A new technique of antibiotic efficiency testing developed

Scientists from the Lomonosov Moscow State University, in cooperation with colleagues, have developed a safe, inexpensive and highly efficient method to research new germicides. They have elaborated a system that measures antimicrobial activity, and determines the mode of action of new materials. The research results are published in Antimicrobial Agents and Chemotherapy. The research has been conducted in collaboration with colleagues from the Moscow Institute of Physics and Technology, the Skolkovo Institute of Science and Technology and Gause Institute of New Antibiotics.

An antibiotic dilemma

Bacteria constantly become resistant to antibiotics, compelling scientists to search for new ones in recent decades. The main research channel is high-throughput screening—namely, testing of a huge number of substances from chemical libraries, as well as new natural compounds. However, such experiments don't provide any data regarding the mode of action. Additionally, scientists seek ways of decreasing reagent costs, process automation and the reduction of steps in the screening process.

Scientists face a dilemma: whether to test many materials simultaneously according to one characteristic, or test many characteristics of one substance in order to understand its impact. Russian scientists have now made an attempt to combine the advantages of these two approaches.

Dr. Ilya Osterman, a researcher of the Chemistry of Natural Compounds Department at the Faculty of Chemistry of the Lomonosov Moscow State University, who is the research author, says, "We've worked out an approach that allows in vivo determination not only of the mode of action of new promising antimicrobial agents, but also their efficiency. Approach automation allows us to analyze thousands of compounds per day."

The resulting method is called a reporter system, a term, used in molecular biology to describe reporter gene or markers inserted into an organism to measure how actively other genes work. The technique is based on genes coding two fluorescent proteins. One of them is red protein Katushka2S, which is a marker for protein synthesis. Its appears when protein production terminates, causing a stop to ribosome movement along the RNA chain.

The system relies on a regulatory component of a tryptophan operon, which contains in its genome instructions of tryptophan amino acid biosynthesis. An operon is a functional group of genes, one part of which codes proteins. The other part regulates the number of these proteins, intensifying suppression of their synthesis when required. A tryptophan operon contains an attenuator—a genome portion, where in case of tryptophan excess, transcription stops.

Genes of tryptophan biosynthesis were replaced with a Katushka2S protein gene. The attenuator itself was also changed—namely, an alanine codon was inserted instead of a three-letter codon of a tryptophan amino acid in the structure of the coded enzymes. This modified attenuator stopped reacting to tryptophan concentration, becoming dependent on antibiotics that disrupt protein synthesis—in their presence, the amount of Katushka2S protein grows.

Ilya Osterman says, "At the first stage, we elaborated a gene engineering design, which included two fluorescent proteins. Expression of the first protein depended on the presence of protein synthesis inhibitors, while expression of the second one depended on the presence of DNA synthesis inhibitors. Afterwards, with the help of a hyperresponsive strain of coliform bacteria, we created a reporter aimed at the detection of corresponding antibiotic types."

Red light for DNA synthesis

What was the second fluorescent protein? It turned out to be RFP, a red fluorescent protein. Both proteins shine in the same range of red and far-red, which is passes through human tissues quite well.

An RFP gene was inserted in such a way that it started working during SOS-response—namely, cell reaction to stress. According to the scientists, this process should start the synthesis of a red fluorescent protein, which, like a red warning light, could give notice that there was something wrong inside a bacterium. The more light detected, the stronger effect the antibiotic has.

The scientists put an RFP gene right after the sulA gene promoter. SulA gene starts at the last stages of SOS-response and suppresses cell division. So once sulA gene starts operating, a red fluorescent protein also begins synthesizing in conjunction with it. Katushka2S operated in the same way in a tryptophan operon; it only warned about a termination of protein synthesis and not DNA synthesis. This system of two reporters has already been tested on activity clarification of a new antibiotic—amicoumacin—along with some other antibiotics. The target, which amicoumacin hits, was hitherto unknown.

Osterman says, "So far, with the help of this approach, we've analyzed more than 50,000 compounds, and discovered new inhibitors of protein and DNA synthesis, which could become the basis of new germicides in the future. The process of screening will be continued."

More information:
Ilya A. Osterman et al, Sorting out antibiotics' mechanisms of action: a double fluorescent protein reporter for high throughput screening of ribosome and DNA biosynthesis inhibitors, Antimicrobial Agents and Chemotherapy (2016). DOI: 10.1128/AAC.02117-16

Provided by Lomonosov Moscow State University