Team:BABS UNSW Australia/project/futurework
Artificial endosymbionts are still far from being a reality. Our project aimed to lay the foundation for enabling the entry of symbionts into cell. However, once the symbiont is safely inside, there are many further hurdles. We had aimed to investigate them, but time constraints mean this work will be left to future teams.
Population Control
Mammalian cells replicate and divide on average once every 24 hours. Most bacteria replicate much more rapidly. This causes the risk of symbionts gradually overwhelming mammalian cells. To combat this, strategies to dynamically control population size are required.
Our idea: AHL-mediated quorum sensing
- Quorum sensing (i.e. inter-bacterial communication through small molecules) allows many natural bacterial communities to regulate traits based on population density. A common example is the regulation of pathogenic traits in Pseudomonas aeruginosa. It will only produce certain virulence factors if the bacterial cell concentration is high enough to actually have an effect on the host.
- AHL-mediated quorum sensing is very well characterised, and several iGEM teams have explored using it as a feedback system for dynamic cell regulation. AHL molecules are constitutively produced by bacteria - if the cell density reaches a threshold concentration, these AHL molecules induce further expression of AHL. This usually results in a “public good” - for example the production of a digestive enzyme, or bioluminescent substrate. In our system, instead of producing a public good, high AHL concentrations would induce expression of an antibiotic targeting the endosymbiont species. This would effectively limit cell densities.
Transmission
For endosymbionts to truly be permanent fixtures of human hosts, they must be transmitted to daughter cells during somatic cell division. We explored linking them cell spindles, but decided this would impede cell division. Instead, we turned to nature for inspiration. In natural endosymbiont-host relations, the are two key mechanism for transfer of endosymbionts between generations - either horizontal or vertical transmission. In vertical transmission, the symbiont is passed directly from mother to host. Due to the lack of selection pressures deleterious mutations build up, leading to loss of gene function. This eventually causes streamlined symbionts genome and an increased reliance and concordance with the host. In horizontal transmission, the symbiont is regained anew from the external environment every generation. This aposymbiotic (external to the cell) phase means the symbiont will never be as host-dependent as vertically transmitted.
This indicates that for sustainable endosymbionts to be cultivated, a guided evolution approach would require them to be vertically transmitted - particularly from cell to cell (not from parent to progeny, at this stage).
This would encourage the symbiont to be retained and for closer links to be forged - until the endosymbiont eventually crossed the semantic boundary into being a synthetic organelle. A logical model is mitochondrial transmission during somatic cell division. It is a poorly characterised mechanism, but recent research indicates that mitochondrial partitioning in symmetrically-dividing cells, while largely a passive process, is also due to host cell cycle-linked mitochondrial membrane fission and interactions with the cell cytoskeleton and endoplasmic reticulum. Little is known about mitochondrial partitioning in asymmetrically-dividing cells [1]. Budding yeast models have shown that plasma membrane anchors in host cell members are able to retain mitochondria when a certain quota of mitochondria have been transmitted to the bud. The mechanism for monitoring levels is unknown.
Engineering endosymbionts to be transmitted in a similar fashion would allow for better understanding of these natural processes - i.e. you can only understand a system once you have built it.
Reference
[1] Mishra, P., & Chan, D. C. (2014). Mitochondrial dynamics and inheritance during cell division, development and disease. Nature Reviews Molecular Cell Biology, 15(10), 634-646.Cell-specific invasion
Invasin proteins allow for entry into cells with beta-integrin receptors. By creating a suite of invasin-like or adhesin proteins that target specific cell types, endosymbionts could be localised to desired tissue only. Medical microbiological research has lead to the discovery of a plethora of specific receptors. While beta-integrins receptors are found embedded in most cellular membranes, certain receptors are far more specific. Salmonella enterica serovar typhibinds to the cystic fibrosis transmembrane receptor (CFTR) on the intestinal epithelium and through this interaction is translocated to the gastric epithelium [1].
Reference
[1] Lyczak, J. B., & Pier, G. B. (2002). Salmonella enterica serovar typhi modulates cell surface expression of its receptor, the cystic fibrosis transmembrane conductance regulator, on the intestinal epithelium. Infection and immunity, 70(11), 6416-6423.Post-Translation Modifications
Post-translational modifications of proteins are different in bacteria and eukaryotes, and these changes contribute significantly to product equivalence and immunogenicity. A range of processes must be considered - glycosylation, gamma-carboxylation, beta-hydroxylation, amidation, sulfidation and more [1]. These modifications are sources of variability, and in the case of therapeutic biologicals, the exact product must be delivered to ensure a non-deleterious effect. The first challenge with regard to post-translation modifications would be to engineer bacterial enzymes capable of catalysing the changes. The second challenge would be to ensure a consistent, accurate final protein product. Rigorous, repeated testing would be necessary prior to use in humans.
Reference
[1] Walsh, G., & Jefferis, R. (2006). Post-translational modifications in the context of therapeutic proteins. Nature biotechnology, 24(10), 1241-1252.