Team:Lund/Description
Introduction
With a global trend of rising life expectancy [1], preventing and treating diseases that develop over time and are diagnosed in the later stages of life, becomes increasingly important. Therefore, Parkinson's and Alzheimer's disease research have been in focus in the last decades. These neurodegenerative diseases affect millions of people worldwide and are expected to grow more prevalent. It is estimated that 50 million people globally have Alzheimer's disease or another type of dementia. In 2050 it is projected that 152 million of the worldwide population will live with dementia [2][3]. These are alarming numbers since neurodegenerative diseases are progressive in nature, and as of 2021, there are no cures available [4][5].
One contributing factor to Parkinson's and Alzheimer's development is the accumulation of amyloid fibrils due to protein misfolding and aggregation [6][7]. Therefore, avoiding the formation of these fibrils is thought to prevent Parkinson's and Alzheimer's disease. We have chosen to focus on the amyloid protein curli and its formation by bacteria in the gut. Curli has been linked to Parkinson's disease. Focusing on a specific bacterial protein allows our team to use synthetic biology to elegantly tackle an issue as important and complex as neurodegenerative diseases while only working with bacterial systems.
Neurodegenerative Diseases, Amyloids and the Gut
There is a wide range of cognitive disorders caused by neurodegeneration, such as Alzheimer’s, Parkinson’s, Creutzfeldt-Jakob, Huntington’s, and multiple sclerosis [8]. Neurodegeneration includes progressively losing the structure and function of neurons and even cell death. Although the exact way the cerebral function is altered is still unknown, several hypotheses have been revised over the years. One is mitochondrial dysfunction. The intrinsic apoptotic mitochondrial pathway involving oxidative stress is one of the most common causes of cellular death during neurodegeneration [6]. Another hypothesis associates some of these diseases with misfolded proteins forming amyloid structures and aggregating [7]. Although the amyloids related to neurodegenerative diseases are generally found in the brain, they might not be the starting point of the diseases. During the past decade, studies have shown that some amyloid proteins found in the gut can cause proteins in the brain to misfold and form amyloid structures through what has been named the gut-brain axis (GBA). The GBA is a bidirectional link between the central nervous system (CNS), the enteric nervous system, and the gut microbiota [9]. The interaction could happen through several pathways, like the vagus nerve (transmitting information from the luminal environment to the CNS), neuroendocrine signaling, or interfering with tryptophan metabolism [10]. Curli has been shown in studies on rodents to enhance the formation of amyloid structures by other proteins [11][12].
Curli
Curli, produced by Escherichia coli and other pathogenic bacteria, is formed by amyloid fibers that are externalized to form a matrix. The fibers mediate host cell adhesion and invasion and, therefore, strongly induce the host’s inflammatory response [13]. In Project Cure-li, curli is addressed by producing a recombinant protein that will act as an inhibitor, reducing the amount or preventing the formation of curli fibers. We chose five different inhibitors through a literature study for this project.
In order to understand how the inhibitors work, a further explanation of the curli fibers is required. Six proteins are encoded dedicated to curli formation. The two structural subunits of curli are CsgA and CsgB. Both of them are secreted outside the cell, where they nucleate into a fiber. CsgB attaches to the outer membrane, where then CsgA subunits elongate the CsgB, forming fibrils. For these subunits to be secreted, a pore or channel is needed; this is CsgG. With the help of CsgE and CsgF, which bind to CsgG, this pore allows the secretion of the curli subunits. In addition to the secretion systems and the main subunits, the bacteria also produce a chaperone called CsgC that inhibits intracellular amyloid formation. [13]
An overview of the curli system can be seen in Figure 1.
Figure 1. A schematic diagram picture of the proteins involved in the curli elongation.
Inhibitors
Five inhibitors were chosen for project Cure-li: degP, CsgC, tANK6, DB3DB3 and the CsgF-peptide. The inhibitors operate in different ways. Two of them are proteins. The protein degP is a protease shown to inhibit E. coli biofilms [14]. The protein CsgC is a chaperone that keeps the CsgA subunits in a conformation incapable of forming the fibrils [15]. The remaining three inhibitors are shorter peptides designed to be expressed in a repeating polypeptide chain. The peptides tANK6 and DB3DB3 inhibit curli fibril elongation by binding to the growing fibril and preventing additional CsgA monomers from binding [16]. Finally, the last inhibitor is a peptide derived from the CsgF protein, and it binds to the CsgG secretion pore to block the secretion of the curli subunits CsgA and CsgB [17]. For more information about the inhibitors, see Engineering.
Inspiration
The intestine and the gut-brain axis is the new frontier of medicine. Every month there is a breakthrough into the field of the microbiome of our gut. We were therefore excited to search for iGEM projects related to the field. Coincidentally, vol 590 of Nature (February 4, 2021) [18] included a passage about how the microbiome could be affecting the development of neurodegenerative diseases. The report brought up studies indicating that mice infected by curli-producing bacteria developed Parkinson’s disease, while infected mice with their vagus nerve cut off did not show any symptoms [18]. The idea of working on a project connecting neurodegenerative diseases, the gut-brain axis, and the intestine microbiome swiftly got hold of the team. We figured that fighting the curli-producing bacteria using genetically engineered probiotic bacteria was a neat approach. From there, Team Lund’s project Cure-li was born.
Why Limosilactobacillus reuteri?
L. reuteri naturally produces reuterin, a broad-spectrum antimicrobial agent that combat enteropathogenic bacteria in the human intestine, such as E. coli [19]. This intrinsic characteristic of L. reuteri and the fact that it has been part of studies led by local researchers in Lund [20] made it an attractive chassis for our project. Pharmaceuticals with non-biological delivery systems require much consideration in terms of chemical and metabolic stability to ensure delivery to the correct place in the gut. As L. reuteri is naturally active in the gut, it provides a convenient and elegant delivery system for our curli inhibitors. Localized secretion of our inhibitors by L. reuteri could also help avoid hydrolysis by peptidases present in the gut. With synthetic biology tools, we can take advantage of all these benefits of using L. reuteri in combatting curli formation.
Goals
Our original goal was to modify L. reuteri to inhibit curli formation due to curli’s link to neurodegenerative diseases. However, difficulties with the shuttle vector and transformation made us focus our efforts on expressing an inhibitor in E. coli. Throughout the project, our team broke down our goals into different objectives that evolved over time.
In the early stages of the project, the first objectives were to identify suitable curli inhibitors and design an expression system for them in L. reuteri. The latter included identifying signal peptides and a shuttle vector that would allow L. reuteri to express and secrete the inhibitors for us to test them. These objectives were achieved through a literature study, and based on this, we chose five inhibitors and three signal peptides to express. For more details on the expression system, see Engineering.
Curli detection was the first objective addressed in the lab, obtaining promising but ultimately inconclusive results. The next objective was cloning using our complete constructs containing all the DNA sequences necessary for the expression and secretion of our various inhibitors. Months later, after failing to transform our inhibitors in E. coli using the L. reuteri shuttle vector, via both restriction enzymes and Gibson assembly at different positions in the vector, the aim pivoted towards inserting one of the constructs into E. coli using pET-11a. The team accomplished this with a plasmid containing the gene for the inhibitor CsgC. As a final objective before terminating the lab work, the team then attempted to express CsgC in a different E. coli culture. While the transformation into this strain was successful, the results of the expression assay were inconclusive. For more information about the results of our lab work, see Results.
References
[1] GHE: Life expectancy and healthy life expectancy [Internet]. Who.int. 2021 [cited 10 October 2021]. Available from: https://www.who.int/data/gho/data/themes/mortality-and-global-health-estimates/ghe-life-expectancy-and-healthy-life-expectancy
[2] Rocca W. The burden of Parkinson's disease: a worldwide perspective. The Lancet Neurology. 2018;17(11):928-929.
[3] Kan D. Alzheimer's Facts and Statistics for 2019: Everything You Need to Know - Being Patient [Internet]. Being Patient. 2021 [cited 10 October 2021]. Available from: https://www.beingpatient.com/alzheimers-disease-facts/
[4] Parkinson's disease - Treatment [Internet]. nhs.uk. 2021 [cited 10 October 2021]. Available from: https://www.nhs.uk/conditions/parkinsons-disease/treatment/
[5] Alzheimer's disease - Treatment [Internet]. nhs.uk. 2021 [cited 10 October 2021]. Available from: https://www.nhs.uk/conditions/alzheimers-disease/treatment/
[6] Hroudová J, Singh N, Fišar Z. Mitochondrial Dysfunctions in Neurodegenerative Diseases: Relevance to Alzheimer’s Disease. BioMed Research International. 2014;2014:1-9.
[7] Friedland R, Chapman M. The role of microbial amyloid in neurodegeneration. PLOS Pathogens. 2017;13(12):e1006654.
[8] What? | JPND [Internet]. JPND | Neurodegenerative Disease Research. 2017 [cited 10 October 2021]. Available from: https://www.neurodegenerationresearch.eu/what/
[9] Carabotti M, Scirocco A, Maselli MA, Severi C. The gut-brain axis: interactions between enteric microbiota, central and enteric nervous systems. Ann Gastroenterol. 2015;28(2):203-209.
[10] A, Pedia P, Us C, Insights P, Gallery V, Conversations P. The Simplified Guide to the Gut-Brain Axis - How the Gut Talks to the Brain [Internet]. Psych Scene Hub. 2021 [cited 10 October 2021]. Available from: https://psychscenehub.com/psychinsights/the-simplified-guide-to-the-gut-brain-axis/
[11] Lundmark K, Westermark G, Olsen A, Westermark P. Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice: Cross-seeding as a disease mechanism. Proceedings of the National Academy of Sciences. 2005;102(17):6098-6102.
[12] Chen S, Stribinskis V, Rane M, Demuth D, Gozal E, Roberts A et al. Exposure to the Functional Bacterial Amyloid Protein Curli Enhances Alpha-Synuclein Aggregation in Aged Fischer 344 Rats and Caenorhabditis elegans. Scientific Reports. 2016;6(1).
[13] Barnhart M, Chapman M. Curli Biogenesis and Function. Annual Review of Microbiology. 2006;60(1):131-147.
[14] Fang K, Jin X, Hong S. Probiotic Escherichia coli inhibits biofilm formation of pathogenic E. coli via extracellular activity of DegP. Scientific Reports. 2018;8(1).
[15] Evans M, Chorell E, Taylor J, Åden J, Götheson A, Li F et al. The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation. Molecular Cell. 2015;57(3):445-455.
[16] Perov S, Lidor O, Salinas N, Golan N, Tayeb- Fligelman E, Deshmukh M et al. Structural Insights into Curli CsgA Cross-β Fibril Architecture Inspire Repurposing of Anti-amyloid Compounds as Anti-biofilm Agents. PLOS Pathogens. 2019;15(8):e1007978.
[17] Yan Z, Yin M, Chen J, Li X. Assembly and substrate recognition of curli biogenesis system. Nature Communications. 2020;11(1).
[18] Willyard C. How gut microbes could drive brain disorders. Nature. 2021;590(7844):22-25.
[19] Cleusix V, Lacroix C, Vollenweider S, Duboux M, Le Blay G. Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria. BMC Microbiology. 2007;7(1).
[20] Chen L. Exploring the Propanediol Utilization Pathway in Lactobacillus reuteri. Lund: Division of Biotechnology, Lund University, 2018. 180 p.