(222e) Bloodmeal-Induced Inhibition of Plasmodium infection in Mosquito Vectors Using the Microbial Symbiont Asaia

Asaia sp. are gram-negative bacteria that have been shown to colonize the Anopheles mosquito midgut. Species of this mosquito are the vectors for malaria transmission throughout the world. Using a paratransgenic strategy in which transgenically modified symbiotic organisms affect their host’s phenotype, Asaia has been engineered to secrete anti-malarial effector molecules into the mosquito midgut, hindering its ability to carry Plasmodium. However, the constitutive expression of these effectors causes a fitness disadvantage to the transgenic Asaia.Therefore, it is desirable to express these molecules only when Plasmodiumis present in the mosquito midgut, namely when a mosquito takes an infected blood-meal. Putative conditional promoters have been cloned into the plasmid pGLR1, which has a promoterless GFP reporter. These plasmids were then transformed into the lab strain Asaia sp.SF2.1 and plated on minimal media enriched with blood. GFP fluorescent colonies were collected and screened both in-vitro, in liquid and solid media, as well as in-vivo, inside the mosquito midgut, to isolate the promoters that were only induced when blood meal conditions were present. Once the conditionality of these promoters was validated, they were cloned into a plasmid secreting the anti-malarial effector scorpine and transformed into Asaia. The increased fitness of these conditional strains was validated against a constitutively expressing control before testing each strain for Plasmodium inhibition. The anti-malarial strains were cultured in mosquitoes that were then fed on Plasmodiuminfected mice. The conditionally active promoter strains showed a significant reduction from the wild-type bacteria in the number of oocysts the pathogen formed as well as a significant reduction in disease prevalence effectively inhibiting the mosquitoes’ ability to transmit malaria.