Implementation

A future perspective for our project.

Infographic

Introduction

As antimicrobial resistances might come up more frequently in the future, we hope to transfer the knowledge gained in research on antimicrobial peptides (AMPs) into promising applications. Our modular screening system ATHELAS can be adjusted quickly to account for different strains of bacteria. Through the different stages of our implementation process, we also want to contribute to fundamental research on antibiotics and microbes in general.

You can see the scheme of our proposed implementation in the infographic to the left with some explanations shown below.

Following the established engineering design cycle, new AMPs get developed while also accumulating knowledge. This stage includes modelling of structures, cloning new constructs, expressing them on a small scale in Nicotiana benthamiana and extracting as well as purifying the peptides.

In order to screen the constructs for their ability to impair bacteria, their predicted tertiary structures were simulated next to a membrane through molecular dynamics software. Furthermore, their simulation has allowed us to derive conclusions about their stability, sparing us from pursuing the expression of instable constructs.

The first step of purification can be done using standard protocols for the extraction of proteins from tobacco leaves. However, one has to think about the scale up process as well, because grinding plant tissue for protein extraction using liquid nitrogen just isn’t economically viable anymore at some point. To counteract this problem, other methods of plant treatment like using a juice extractor are tested to ensure that enough plant material can get processed in order to move on to the next stage.

Purification of the AMPs gets explored for a fast and responsive application inside the laboratory. Additionally, several hurdles regarding large batches of extracts which might come up later when scaling up the production will get addressed.

To check for antimicrobial properties, several assays will be carried out, which also help in the design process as they reveal parts of the mechanism of action.

Constructs with reliable antimicrobial properties enter the next stage: The safety assessment. Important data like information on haemolytic activity, cytotoxicity and the quantification of antimicrobial properties are collected and fed back into the engineering design cycle.

While our main focus is on screening AMP-cyclotide-constructs and establishing them as new potential antimicrobial agents, we would also want to use the data to learn more about the mechanism of action of AMPs. This would be especially relevant for novel AMPs, which have not yet been characterized that well.

This can be done using the crude extract as well as purified protein solutions in order to continuously check the existence and activity of our constructs. At this stage, several chemical and physical properties are assessed as well in order to determine the correct conditions when working with our constructs inside the laboratory or on a large scale when moving on towards a possible implementation of our project.

If we want to use our constructs in clinical trials, we would also need to check if the His-tag influences these properties and if replacing it would enhance or reduce antimicrobial activity. To further guarantee an efficient purification and detection process, monoclonal antibodies which target the cyclotide backbone can be used.

One of the advantages of using cyclotides as scaffolds for different AMPs is their outstanding stability ³ . Although this is highly preferable for storage and distribution purposes, it can ultimately lead to pollution of the environment. To account for this, the degradation of our AMPs will be assessed. It is also important to check the bioavailability of our peptides, as this information will decide the pharmaceutical form and dosage.

Before licensing, it is checked if our transiently expressed peptides contain any agrobacteria in addition to the measurements of activity levels and purity. If these examinations turn out promising and other legal regulations are fulfilled as well, the official licensing can be proposed.

At first, we want to rely on transient expression of tobacco plants inside greenhouses, but if the peptides are expressed reliably, it is possible to create stable transgenic plants which can be cultivated on an open field. However, the peptides produced in these plants would only be suitable for non-medical applications. This open-field cultivation would take place in Spain or the USA, because regulations in Germany do not allow for such field tests. Transgenic plants would be modified to express the gene of interest only if ethanol is applied, which is called ethanol-induced expression ⁴ .

AMPs are considered an alternative to antibiotics because it is considered unlikely for bacteria to develop resistances against them ⁴ . Through large scale application of AMPs, we can collect data on this hypothesis and check whether it holds up or not.

Additionally, we can improve our methods of plant treatment and protein purification as they are used at an industrial level.

We chose N. benthamiana as expression platform partly to enable an easily scaled up production of our peptides. At this stage, enclosed greenhouses containing tobacco plants produce raw plant material, which can be treated afterwards to extract and purify the AMPs. For these steps, we can rely on our knowledge from the research stage as well as the field trip to Nomad Bioscience.

In comparison to the laboratory, different methods and protocols get applied, which have been examined during the research stage. Cloning and transformation of E. coli and A. tumefaciens can get scaled up easily, while in contrast, the infiltration of tobacco plants is done using vacuum infiltration or spraying the plants rather than infiltration by syringe ¹ .

Infected plants are grown inside greenhouses or possibly, as mentioned beforehand, in an open field environment. Protein extraction can be done using juice extractors and food mills or by letting the plants dry with subsequent grinding. Cell debris and cellulose are separated via depth filters or continuous flow centrifugation.

For purifying our peptides, traditional methods using chromatography can get difficult to implement on an industrial scale. Therefore, several steps of precipitation and filtering are applied. As our peptides should be heat-resistant, a simple treatment at around 50°C is a quick and easy way of removing impurities like other proteins.

The overall stability of cyclic peptides could also help in cheapening the overall operating procedure. If our constructs prove to be stable at room temperature, this would require less cooling throughout the production facility and therefore lower overall costs. The concentrated construct can be used for drug formulation or integrated into other products.

There are several applications for AMPs in a medical environment, individual research or waste treatment when facing resistant strains of bacteria.

This is especially relevant with Gram-negative bacteria (Klebsiella spp., Escherichia coli, Pseudomonas spp.) that are resistant to single or multiple antibiotics. Current therapeutic treatment strategies heavily rely on broad spectrum antibiotics with associated side effects, because antibiotics can have an impact on the gut microbiome ² and therefore weaken the immune response.

This problem calls for specific anti-bacterial agents like bacteriocins or in our case, AMPs. As we use a cyclotide scaffold to incorporate multiple peptides with different anti-microbial properties, we can design anti-bacterial agents to accommodate various needs and applications.

Outside of clinical use, it would also be possible to treat waste or waste water with our AMPs, which can harbour pathogenic and possibly resistant bacteria to stop them from contaminating the environment. However, for this application, we would have to show the biodegradability of our constructs in advance.

Additionally, research groups from around the world can create their own constructs or try out different AMPs on their strains of bacteria while using our expression platform.

References

¹ Marillonnet, S., Thoeringer, C., Kandzia, R. et al. Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol 23, 718–723 (2005). https://doi.org/10.1038/nbt1094

² Ramirez, J., Guarner, F., Bustos Fernandez, L., Maruy, A., Sdepanian, V. L., & Cohen, H. (2020). Antibiotics as Major Disruptors of Gut Microbiota. Frontiers in cellular and infection microbiology, 10, 572912. https://doi.org/10.3389/fcimb.2020.572912

³ Koehbach J, Gani J, Hilpert K, Craik DJ. Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens. Microorganisms. 2021; 9(6):1249. https://doi.org/10.3390/microorganisms9061249

⁴ Schneider, T., Hahn-Löbmann, S., Stephan, A. et al. Plant-made Salmonella bacteriocins salmocins for control of Salmonella pathovars. Sci Rep 8, 4078 (2018). https://doi.org/10.1038/s41598-018-22465-9

⁵ Abdi, M, Mirkalantari, S, Amirmozafari, N. Bacterial resistance to antimicrobial peptides. J Pep Sci. 2019; 25:e3210. https://doi.org/10.1002/psc.3210

About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.