Prevention of insect-borne disease: an approach using transgenic symbiotic bacteria

Abstract

Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting insects may serve as a powerful approach to control certain arthropod-borne diseases. The endosymbiont of the Chagas disease vector, Rhodnius prolixus, has been transformed to express cecropin A, a peptide lethal to the parasite, Trypanosoma cruzi. In insects carrying the transformed bacteria, cecropin A expression results in elimination or reduction in number of T. cruzi. A method has been devised to spread the transgenic bacteria to populations of R. prolixus, in a manner that mimics their natural coprophagous route of symbiont acquisition.

Figure 1

Concentrations of synthetic cecropin A lethal for E. coli, the R. rhodnii symbiont, and three strains for T. cruzi. Values for E. coli and R. rhodnii are minimum bactericidal concentrations (MBC). The values for T. cruzi (epimastigote forms derived from cell culture) are concentrations needed to kill 100% of parasites at 24 h of incubation (LC). The DM28 strain was used for our in vivo experiments and is a common human Chagas disease pathogen in Brazil.

Figure 2

The shuttle plasmid pRr Thio Cec.

Figure 3

Western blot. Lane 1, synthetic cecropin A, 5 μl of a 1.0 mM aqueous solution (4.1 kDa). Lanes 2–6, hind gut preparations from five third-instar nymphs of R. prolixus (R₁–R₅) carrying R. rhodnii, which has been transformed to express the cecropin A gene product. Bioassays (Fig. 4) showed that all five specimens (R₁–R₅) had significant biological activity attributable to cecropin A, though only R₄ and R₅ had sufficient cecropin A to be detected in our Western blot system.

Figure 4

Assays for biological activity. GA, samples from R. prolixus nymphs carrying the R. rhodnii symbiont transformed to express the cecropin A gene product. WT, samples from R. prolixus nymphs carrying native symbionts. Synthetic CecA, control incubations with 10 μl sterile Luria–Bertani medium. Gut contents from insects carrying the cecropin A-expressing symbionts markedly inhibited the growth of E. coli.

Figure 5

Number of T. cruzi in the hind gut of R. prolixus in control (black bars) R. prolixus carrying native symbiotic R. rhodnii in the hind gut, and (hatched bars) recombinant group where R. prolixus carry genetically modified R. rhodnii, which express the gene for cecropin A peptide. Unstained metacyclic trypomastigotes were counted, using a Neubauer hemocytometer, and the count number is expressed as log₁₀. All values are the mean of four measurements. No trypanosomes were seen in recombinant insects 2, 3, 4, 6, and 7, and the hatched bars in these columns indicate that counts were performed.

Figure 6

Survival of aposymbiotic first-instar nymphs of R. prolixus exposed to CRUZIGARD. Thirty nymphs were exposed to CRUZIGARD impregnated with genetically transformed R. rhodnii. Fifteen nymphs were exposed to CRUZIGARD containing no added bacteria. Aposymbiotic first-instar nymphs exposed to natural feces in our insectary experience, on average, 50% mortality to the adult stage.