullet,” explains Bandow.
Bandow’s project partners are currently studying four substances in animal experiments. If the potential drugs pass this test successfully, they will be tested in clinical studies prior to being released to the market. In addition, the RUB chemists are currently developing other new substances. “We want to identify the mechanisms of action of at least two more compounds,” says Bandow. As the project’s funding period draws to an end, the scientists are currently looking for follow-up funding. If the InA project research activities are to continue on the promising path, a long search might eventually result in the discovery of a new antibiotic.
DEVELOPMENT OF RESISTANCE
Bacteria reproduce very quickly and are very good at adapting to their environment. Through mutations, they coincidentally develop mechanisms that disrupt or inhibit the efficiency of antibiotics. For example, they may identify the drugs as toxic substances and pump them out of the cell. In accordance with the principle of evolutionary selection, in the presence of the antibiotic those mutated cells have the best chances of survival and may be the only ones to reproduce. In the course of this process, the bacteria gradually build up resistance against multiple antibiotics. Alexander Fleming who discovered penicillin already warned as early as at the beginning of the 20th century that antibiotic drugs will lose their effectiveness if misused or overused.
ANTIBIOTIC RESEARCH IN NORTH RHINE-WESTPHALIA
The project “Innovative Antibiotics from NRW” (InA) is financed as part of the “BIO.NRW.red” programme that the state government of North Rhine-Westphalia launched in the biotech sector. With this project, North Rhine-Westphalia has cemented its position as an important centre for antibiotic research. The consortium consists of six partners: participants from the Ruhr-Universität are the junior research group Microbial Antibiotic Research, headed by Junior Professor Dr Julia Bandow, and the Department of Inorganic Chemistry I, headed by Prof Dr Nils Metzler-Nolte. Further partners are Prof Dr Heike Brötz-Oesterhelt from the Heinrich-Heine-Universität Düsseldorf, Prof Dr Hans-Georg Sahl from the Friedrich-Wilhelms-Universität Bonn as well as the two enterprises Squarix biotechnology in Marl and AiCuris in Wuppertal, the latter a spin-off of the Bayer Pharma AG.
Further information: www.rub.de/ina