SYMBIOTIC CHIMERAS: NOVEL IMMUNOLOGICAL PROPERTIES OF ASAIA SYMBIONTS ENGINEERED TO BE COVERED BY THE WOLBACHIA SURFACE PROTEIN

Vector-borne diseases represent one-sixth of all infectious diseases and cause more than 1,000,000 deaths every year. The World Health Organization (WHO) proposes the adoption of sustainable integrated vector management, which includes strategies such as environmental remediation, information and education of the population, but also the integration of classical chemicals (e.g. insecticides) with non-chemical control methods for the control of arthropod vectors. Among these strategies, the symbiotic control (SC), which exploits microorganism symbionts of the insects, is getting more and more attention as it is particularly promising for the control of vector-borne diseases. Particularly, in this thesis, I studied the potentiality of bacteria belonging to two genera: Asaia and Wolbachia. The former includes acetic acid bacteria, symbionts of many insects such as mosquitoes and recently proposed as good candidates for paratransgenesis-based control strategies. The latter includes symbionts of arthropods and nematodes, with the capability to stimulate innate immune responses in mosquitoes with a reduction of their vectorial capability. We thus engineered the bacterium Asaia to express the Wolbachia surface protein (WSP), a potential inductor of innate immunity, derived from Wolbachia infecting the nematode Dirofilaria immitis (AsaiaWSP). My project has two main aims: i) to test the capability of the bacterium AsaiaWSP to stimulate the immune system of mosquitoes and thus to interfere with D. immitis development after infection; ii) to verify if AsaiaWSP is able to induce a macrophage polarization during the immune response towards the M1/Th1 phenotype and if this polarization could determine an anti-Leishmania effect. The obtained results to achieve these aims have been summarized in two articles: Article 1 The first study, here presented, consolidates the previous evidence on the immune-stimulating property of WSP. In fact, WSP from Wolbachia of D. immitis was shown to stimulate the immune response in mosquitoes and mammals (humans, rodents, dogs) in vitro, but here for the first time an engineered bacterium expressing WSP was tested. AsaiaWSP activated the expression of immune genes coding for effector molecules in Ae. aegypti and An. stephensi mosquitoes. Once verified the stimulation of the immune system of the mosquitoes, the capability to inhibit the development of the nematode D. immitis, ethiological agent of dirofilariasis, in the vector Ae. aegypti was investigated. We obtained a first evidence of an inhibition of D. immitis larval development after the infection of mosquitoes with the engineered bacterium AsaiaWSP. Article 2 In the second paper, I focused my attention on leishmaniases, vector-borne diseases widespread in more than one hundred countries in tropical, sub-tropical and temperate zones and caused by the protozoan parasite Leishmania. I investigated the capability of the chimeric bacterium AsaiaWSP to polarize the immune response towards the M1/Th1 phenotype, which is protective for the host; in fact, macrophage polarization towards the M1/Th1 or M2/Th2 side is crucial for the outcome of the visceral leishmaniasis. The chimeric bacterium AsaiaWSP acted as a polarizing agent, stimulating the phagocytosis and inducing the release of M1/Th1 cytokines, ROS and the expression of iNOS. Then, AsaiaWSP determined an anti-leishmanial effect with a reduction of the number of intracellular parasites. In conclusion the modified bacterium Asaia, here proposed, appears as a promising candidate for paratransgenesis-based control strategies for the control of vector-borne diseases in general, and as a successful immunomodulator, which could be used in combination with the classical chemotherapeutic agents for the treatment of leishmaniases and other M1-impaired diseases.