Team:Heidelberg/Description


Description





Project Description

Once you have looked into the incredible world of the microbiome, it won't let you go. We have microbiomes in our digestive system, on our skin and even in unexpected places like the lung. A balanced gut microbiome can not only help us digest food, but to improve one's health as well. With our project, we conduct basic research for a new therapeutic approach to treat microbiome-associated and inherited one gene diseases.

The current state-of-the-art treatments for microbiome-associated diseases range from pre-, pro- and synbiotics to faecal microbiota transplantation (FMT). These methods have shown promising results in some patients, but also have disadvantages. Choosing the right donor for FTM is not trivial, and it means a serious intervention in a patient's microbiome. Furthermore, rejection during re-cultivation of new bacterial strains can occur. Additionally, since the onset of the Covid-19 pandemic, FTM has been almost completely suspended because the risk of infection through fecal-oral transmission cannot be ruled out.

Our solution to this problem is a personalized treatment through transformation of the patient's gut microbiome. For this purpose, we make use of the ability of some specific bacteria for natural transformation [1]. These can take up plasmid DNA from their surroundings and incorporate them instead of digesting. In the future, our method should be able to treat diseases without disrupting the microbiome, by giving them capabilities the host is lacking. We would like to develop a capsule that contains plasmids with the target gene, and transports them to the intestine. Intestinal bacteria will then take up these plasmids by natural transformation in order to express and spread them by conjugation for long-term efficiency. The target gene product is expected to specifically cure diseases or alleviate symptoms.

Figure 1: Planned transport of the transformed DNA into the gut microbiome. After the capsule has been taken by the patient it should resist the stomach acid and release the plasmids inside in the intestine. The plasmids are then taken up by natural competent bacteria, that will express the genes to produce the wanted proteins. Created with BioRender.com

Our drylab team compared microbiome 16S rRNA data from different microbiome studies in order to find natural competent bacteria and define key features of a bacterial strain with this ability. The differences in the composition of patients which have been diagnosed with phenylketonuria and patients who are deemed to be healthy have been analysed in order to single out specific strains whose population is much higher in the diseased patients. While the data of the healthy cohort couldn’t be extracted very specifically, it was paid attention to the fact that the diseased patients should all be children as these are the main target group.

In the laboratory, we have designed a workflow for creating multi copy and linear plasmids suitable for Acinetobacter baylyi ADP1 (A. baylyi), one of the natural competent strains. As a proof of concept we did a natural transformation of A. baylyi with a plasmid containing a suitable origin of replication. Starting from this, we focused on more aspects that need to be considered. In a person's gut, not only one, but multiple strains coexist. Because of that, we compared co-cultures of various strains transformed with fluorescence proteins for easier distinction. Additionally to the lab work, we compared modelled data versus our own co-culture results. Of course our successfully transformed bacteria have higher costs compared to wildtype strains. For this reason we looked into novel natural selection advantages for our bacteria. Introducing antibiotic resistances during medical treatments is obviously not an option. The most promising enzymes for selection advantages in the human gut are the ones that give the bacteria an advantage in degrading special sugars. These might be supplemental for bacterial metabolism. Therefore, we focused on ß-glucosidase and ß-agarase [2]. In addition to the previous experiments we compared natural transformation efficiency in mono- and co-cultures of A. baylyi with other gut-associated bacterial strains such as Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). With these experiments we implemented a more lifelike environment because growing co-cultures is more similar to the natural gut microbiome.

Regarding our therapeutic aspect, which are the inherited one gene diseases, we tested the efficiency of the phenylalanine ammonia lyase (PAL). We tested its ability to degrade phenylalanine (phe) when cloned in gut microbiome related species such as E. coli. A lack of the phenylalanine degrading enzyme phenylalanine hydroxylase (PAH) is a problem in the disease phenylketonuria (PKU) [3].

With the help of experts, we started looking out of the box. Why only focus on the gut, when there are multiple microbiomes in other parts of the human body. As an example, patients with cystic fibrosis could also benefit from bacteria with mucus degradation abilities in the lung microbiome. If we had had more time to focus on this interesting area of application in the laboratory, we would have tested enzymes for mucus degradation [4]. But so, we considered the possibilities in theory.

To sum it up, we have established a proof of concept of how natural transformation can be used to equip bacteria with functions a potential host is lacking. This gives us courage to hope that our idea can be implemented one day and help patients train their microbiome.

In our project promotion video we provide a summary of our project:

References

[1] Jiang, X., Palazzotto, E., Wybraniec, E., Munro, L. J., Zhang, H., Kell, D. B., Weber, T., & Lee, S. Y. (2020). Automating Cloning by Natural Transformation. ACS synthetic biology, 9(12), 3228–3235. https://doi.org/10.1021/acssynbio.0c00240

[2] Chi, W. J., Chang, Y. K., & Hong, S. K. (2012). Agar degradation by microorganisms and agar-degrading enzymes. Applied microbiology and biotechnology, 94(4), 917–930. https://doi.org/10.1007/s00253-012-4023-2

[3] Durrer, K. E., Allen, M. S., & Hunt von Herbing, I. (2017). Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU. PloS one, 12(5), e0176286. https://doi.org/10.1371/journal.pone.0176286

[4] Hodson, M. E., & Shah, P. L. (1995). DNase trials in cystic fibrosis. The European respiratory journal, 8(10), 1786–1791. https://doi.org/10.1183/09031936.95.08101786