The founding of GENAWIF is tightly linked to the history of the so called „garlic group“ which did its research in the plant physiology department at the RWTH Aachen university. Recently, the garlic group said good bye due to the retirement of the group leader, Professor Alan Slusarenko, who was also the head of the plant physiology department. As the group name implies, most of the research was focused on garlic, and more precisely, on the antibiotic allicin, a defense substance which is released when the garlic cells are damaged. Interestingly, almost everyone knows allicin, because this substance is responsible for the typical smell of freshly cut-, smashed, or raw eaten 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 milliliter of juice with three people cutting garlic and me working with the juice under the fume cupboard, but it (and me) smelled nonetheless. Especially on hot summer days, not every colleague made jumps of pure joy when confronted with the intense garlic odour lurking its way through the floors. Coming back to garlic juice, it could be well used as a source of allicin because High Pressure Liquid Chromatography (HPLC) analysis could show, that the main component was indeed allicin. The idea at that time was also that garlic juice might be a low cost alternative compared to high cost alternatives and synthetic chemicals.

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 defense substance, several plant pathogenic microbes were tested. Allicin proofed to be potent against many pathogens, some prominent examples one might heard of are Botrytis cinerea (causing gray mould, e.g. on grapevine), Phytophtora infestans (causing potato blight), or Magnaporthe grisea (causing blast disease on rice) (Curtis et al., 2004).

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 revealed the exact reaction mechanisms that occured during this synthesis. Purities up to 98% with a yield of 91% were achieved and proofed by HPLC, LC-MS (liquid cromatographie coupled with mass spectrometry) and 1H-NMR (nuclear magnetic resonance) analysis (Albrecht et al., 2017). It is fair to say that the garlic group became a highly demanded supplier for allicin and expertise, leading to various cooperations with national and international working groups. 

With the pure allicin in hand, experiments were performed to get an idea of the molecular mechanism behind the antibiotic activity of allicin. For this, various model organisms like Saccharomyces cerevisiae, also known as bakers yeast (Gruhlke et al., 2010, 2017), Pseudomonad bacteria (Borlinghaus et al., 2020; Reiter et al., 2017, 2020a), plants (Borlinghaus et al., 2014; Leontiev et al., 2018), and even human cell lines were used (Gruhlke et al., 2019; Reiter et al., 2017) and investigated with genetic and proteomic techniques. Other cooperation partners did research on the mode of action of allicin on Escherichia coli, Staphylococcus and Bacillus bacteria (Chi et al., 2019, 2019; Müller et al., 2016; Wüllner et al., 2019).  In the course of this research approaches, special techniques were developed like genetically constructed biosensors to monitor oxidative stress with living cells in real time (Gruhlke et al., 2017), or the usage of yeast mutant libraries to screen for genes important for tolerance against natural oxidants (Roman  Leontiev and   Alan   J. Slusarenko*, 2017). These investigations led to the working model for allicin that we know today. To put 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 would prevent oxdidative damage, but since the main buffer systems also contain thiols, they are targeted by allicin as well. One can imagine this as a broad range attack on so many different targets while simultaneosly weaking the cellular defense, so that a cell can hardly defend itself against allicin. This ultimately leads to cell death, even in higher human cells and tissues (Borlinghaus et al., 2021). Other antibiotics are often affecting special cellular targets, which is why cells can more easily evolve to obtain counter measures against such specific modes of action.

The fundamental research on allicin developed to a more practical research during the last few years. For example, since allicin can be smelled, 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, succesfully showing that bacteria in this model could be killed using allicin in an air flow close to normal inhalation of air (Reiter et al., 2020b). Simultaneously, studies on the effect of allicin on human cell lines implicated that allicin might be a potent anti cancer drug (Gruhlke et al., 2019; Schultz et al., 2020). Just recently, a study performed in cooperation with the working group of Professor Drosten demonstrated that Sars-CoV2 infected human cells produced less viral RNA, less viral protein and less infectious viral particles when treated with allicin concentrations that could be endured by the human cells without being killed (Mösbauer et al., 2021). As a safety precaution, it needs to be clearly stated that one should not try selftreatment with garlic or allicin, because allicin is a dose-dependent toxin that can also kill human cells and tissue! An ongoing part of research adresses how allicin can be applied where it needs to be active, because oral application won´t affect cancer or infections in the body since 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. It was clear that his or her successor would direct the research in other directions. Thus, the idea of GENAWIF was born, not just to continue with allicin research, but also to expand our know how and developed 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 „good bye“ 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 adress these in more detail, especially in context how our research from the past will develop in the coming future!

References

Albrecht, F., Leontiev, R., Jacob, C., and Slusarenko, A.J. (2017). An Optimized Facile Procedure to Synthesize and Purify Allicin. Molecules 22. https://doi.org/10.3390/molecules22050770.

Borlinghaus, J., Albrecht, F., Gruhlke, M.C.H., Nwachukwu, I.D., and Slusarenko, A.J. (2014). Allicin: Chemistry and Biological Properties. Molecules 19, 12591–12618. https://doi.org/10.3390/molecules190812591.

Borlinghaus, J., Bolger, A., Schier, C., Vogel, A., Usadel, B., Gruhlke, M.C., and Slusarenko, A.J. (2020). Genetic and molecular characterization of multicomponent resistance of Pseudomonas against allicin. Life Sci. Alliance 3, e202000670. https://doi.org/10.26508/lsa.202000670.

Borlinghaus, J., Foerster (née Reiter), J., Kappler, U., Antelmann, H., Noll, U., Gruhlke, M.C.H., and Slusarenko, A.J. (2021). Allicin, the Odor of Freshly Crushed Garlic: A Review of Recent Progress in Understanding Allicin’s Effects on Cells. Molecules 26. https://doi.org/10.3390/molecules26061505.

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., et al. (2019). 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) 8, 605. https://doi.org/10.3390/antiox8120605.

Curtis, H., Noll, U., Störmann, J., and Slusarenko, A.J. (2004). 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 65, 79–89. https://doi.org/10.1016/j.pmpp.2004.11.006.

Gruhlke, M.C., Portz, D., Stitz, M., Anwar, A., Schneider, T., Jacob, C., Schlaich, N.L., and Slusarenko, A.J. (2010). Allicin disrupts the cell’s electrochemical potential and induces apoptosis in yeast. Free Radical Biology and Medicine 49, 1916–1924. .

Gruhlke, M.C.H., Schlembach, I., Leontiev, R., Uebachs, A., Gollwitzer, P.U.G., Weiss, A., Delaunay, A., Toledano, M., and Slusarenko, A.J. (2017). 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. 108, 793–802. https://doi.org/10.1016/j.freeradbiomed.2017.05.004.

Gruhlke, M.C.H., Antelmann, H., Bernhardt, J., Kloubert, V., Rink, L., and Slusarenko, A.J. (2019). The human allicin-proteome: S-thioallylation of proteins by the garlic defence substance allicin and its biological effects. Free Rad. Biol. Med. 131, 144–153. https://doi.org/10.1016/j.freeradbiomed.2018.11.022.

Leontiev, R., Hohaus, N., Jacob, C., Gruhlke, M.C.H., and Slusarenko, A.J. (2018). A Comparison of the Antibacterial and Antifungal Activities of Thiosulfinate Analogues of Allicin. Sci. Rep. 8, 6763–6763. https://doi.org/10.1038/s41598-018-25154-9.

Loi, V.V., Huyen, N.T.T., Busche, T., Tung, Q.N., Gruhlke, M.C.H., Kalinowski, J., Bernhardt, J., Slusarenko, A.J., and Antelmann, H. (2019). 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 Radic Biol Med 139, 55–69. https://doi.org/10.1016/j.freeradbiomed.2019.05.018.

Mösbauer, K., Fritsch, V.N., Adrian, L., Bernhardt, J., Gruhlke, M.C.H., Slusarenko, A.J., Niemeyer, D., and Antelmann, H. (2021). The Effect of Allicin on the Proteome of SARS-CoV-2 Infected Calu-3 Cells. Front Microbiol 12, 746795. https://doi.org/10.3389/fmicb.2021.746795.

Müller, A., Eller, J., Albrecht, F., Prochnow, P., Kuhlmann, K., Bandow, J.E., Slusarenko, A.J., and Leichert, L.I.O. (2016). Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of  Protein Cysteines. J. Biol. Chem. 291, 11477–11490. https://doi.org/10.1074/jbc.M115.702308.

Reiter, J., Levina, N., van der Linden, M., Gruhlke, M., Martin, C., and Slusarenko, A.J. (2017). Diallylthiosulfinate (Allicin), a Volatile Antimicrobial from Garlic (Allium sativum), Kills Human Lung Pathogenic Bacteria, Including MDR Strains, as a Vapor. Molecules 22, 1711. https://doi.org/10.3390/molecules22101711.

Reiter, J., Hübbers, A.M., Albrecht, F., Leichert, L.I.O., and Slusarenko, A.J. (2020a). Allicin, a natural antimicrobial defence substance from garlic, inhibits DNA gyrase activity in bacteria. International Journal of Medical Microbiology 310, 151359. https://doi.org/10.1016/j.ijmm.2019.151359.

Reiter, J., Borlinghaus, J., Dörner, P., Schröder, W., Gruhlke, M.C.H., Klaas, M., and Slusarenko, A.J. (2020b). Investigation of the deposition behaviour and antibacterial effectivity of allicin aerosols and vapour using a lung model. Exp Ther Med 19, 1541–1549. https://doi.org/10.3892/etm.2019.8387.

Roman  Leontiev and   Alan   J. Slusarenko* (2017). Finding the Starting Point for Mode-of-Action Studies of Novel Selenium Compounds: Yeast as a Genetic Toolkit. Current Organic Synthesis 14, 1102–1108. https://doi.org/10.2174/1570179414666170525112446.

Schultz, C.R., Gruhlke, M.C.H., Slusarenko, A.J., and Bachmann, A.S. (2020). Allicin, a Potent New Ornithine Decarboxylase Inhibitor in Neuroblastoma Cells. J Nat Prod 83, 2518–2527. https://doi.org/10.1021/acs.jnatprod.0c00613. Wüllner, D., Haupt, A., Prochnow, P., Leontiev, R., Slusarenko, A.J., and Bandow, J.E. (2019). Interspecies Comparison of the Bacterial Response to Allicin Reveals Species-Specific Defense Strategies. PROTEOMICS 19, 1900064. https://doi.org/10.1002/pmic.201900064.

The garlic group, the foundation of GENAWIF, and the farewell from the RWTH Aachen

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