Team:UCC Ireland/Limitless Lactis
Our L.lactis strain
Numerous features of our L.lactis strain make it ideal as a platform for producing therapeutic proteins. First of all, it is extremely safe for human consumption. L.lactis is a Generally Recognised as Safe (GRAS) bacterium which is routinely used in the production of dairy products. It is naturally non-pathogenic, non-invasive and non-colonising, meaning that there is virtually no risk of it causing an infection in humans. This gives it an advantage over attenuated pathogenic bacteria used for similar purposes (such as Listeria Monocytogenes and Salmonella Typhimurium) as there is no chance of it reverting to a pathogenic form. Furthermore, L.lactis has been shown not to have any determinants of antibiotic resistance commonly used in humans. Thus, it is ideal for use in a clinical setting, particularly given that the individuals receiving the L.lactis will be weakened or immunocompromised.
Our L.lactis strain is also ideal for therapeutic protein production due to its numerous genomic modifications. First of all, it has the hlyA gene integrated into its chromosome. Originally found in Listeria Monocytogenes, the hlyA gene encodes a pore forming protein which allows escape of the bacterium from phagosomes.(1) This allows the L.lactis to escape from vesicles once phagocytosed and secrete the therapeutic protein into the cytosol of the phagocyte, as opposed to being degraded within the phagosome.
Another advantageous genomic modification of our L.lactis strain is the deletion of the Hrt A gene. This gene encodes a protease which is attached to the extracellular surface of L.lactis. It has been shown to reduce the secretion efficiency of protein secretion by degrading proteins once they have been released from the cell.(2) As a results, strains of L.lactis from which it has been removed or degraded are more efficacious at releasing recombinant proteins into their extracellular environment. This modification of our L.lactis strain offers another key benefit to the system. It ensures that proteins produced in the bacterium can leave the cell properly and reach the cytosol off the phagocyte which has taken it in.
Our L.lactis also has also been genetically modified to allow for biological containment. These are important features as accumulation of the bacteria in the healthcare environment in which it would be used could raise safety concerns. One important modification is knockout of the PyrG gene. This encodes a CTP synthase which is unique to L.lactis, catalysing the conversion of UTP to CTP.(3) Knockout of this gene means that the L.lactis cannot replicate in an environment with no free cytidine, thus contributing to the cessation of its growth in the wild.
Finally, our L.lactis strain is suitable for oral administration. This property is highly important for our project. If it is to be used for the delivery of a vaccine in the third world, administration must be simple and inexpensive. Of course, oral administration is the obvious choice for simplicity and economic purposes. L. lactis is regularly ingested by humans in milk and cheese, and thus is extremely safe. Furthermore, with other potential modifications, such as the addition of genes which cause trehalose accumulation (and thus increased viability in acidic conditions such as the gastric mucosa), our L.lactis strain could be extremely useful as a vaccine delivering platform.(4) Thus, our protein producing platform has great potential for oral administration in disadvantaged regions. Bibliography
1. Hernandez-Flores KG, Vivanco-Cid H. Biological effects of listeriolysin O: implications for vaccination. BioMed research international. 2015;2015:360741.
2. Sriraman K, Jayaraman G. HtrA is essential for efficient secretion of recombinant proteins by Lactococcus lactis. Applied and environmental microbiology. 2008;74(23):7442-6.
3. Bahey-El-Din M, Casey PG, Griffin BT, Gahan CG. Efficacy of a Lactococcus lactis DeltapyrG vaccine delivery platform expressing chromosomally integrated hly from Listeria monocytogenes. Bioengineered bugs. 2010;1(1):66-74.
4. Carvalho AL, Cardoso FS, Bohn A, Neves AR, Santos H. Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance. Applied and environmental microbiology. 2011;77(12):4189-99.
