HARNESSING THE WISDOM OF PLANTS

The rise of antibiotic resistance is a major public health concern, which threatens to make even common infections much harder and more expensive to treat. Researchers at Wageningen University & Research are investigating whether secondary metabolites from plants could act as efflux pump inhibitors and help restore antimicrobial efficiency, as Dr Carla Araya-Cloutier explains.

The growth of antibiotic resistance represents a significant threat to public health, with more and more infections becoming resistant to drugs, making them harder to treat. One reason is the over-expression of efflux pumps, transporter proteins intrinsically present in all bacteria, which extrude substances toxic to the bacteria, such as antibiotics. “When bacteria are over-exposed to antibiotics, they create new mechanisms of resistance, but one of the first responses is to over-express these pumps,” explains Dr Carla Araya-Cloutier, Assistant Professor in the Laboratory of Food Chemistry at Wageningen University & Research. A method of blocking the activity of these pumps could help restore the effectiveness of existing antibiotic treatments, a topic Dr Araya-Cloutier is exploring in her research. “I am the head of the Plant Bioactives group. We aim to search for adjuvants or ‘helpers’ – some people call them resistancemodifying agents – in the plant kingdom,” she explains. “An adjuvant can be combined with an antibiotic to make it less susceptible to the resistance mechanisms of the bacteria.”

Efflux pump inhibitors

This research involves looking for novel plant compounds or phytochemicals that can act as efflux pump inhibitors (EPIs) in gram negative bacteria. These are more challenging microbes than gram positive bacteria, with very complex efflux pumps that span through a double membrane. “There’s an inner membrane, a periplasm and an outer membrane, and these pumps go all the way from inside to outside,” explains Dr ArayaCloutier. These pumps are poly-specific and can extrude different kinds of compounds or ‘substrates’, so Dr Araya-Cloutier says they are the most challenging to inhibit. “They are ‘smart’ if I could put it like that. These pumps recognise a great diversity of compounds, hence their poly-specificity, and extrude them in a very efficient way,” she continues. “It’s already difficult for antibiotics to get inside these gram-negative bacteria due to their double membrane. Once they get inside, they can then be taken by the efflux pumps again, to the outside.”

As the head of a research project backed by the Dutch Research Council (NWO), Dr Araya-Cloutier is studying different types of phytochemicals in the search for new EPIs, or phytoEPIs. As sessile organisms, lacking the ability to move under their own power, plants need to have strong and resilient defence mechanisms. “They have to withstand predators, bacteria, and other threats in the same location,” explains Dr Araya-Cloutier. The secondary metabolism of plants is very rich, with different families of molecules, structures and functional groups, which Dr Araya-Cloutier and her colleagues in the project are now trying to harness and exploit. “Normally plants will have a set of constitutive metabolites. If you grow a soya bean plant, it is normally meant to grow efficiently and healthily, thus secondary metabolism and diversity of phytochemicals is rather limited or narrow. Here, we exploit the defence metabolism in plants which is activated upon exposure to stress.”

The idea is to stress the plant using biotic

The Plant (Bio)Actives group

or abiotic agents in such a way that the plant reacts and creates a new set of secondary or defence metabolites, with researchers also aiming to relate the structure of the metabolites that are produced to the bioactivity, opening up the possibility of rational design. Previous research has shown that these defence metabolites are more potent antimicrobials than the constitutive metabolites, now Dr Araya-Cloutier is looking to build on these earlier findings. “We stress plants to create these novel sets of more potent defence metabolites,” she outlines. Researchers are stressing different plants, including some from the Fabaceae family such as soy beans, which produce prenylated phenolic compounds as a defence response and can act as phytoEPIs. “We germinate the beans and create sprouts, and at a certain point we induce stress by adding a fungi. This is an efficient way of stimulating the sprouts to create these new metabolites” continues Dr Araya-Cloutier. “We can also use specific rhizobacteria, which is normally present in the soil, while another option is using certain chemicals as abiotic stressors.” model, gram-negative organism, frequently involved in antibiotic-resistance infections. “We test these molecules on different strains of E. coli. One is the wild type, while another is a mutant strain, which has over-expression of these efflux pumps, and is basically an antibiotic resistant strain, as it’s able to extrude out most of the antibiotics that you put in,” she outlines. A couple of candidate compounds have been identified and their effectiveness is being investigated, while there are also several other lines of research in the project. “We are collaborating with the biochemistry group of Prof. Dr. K.M. Pos in Germany where we can essentially test the binding of the candidate phytoEPIs with the actual efflux pump,” says Dr Araya-Cloutier. This will open up further insights into how EPIs actually work. Through a collaboration with the physics group of Prof. Dr. P. Ruggerone in Italy molecular modelling tools, such as molecular dynamics simulations, will be used to model this binding and inhibition process.

The final aim in the project is to propose a couple of natural lead compounds for potential use as EPIs, which Dr Araya-Cloutier says could

We know that plants have defended themselves

for millions of years, and they are able to trick these efflux pumps into not working properly.

Inducing defence metabolites in plants

These plants are placed under stress at different time points over a period of around 10 days, and differences have been observed in terms of the amount and composition of the metabolites that are produced. Once the metabolites have been produced, the next step is to extract and characterise them. “We extract the metabolites with different solvents. We can then characterise the compounds via chromatography and mass spectrometry,” says Dr Araya-Cloutier. A combination of phytochemistry and microbiology expertise is then used to assess how effective these compounds will be as EPIs. “We use our chemical knowledge of the structural features present in the compounds to look at the structure-activity relationships,” explains Dr Araya-Cloutier. “With knowledge of the structure of the molecules, and of their activity as EPIs, we can then look to identify the structural motifs that are key to this bioactivity of efflux pump inhibition.”

The purified candidate phytoEPIs are combined with certain antibiotics, then used to treat antibiotic-resistant bacterial strains of common infections, which will provide a clearer picture of their effectiveness. Dr Araya-Cloutier is using E. coli, which is a then be developed further on the basis of the information available. “Information about the structure-activity relationships of the compounds and their inhibition mechanism would be very valuable for pharmaceutical companies, so they can then identify which molecules could be optimised further,” she explains. There are still many more hurdles to negotiate before these EPIs can be applied, but Dr Araya-Cloutier believes there is exciting potential in this area. “We are still at an early stage, but we know that plants have defended themselves for millions of years, thus are able to trick these efflux pumps into not working properly,” she says.

An effective means of restoring antimicrobial efficiency will have dramatic effects on the healthcare sector, helping both counteract the rise of antibiotic resistance and reduce the cost of treatment, while it could also have a wider impact. Antibiotic resistance affects not just healthcare, but also our food and farm systems and the environment more generally, so the compounds could also be used in other areas. “We could re-purpose any EPI compounds for other situations where antimicrobial resistance is of concern and where efflux pump inhibition might be beneficial,” says Dr Araya-Cloutier.

HARNESSING THE WISDOM OF PLANTS

Harnessing the wisdom of plants: Blocking pathogen’s emergency exit to restore antimicrobial efficiency Project Objectives

The rise of antibiotic resistance threatens to make even common infections much harder to treat. One of the main strategies gramnegative bacteria use to evade antibiotics is through efflux pumps, which enable them to remove (or extrude) antibiotics and other toxic substances. The aim in the project is to harness plant secondary metabolism to identify and characterize phytochemicals capable of effectively disarming bacterial efflux pumps (phytoEPIs). By combining expertise in chemistry, microbiology and molecular modelling fundamental understanding of their structure-function relationships and mechanism of inhibition will be obtained. Candidate phytoEPIs can serve as lead compounds for the development of adjuvants to restore the effectiveness of antibiotic treatments.

Project Funding

This project has received a VENI grant (VI. Veni.192.095) from the Dutch Research Council (NWO)

Project Partners

• Prof. Klaas M. Pos, Institute of

Biochemistry, Goethe-University Frankfurt • Prof. Paolo Ruggerone, Department of

Physics, University of Cagliari

Contact Details

Project Coordinator, Dr. Carla Araya-Cloutier Laboratory of Food Chemistry Department of Agrotechnology and Food Sciences Wageningen University & Research The Netherlands T: +31 317 48 48 63 E: carla.arayacloutier@wur.nl W: https://research.wur.nl/en/persons/ carla-araya-cloutier W: www.fch.wur.nl

Dr. Carla Araya-Cloutier

Dr. Carla Araya-Cloutier is an Assistant Professor in the Laboratory of Food Chemistry at Wageningen University & Research. Her Plant (Bio)Actives group focusses on phytochemicals from elicited plants as antimicrobial agents. The main goal is to understand and predict their structure-activity relationships and mechanism of action, using a combination of in-vitro and in-silico techniques.