Design And Application Of Bacterial Regulation Systems Responsed To Quorum Sensing And In Vivo Signals

Bacterial vector vaccine has become a hotspot in vaccine research field because of its superiorities of including mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes. High expression efficiency and biosafety are crucial for bacterial vaccine design. In order to improve antigen expression and reduce metabolic burden, alternative expression strategies, such as the use of in vivo inducible expression systems, have been proposed. This work focused on the design of antigen expression systems by rewiring or integrating quorum sensing gene circuits with in vivo inducible regulatory elements. These established systems presented many advantages such as high expression efficacy, rigorous regulation and broad host range and performed well both in in vitro cultivation and in in vivo systems including cell and animal models. In order to explore the potential use of the "bacterial density- in vivo signal" dual-regulated expression systems in biotechnology, the dual-regulated expression systems were used to express a heterologous protective antigen GAPDH from Aeromonas hydrophila in an attenuated fish-originated bacterium Edwardsiella tarda, and the resultant recombinant E. tarda strain, being administrated to fish host by injection, could evoke an effective immune protection against the challenge of both Edwardsiella tarda and Aeromonas hydrophila. Our data suggested that the dual-regulated expression system had great potential in establishing multi-valence bacterial vector vaccine. Besides, in order to improve the biosafety of bacterial vaccines, some lysis systems were designed by integrating lysis genes with quorum sensing or in vivo inducible regulatory elements, and their applications in bacterial vaccines were preliminary investigated.Firstly, based on luxI-luxR quorum sensing system in Vibrio fischeri, a series of expression systems were constructed and tested in many respects such as efficiency, yield and host adaptation. The ideal candidate screened from above systems possessed preeminent characteristics such as a high expression efficiency, a high strictness regulated by cell density and broad host adaptability.Then, based on in vivo signal characteristics lack of free Fe2+ and arabinose, genetic components were added into the quorum sensing expression circuits to form protein expression systems controlled by in vivo signal and bacterial density. (1) An iron-density dual-regulated system was constructed by integrating iron-limiting genetic elements (such as iron-limiting promoter PviuA and iron-uptake protein binding site Fur box) with quorum sensing gene. (2) An arabinose-density dual-regulated system was also constructed via expressing quorum quenching protein AiiA controlled by ParaBAD.Both of the two systems could shut down strictly during in vitro cultivation, and once simulating in vivo environment (iron-limiting or arabinose-limiting) after reaching a threshold of cell density, they would unlock quorum sensing systems and start protein expression immediately. In in vivo circumstance, the systems carrying reporter gene katushka presented strong red fluorescence after infecting J774 macrophage or zebrafish larvae. Fluorescence value enhanced with bacterial multiplication, reflecting favorable dual-regulating feature.To prepare bacterial vector vaccines, two systems carrying gap A34 gene of Aeromonas hydrophila LSA34 encoding protective antigen GAPDH were introduced into Edwardsiella tarda WED, respectively. The resultant vaccines were evaluated through turbot(Scophtalmus maximus) vaccination. Most vaccinated fish could defense the challenge with both A. hydrophila and E. tarda, indicating these two in vivo-inducible expression systems performed well in the application of bacterial vector vaccine.Finally, based on the concept of inducible suicide of bacterial host to improve its biosafety, several in vivo-inducible lysis systems were designed and their applications in bacterial vaccines were preliminary investigated. Quorum sensing circuit and iron-regulated promoters were respectively integrated with lysis genes to construct in vivo-inducible lysis systems. The resultant system remained closed during in vitro cultivation and presented satisfactory lysis in vivo. Vibrio anguillarum wildtype MVM425 loaded with this system lysed in response to the iron-limiting signal after infecting zebrafish. Further, the controllable bacterial lysis system, after being transformed into a live attenuated V. anguillarum vaccine strain MVAV6203, was confirmed to significantly improve biological safety of the live attenuated vaccine without impairing its immune protection efficacy.