The founding of GENAWIF is tightly linked to the history of the Department of Plant Physiology at the RWTH Aachen University and the so-called garlic group. To get an idea about Plant Physiology, it is basically the science about understanding how plants function and react to their environment. From the subcellular to the whole organism level of complexity, including the interactions of plants with other organisms like humans, animals, insects, and microorganisms.
Since the beginning of the department in 1974, a major topic has always been how plants react to stress like heat, drought, nutrient deficiency or pathogens, and how these factors influence the health of the plant. One is often not aware that this science is quite beneficial for us. For example, from the consumer point of view, we can address and optimize needs of plants to increase or ensure crop yields with the knowledge obtained from research into plant physiology. With decreasing availability of land mass, we also might find ways to turn hostile environments into fertile farming land.
During the course of evolution, plants developed intriguing strategies to cope with environmental stresses. Especially, the arsenal of biochemical substances with different biological modes of action is remarkable. Indeed, mankind benefits from plant metabolites in many ways, often without being aware of it. For example, salicylic acid is a plant metabolite and the origin of aspirin (acetyl salicylic acid), one of the most used drugs against headaches. Before sophisticated drugs had been developed, people could chew on willow bark for intake of this natural painkiller.
There is a story quite similar to that of salicylic acid and willow bark about garlic, a plant which almost everyone uses in the kitchen, but probably without knowing about the biochemical power that resides within it. Recently, the so-called Garlic Group said “farewell” to the RWTH due to the retirement of the group leader, Professor Alan Slusarenko, who was also the Head of the Plant Physiology Department at the RWTH Aachen from 1995 until 2022.
As the group name implies, most of the research was focused on garlic, and more precisely, on the astonishing antibiotic allicin, a natural defence substance which is released when garlic cells are damaged. Interestingly, almost everyone is familiar with allicin, because this substance is responsible for the typical smell of freshly cut or crushed raw garlic. Allicin is special because of its very strong antibiotic activity against bacteria and fungi, which was the reasoning why Professor Slusarenko developed his interest for this very odorous compound approximately 20 years ago. Thus, the question arose what mode of action allicin has in comparison to other antibiotics with a rather narrow range of target organisms.
The first experiments were done with garlic juice, using a conventional juicer from the kitchen and a lot of garlic bulbs. For a larger experiment, I remember that we prepared 600 millilitres of juice with three people cutting garlic and me working with the juice under the fume hood, but the lab (and myself) smelled nonetheless. Especially on hot summer days, not every colleague made jumps of pure joy when confronted with the intense garlic odour lurking in the corridors and doors were sometimes demonstratively slammed shut! Allicin is not the only component in garlic juice, but the major one, as we could prove with High Pressure Liquid Chromatography (HPLC) analysis. Additionally, since the juice could be prepared cheaply and easily, we had the idea that the juice might work as a very low tec alternative to cost intensive or synthetic substances.
The early experiments were aimed at getting an idea on the spectrum of microbes that were sensitive to allicin, and since we dealt with a plant defence substance, several plant pathogenic microbes were tested. Allicin proved to be potent against many pathogens, some important examples are Botrytis cinerea (causing grey mould, e.g., on grapevine), Phytophthora infestans (causing potato blight), or Magnaporthe grisea (causing blast disease on rice) 1.
Since garlic juice is a mixture from different ingredients, extraction procedures were developed to enrich allicin, and garlic extract was used until the garlic group worked on the chemical synthesis of allicin. The procedure to oxidise diallyl disulfide (DADS, a natural degradation product from allicin) back to allicin was optimized to a very efficient protocol, which also clarified the exact reaction mechanisms that occurred during the synthesis. Purities up to 98% with a yield of 91% were achieved and confirmed by HPLC, LC-MS (liquid chromatography coupled with mass spectrometry) and 1H-NMR (nuclear magnetic resonance) analysis 2. It is fair to say that the garlic group was in high demand to supply allicin and expertise to several labs, leading to many successful national and international cooperations.
With pure allicin at hand, experiments were performed to get an idea of the molecular mechanisms behind the antibiotic activity of allicin. For this, various model organisms like Saccharomyces cerevisiae, also known as baker’s yeast 3,4, pseudomonad bacteria 5–7, plants 8,9, and even human cell lines were used 6,10 and investigated with genetic and proteomic techniques. We collaborated with other cooperation partners to research the mode of action of allicin in Escherichia coli, Staphylococcus and Bacillus bacteria 11–14. In the course of this research, special techniques were developed, for example genetically constructed biosensors to monitor oxidative stress with living cells in real time 4, or the use of yeast mutant libraries to screen for genes important for tolerance against natural oxidants 15. These investigations led to the working model for allicin that we know today.
To make a long story short, allicin has various modes of action, the most important one, however, is its reactivity with free thiol groups, altering protein activities by adding so called allyl groups via oxidation. Normally, the cellular redox buffer glutathione would prevent oxidative damage, but it is targeted by allicin as well. It can be viewed as a kind of “redox toxin” in the cell. One can imagine this as a broad range attack on many different targets while simultaneously weakening the cellular defences, so that a cell can hardly defend itself against allicin. This ultimately leads to cell death, even dose-dependently, in higher human cells and tissues 16. Other antibiotics often affect one specific cellular target, which is why cells can more easily evolve counter measures against such specific modes of action.
The fundamental research on allicin’s mode of action developed to more applied research during the last few years. For example, since allicin can be smelled, it is volatile, and ideas came up to use allicin as a vapour in lungs infected with bacteria. A lung model was developed together with the Aerodynamic Institute of the RWTH Aachen University, successfully showing that bacteria in this model could be killed using allicin in an air flow mimicking normal inhalation of air 17. Simultaneously, studies on the effect of allicin on human cell lines implicated that allicin might be a potent anti-cancer drug 10,18. Just recently, a study performed in cooperation with the Institute for Virology at the Charité – Universitätsmedizin Berlin demonstrated that SARS-CoV-2 infected human cells produced less viral RNA, less viral protein and less infectious viral particles when treated with allicin concentrations physiologically tolerated by human cells 19.
As a safety precaution and disclaimer, it needs to be clearly stated that one should not try self-treatment with garlic or allicin, because allicin is a dose-dependent toxin that can also kill human cells and tissue! An ongoing part of research addresses how allicin can be applied where it needs to be active at the appropriate concentrations, because upon oral application allicin is readily converted to other sulfur compounds once inside the stomach.
All these practical approaches are promising to address serious human diseases, thus we thought about possibilities to continue this research even after the retirement of Professor Slusarenko. Thus, the idea of GENAWIF was born, not just to continue with allicin research, but also to expand our knowhow and techniques to other research areas with the focus on natural compounds. GENAWIF was already founded in 2020 to have enough time to prepare the transition from the RWTH Aachen into privately driven research.
The farewell party on the 25th of march was not only a “farewell“ from the garlic group and the RWTH Aachen, but also a new beginning with GENAWIF. In this background story to GENAWIF, a lot of topics were touched upon, and we are happy to announce that we will address these in more detail, especially in context how our research from the past will develop in the future!
References
(1) Curtis, H.; Noll, U.; Störmann, J.; Slusarenko, A. J. Broad-Spectrum Activity of the Volatile Phytoanticipin Allicin in Extracts of Garlic (Allium Sativum L.) against Plant Pathogenic Bacteria, Fungi and Oomycetes. Physiological and Molecular Plant Pathology 2004, 65 (2), 79–89. https://doi.org/10.1016/j.pmpp.2004.11.006.
(2) Albrecht, F.; Leontiev, R.; Jacob, C.; Slusarenko, A. J. An Optimized Facile Procedure to Synthesize and Purify Allicin. Molecules 2017, 22 (5). https://doi.org/10.3390/molecules22050770.
(3) Gruhlke, M. C. H.; Portz, D.; Stitz, M.; Anwar, A.; Schneider, T.; Jacob, C.; Schlaich, N. L.; Slusarenko, A. J. Allicin Disrupts the Cell’s Electrochemical Potential and Induces Apoptosis in Yeast. Free Rad. Biol. Med. 2010, 49 (12), 1916–1924. https://doi.org/10.1016/j.freeradbiomed.2010.09.019.
(4) Gruhlke, M. C. H.; Schlembach, I.; Leontiev, R.; Uebachs, A.; Gollwitzer, P. U. G.; Weiss, A.; Delaunay, A.; Toledano, M.; Slusarenko, A. J. Yap1p, the Central Regulator of the S. Cerevisiae Oxidative Stress Response, Is Activated by Allicin, a Natural Oxidant and Defence Substance of Garlic. Free Rad. Biol. Med. 2017, 108, 793–802. https://doi.org/10.1016/j.freeradbiomed.2017.05.004.
(5) Borlinghaus, J.; Bolger, A.; Schier, C.; Vogel, A.; Usadel, B.; Gruhlke, M. C.; Slusarenko, A. J. Genetic and Molecular Characterization of Multicomponent Resistance of Pseudomonas against Allicin. Life Sci. Alliance 2020, 3 (5), e202000670. https://doi.org/10.26508/lsa.202000670.
(6) Reiter, J.; Levina, N.; van der Linden, M.; Gruhlke, M.; Martin, C.; Slusarenko, A. J. Diallylthiosulfinate (Allicin), a Volatile Antimicrobial from Garlic (Allium Sativum), Kills Human Lung Pathogenic Bacteria, Including MDR Strains, as a Vapor. Molecules 2017, 22 (10), 1711. https://doi.org/10.3390/molecules22101711.
(7) Reiter, J.; Hübbers, A. M.; Albrecht, F.; Leichert, L. I. O.; Slusarenko, A. J. Allicin, a Natural Antimicrobial Defence Substance from Garlic, Inhibits DNA Gyrase Activity in Bacteria. International Journal of Medical Microbiology 2020, 310 (1), 151359. https://doi.org/10.1016/j.ijmm.2019.151359.
(8) Borlinghaus, J.; Albrecht, F.; Gruhlke, M. C. H.; Nwachukwu, I. D.; Slusarenko, A. J. Allicin: Chemistry and Biological Properties. Molecules 2014, 19 (8), 12591–12618. https://doi.org/10.3390/molecules190812591.
(9) Leontiev, R.; Hohaus, N.; Jacob, C.; Gruhlke, M. C. H.; Slusarenko, A. J. A Comparison of the Antibacterial and Antifungal Activities of Thiosulfinate Analogues of Allicin. Sci. Rep. 2018, 8 (1), 6763–6763. https://doi.org/10.1038/s41598-018-25154-9.
(10) Gruhlke, M. C. H.; Antelmann, H.; Bernhardt, J.; Kloubert, V.; Rink, L.; Slusarenko, A. J. The Human Allicin-Proteome: S-Thioallylation of Proteins by the Garlic Defence Substance Allicin and Its Biological Effects. Free Radical Biology and Medicine 2019, 131, 144–153. https://doi.org/10.1016/j.freeradbiomed.2018.11.022.
(11) Chi, B. K.; Huyen, N. T. T.; Loi, V. V.; Gruhlke, M. C. H.; Schaffer, M.; Mäder, U.; Maaß, S.; Becher, D.; Bernhardt, J.; Arbach, M.; Hamilton, C. J.; Slusarenko, A. J.; Antelmann, H. The Disulfide Stress Response and Protein S-Thioallylation Caused by Allicin and Diallyl Polysulfanes in Bacillus Subtilis as Revealed by Transcriptomics and Proteomics. Antioxidants (Basel) 2019, 8 (12), 605. https://doi.org/10.3390/antiox8120605.
(12) Loi, V. V.; Huyen, N. T. T.; Busche, T.; Tung, Q. N.; Gruhlke, M. C. H.; Kalinowski, J.; Bernhardt, J.; Slusarenko, A. J.; Antelmann, H. Staphylococcus Aureus Responds to Allicin by Global S-Thioallylation – Role of the Brx/BSH/YpdA Pathway and the Disulfide Reductase MerA to Overcome Allicin Stress. Free Rad. Biol. Med. 2019, 139, 55–69. https://doi.org/10.1016/j.freeradbiomed.2019.05.018.
(13) Müller, A.; Eller, J.; Albrecht, F.; Prochnow, P.; Kuhlmann, K.; Bandow, J. E.; Slusarenko, A. J.; Leichert, L. I. O. Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines. J. Biol. Chem. 2016, 291 (22), 11477–11490. https://doi.org/10.1074/jbc.M115.702308.
(14) Wüllner, D.; Haupt, A.; Prochnow, P.; Leontiev, R.; Slusarenko, A. J.; Bandow, J. E. Interspecies Comparison of the Bacterial Response to Allicin Reveals Species-Specific Defense Strategies. PROTEOMICS 2019, 19 (24), 1900064. https://doi.org/10.1002/pmic.201900064.
(15) Leontiev, R.; Slusarenko, A. J. Finding the Starting Point for Mode-of-Action Studies of Novel Selenium Compounds: Yeast as a Genetic Toolkit. Current Organic Synthesis 2017, 14 (8), 1102–1108. https://doi.org/10.2174/1570179414666170525112446.
(16) Borlinghaus, J.; Foerster (née Reiter), J.; Kappler, U.; Antelmann, H.; Noll, U.; Gruhlke, M. C. H.; Slusarenko, A. J. Allicin, the Odor of Freshly Crushed Garlic: A Review of Recent Progress in Understanding Allicin’s Effects on Cells. Molecules 2021, 26 (6). https://doi.org/10.3390/molecules26061505.
(17) Reiter, J.; Borlinghaus, J.; Dörner, P.; Schröder, W.; Gruhlke, M. C. H.; Klaas, M.; Slusarenko, A. J. Investigation of the Deposition Behaviour and Antibacterial Effectivity of Allicin Aerosols and Vapour Using a Lung Model. Exp Ther Med 2020, 19 (2), 1541–1549. https://doi.org/10.3892/etm.2019.8387.
(18) Schultz, C. R.; Gruhlke, M. C. H.; Slusarenko, A. J.; Bachmann, A. S. Allicin, a Potent New Ornithine Decarboxylase Inhibitor in Neuroblastoma Cells. J. Nat. Prod. 2020. https://doi.org/10.1021/acs.jnatprod.0c00613.
(19) Mösbauer, K.; Fritsch, V. N.; Adrian, L.; Bernhardt, J.; Gruhlke, M. C. H.; Slusarenko, A. J.; Niemeyer, D.; Antelmann, H. The Effect of Allicin on the Proteome of SARS-CoV-2 Infected Calu-3 Cells. Front Microbiol 2021, 12, 746795. https://doi.org/10.3389/fmicb.2021.746795.
Jan Borlinghaus, 27.04.2022
