Listeria monocytogenes is a gram-positive, foodborne pathogen responsible for the disease listeriosis. It is a particular threat as it can survive a range of pHs as well as temperatures as low as 0°C allowing it to survive in industrial and domestic refrigerators. This pathogen commonly contaminates unpasteurised dairy products, particularly soft cheeses, as well as fresh vegetables, cooked meats and seafood (Gandhi and Chikindas, 2007).
The presence of as little as 100 L. monocytogenes cells per gram of food can lead to symptoms ranging from fever, diarrhoea, and muscle aches to more severe ailments such as meningitis and sepsis (Vázquez-Boland, J.A. et al., 2001). Whilst in the majority of healthy people listeriosis manifests itself with flu-like symptoms, it can be fatal for immunosuppressed individuals like people suffering from AIDS or pregnant women who can suffer from fetal infections and miscarriage.
The Problem & Our Original Solution
Currently, there are tests to detect the presence of Listeria. These include the Anton Test (Hitchens, D. et al., 2017) or the Rapid Neogen Test which both have their own drawbacks. The Anton Test is time-consuming, taking 24 hours and involves inoculating the conjunctiva of a guinea pig or rabbit to invoke an immune response. In contrast, the ANSR® Listeria Right Now™ test by Neogen only takes 18 minutes, however, the machinery required is expensive. To buy the 16-tube reader only used with the test kits costs USD10,918.00, while purchasing a basic system including all equipment required for testing costs USD14,420.00. The test kits themselves are available to buy for USD1,483.00 for a pack of 96 (Neogen Ordering Website). Though these products would be invaluable, these costs may be prohibitive for small-scale businesses such as artisan cheesemakers.
Our project aims to improve Listeria testing, particularly in soft cheeses, by developing a biosensor that is fully integrated into the soft cheese starter culture. The biosensor would be based on Listeria's quorum sensing mechanism and would detect the signalling molecule released by the pathogen. The signalling molecule, an autoinducing peptide (AIP), will trigger a significant colour change (to purple) in the cheese product, allowing for fast detection of Listeria either during the cheese making process or at any point of the product's shelf life. We hope to accomplish this by transferring the AIP responsive genes from Listeria monocytogenes into Lactococcus lactis, the most common lactic acid bacteria species utilised as a starter culture for cheese manufacturing.
Agr Quorum Sensing in Listeria monocytogenes
The agr gene regulatory system is involved in the biofilm formation and virulence in L. monocytogenes (Zetzmann, M. et al., 2016). This agr quorum sensing system is highly specific to L. monocytogenes and relies on the production of AIP through genes agrA/B/C/D. AgrC is a membrane protein which detects extracellular AIP and induces intracellular cascade leading to the phosphorylation of a transcription factor AgrA. Once phosphorylated, AgrA upregulates transcription of agrD/B which produces and secretes more AIP (Figure 1).
Figure 1. Native Listeria monocytogenes agr regulatory system.
What is AIP?
The AIP encoded by agrD is highly specific to L. monocytogenes despite the agr operon being present in other pathogenic species such as S. aureus (Le and Otto, 2015). AIP is a cyclic pentapeptide with the amino acid sequence CFMFV forming a thiolactone ring and acts as a signalling molecule in a quorum sensing mechanism in Listeria.
Detection & Response
Modified detection strain will contain agrC and agrA to detect AIP and cause a response. Activation of AgrA through histidine kinase phosphorylation will then cause AgrA binding to the PII promoter and induce transcription of chromoprotein amilCP as a reporter (Figure 2).
Figure 2. Lactococcus lactis detection and response system
Since we are not allowed to work with Listeria in the lab, we still need to be able to check if the response by L. lactis bacteria is produced. For this purpose, we designed Escherichia coli which is able to synthesise the Listeria specific AIP molecule to act as a 'pseudo-Listeria' (Figure 3).
Figure 3. AIP production in Escherichia coli
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Neogen Ordering Website (accessed 17/10/2018) https://order.neogen.com
Le, K. and Otto, M. (2015). Quorum-sensing regulation in staphylococci—an overview. Frontiers in Microbiology, 6.
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