falciparum and the rodent parasite P. berghei in the mosquito was efficiently suppressed by up to 98%.
Results
Administration of Transgenic P. agglomerans via Sugar Meals and Rapid Proliferation After a Blood Meal. P. agglomerans is a natural symbiotic bacterium whose occurrence is widespread in anopheline mosquitoes (15–17). Our strain was originally isolated from midguts of an Anopheles gambiae laboratory colony and subse- quently further selected for improved adaptation to the A. gambiae and A. stephensi midgut environments (17). To examine recombi- nant P. agglomerans colonization and survival in the mosquito midgut, we transformed P. agglomerans with a highly stable plasmid pPnptII:gfp (19) that expresses green fluorescent protein (GFP). We found the GFP-tagged bacteria were easily administered to mosquito midguts via sugar meals (Fig. S1). Midguts of female mosquitoes that had been fed GFP-tagged P. agglomerans became strongly fluorescent after ingestion of a blood meal (Fig. 1 A and B), indicating rapid bacteria proliferation. To quantify the dynamics of bacteria numbers, mosquitoes that had been fed GFP-tagged P. agglomerans were dissected at different times after a blood meal and midgut homogenates were plated on se- lective kanamycin-containing plates. Bacteria numbers increased dramatically by more than 200-fold during the first 2 d after a blood meal (Fig. 1C). Thereafter, bacteria numbers decreased to about prefeeding levels, presumably because the majority was excreted together with the remnants of blood digestion.
Recombinant P. agglomerans Strains Efficiently Secrete Anti-Plasmodium Effector Molecules. As mentioned previously, for maximum effec- tiveness of intervention, it is crucial that the effector molecules be secreted from the engineered bacteria to allow diffusion to the
falciparum and the rodent parasite P. berghei in the mosquito was efficiently suppressed by up to 98%.ResultsAdministration of Transgenic P. agglomerans via Sugar Meals and Rapid Proliferation After a Blood Meal. P. agglomerans is a natural symbiotic bacterium whose occurrence is widespread in anopheline mosquitoes (15–17). Our strain was originally isolated from midguts of an Anopheles gambiae laboratory colony and subse- quently further selected for improved adaptation to the A. gambiae and A. stephensi midgut environments (17). To examine recombi- nant P. agglomerans colonization and survival in the mosquito midgut, we transformed P. agglomerans with a highly stable plasmid pPnptII:gfp (19) that expresses green fluorescent protein (GFP). We found the GFP-tagged bacteria were easily administered to mosquito midguts via sugar meals (Fig. S1). Midguts of female mosquitoes that had been fed GFP-tagged P. agglomerans became strongly fluorescent after ingestion of a blood meal (Fig. 1 A and B), indicating rapid bacteria proliferation. To quantify the dynamics of bacteria numbers, mosquitoes that had been fed GFP-tagged P. agglomerans were dissected at different times after a blood meal and midgut homogenates were plated on se- lective kanamycin-containing plates. Bacteria numbers increased dramatically by more than 200-fold during the first 2 d after a blood meal (Fig. 1C). Thereafter, bacteria numbers decreased to about prefeeding levels, presumably because the majority was excreted together with the remnants of blood digestion.Recombinant P. agglomerans Strains Efficiently Secrete Anti-Plasmodium Effector Molecules. As mentioned previously, for maximum effec- tiveness of intervention, it is crucial that the effector molecules be secreted from the engineered bacteria to allow diffusion to the
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