Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria

Abstract

The huge burden of malaria in developing countries urgently demands the development of novel approaches to fight this deadly disease. Although engineered symbiotic bacteria have been shown to render mosquitoes resistant to the parasite, the challenge remains to effectively introduce such bacteria into mosquito populations. We describe a Serratia bacterium strain (AS1) isolated from Anopheles ovaries that stably colonizes the mosquito midgut, female ovaries, and male accessory glands and spreads rapidly throughout mosquito populations. Serratia AS1 was genetically engineered for secretion of anti-Plasmodium effector proteins, and the recombinant strains inhibit development of Plasmodium falciparum in mosquitoes.

Fig. 1.. Serratia AS1 bacteria stably colonize the midgut and rapidly proliferate after a blood meal.

Serratia AS1 tagged with eGFP (AS1-GFP) was fed to 3-day-old A. stephensi mosquitoes in a sugar meal for 24 hours, then mosquitoes were allowed to feed on a blood meal. (A) Population dynamics of AS1-GFP. Fluorescent bacteria colony-forming units (CFUs) were determined by plating serially diluted homogenates of midguts on Luria-Bertani (LB) agar plates containing 100 μg/ml of kanamycin. Data were pooled from three biological replicates (shown are means ± SEM). **P < 0.01 (Student’s t test). The maximum bacteria number is reached when Plasmodium ookinetes would be invading the midgut if the blood were infected with the parasite. h, hours; d, days. (B) AS1-GFP in the midgut of a female at 24 hours after blood ingestion (left). On the right is a control mosquito.

Fig. 2.. Serratia AS1 colonizes the reproductive organs of A. gambiae.

(A) Serratia AS1 colonization of female ovaries. AS1-GFP was fed to 2-day-old A. gambiae mosquitoes. Three days after females ingested blood, ovaries were dissected and visualized by fluorescent microscopy. Females not infected with AS1-GFP (right) were used as controls. (B) Serratia AS1 attaches to laid eggs. The egg in the upper image was laid by a mosquito that had been fed AS1-GFP and shows bacteria attachment to the chorion ridges. (C) Serratia AS1 colonization of male accessory glands. AS1-GFP was fed to newly emerged male mosquitoes. Three days later, the male reproductive organs were dissected and visualized by fluorescent microscopy. Scale bars, 100 μm.

Fig. 3.. Serratia AS1 bacteria efficiently spread throughout multiple mosquito generations.

A total of 190 virgin females, 190 virgin males, 10 virgin females fed with AS1-mCherry, and 10 virgin males fed with AS1-GFP were added to a cage. After 3 days, these mosquitoes were fed blood and allowed to lay eggs. The resulting larvae were reared to adulthood following standard protocol. Bacterial load (CFUs) was determined by plating serial dilutions of tissue homogenates on LB agar plates containing 100 μg/ml of kanamycin and counting fluorescent colonies (fig. S9). (A) CFUs per fourth-instar larva gut. (B) CFUs per male midgut. (C) CFUs per male accessory gland. (D) CFUs per female midgut. (E) CFUs per female ovary. G1, G2, and G3 stand for first, second, and third generation, respectively. Values are means ± SEM from 10 to 15 mosquitoes in one experiment. The experiments were repeated three times with similar results. *P < 0.05; ns, not significant (Student’s t test).

Fig. 4.. Inhibition of P. falciparum infection of A. gambiae by recombinant Serratia strains that are engineered to secrete anti-Plasmodium effector molecules.

(A) Western blot analysis of protein secretion. Culture supernatants were concentrated using Amicon Ultra-4 centrifugal filter units. Aliquots originating from equal supernatant volumes were analyzed by Western blotting using an antibody against E-tag. The culture supernatant of wild-type Serratia AS1 (WT) served as a negative control. HasA, secretion leader; Shiva1, a cecropin-like synthetic antimicrobial peptide; EPIP, enolase-plasminogen interaction peptide (lysine-rich enolase peptide); MP2, midgut peptide 2; mPLA2, inactive bee venom phospholipase A2; scorpine, scorpion Pandinus imperator venom antimicrobial peptide. (B) Western blot analysis of the multiprotein secretion encoded by five different effector genes (fig. S10). (C) Inhibition of P. falciparum infection of A. gambiae by recombinant Serratia strains. The recombinant bacteria were introduced into A. gambiae females in a sugar meal, except in the case of controls, which were fed no AS1 bacteria (–Bact). Multi, a fusion protein composed of MP2, scorpine, (EPIP)₄, Shiva1, and (SM1)₂ peptides (fig. S10). In the graph, circles represent the number of oocysts in individual midguts, and horizontal lines indicate the median number of oocysts per midgut (also listed in the table below). Data were pooled from two biological replicates. N, number of mosquitoes analyzed; inhibition %, percent inhibition of oocyst formation relative to the –Bact control. Statistical comparisons of oocyst intensities to those in the –Bact control were performed using the Mann-Whitney test.