We engineered autotransporters Antigen43 (Ag43) and Ice-Nucleation protein (INP) to successfully express surface epitopes in E.coli, that mimic the two different trypanosomal antigens. Therefore, we created standardised and user-friendly Ag43 and INP BioBricks ready for peptides of choice to be cloned in. Two E.coli strains were generated, each expressing a distinct surface epitope and either a quorum sensing sender or receiver module respectively. We delivered a proof-of-principle demonstration of our project, by showing that the quorum-based system is capable of detecting cell surface Ag43-FLAG ‘Receiver’ E.coli in an anti-FLAG bead pull-down. These ‘Receivers’ co-cultured with AHL-synthesising ‘Senders’ produced a strong –FLAG-tag dependent, AHL-inducible GFP response. This system if applied in a real-life scenario could effectively diagnose a neglected tropical disease such as Trypanosomiasis, using patient serum-derived antibodies, with minimal facilities.

The user-friendly, portable, inexpensive GFP detection device

We constructed a cheap, Raspberry Pi computer-controlled fluorimeter suitable for developing countries, showing it could detect QS fluorescence output. The detection device costs around $100 and could replace heavy and expensive microscopes, to make the detection system affordable to remote populations in Africa.

The user-friendly, portable, inexpensive GFP detection device

This novel application to diagnose patient exposure to tropical pathogens integrates antigen display and quorum sensing to implement AND logic sensing. It facilitates cheap, simple diagnosis of neglected tropical diseases such as HAT. Future diagnosis kits can be constructed and modularized using the cheap and self-replenishing E. coli based detection system we created as a platform for displaying two different mimotopes. Mimotopes are shortened versions of real epitopes identified for a panel of diseases, all of which are currently stored in a universal database, available to everyone and known as MimoDB [http://immunet.cn/mimodb]. Moreover, it was recently shown that upon heat-treatment of Ag43 autotransporter-foreign peptide complex at 60°C, a chimeric form of the expressed foreign protein can be isolated and remain fully active. The chimeric protein was then used to induce an immune response and to develop a novel vaccine for cancer therapy ((Huang et al., 2014)). Therefore, we suggest that further research can be done on Ag43-trypanosomal mimotope/ other mimotope complexes for a potential first vaccine for HAT/ other diseases.

Reference: Huang, F., Li, L., Liu, Q., Li, Y., Bai, R., Huang, Y., Zhao, H., Guo, J., Zhou, S., Wang, H. and others, (2014). Bacterial surface display of endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis. International Journal of Cancer, 134(8), pp.1981--1990.