Scientists say engineered mosquitoes that overexpress a transcription factor controlling natural malarial parasite immune responses could represent a viable approach to preventing the spread of human malaria parasites in the field. A team at the W. Harry Feinstone Department of Molecular Microbiology and Immunology at Johns Hopkins University Bloomberg School of Public Health developed transgenic Anopheles stephensi mosquito lines that overexpressed Rel2 when they took a blood meal. The vectors were found to be resistant to human Plasmodium falciparum malarial parasites and to microbial infection without any significant reduction in survival fitness.
Reporting in PLoS Pathogens, George Dimopoulos, Ph.D., and colleagues suggest that their work shows, for the first time, that the mosquito’s innate immune system can be harnessed in a genetic engineering approach to develop a control strategy for human malaria. Importantly, this approach also doesn’t involve the introduction of foreign genes into the vectors. The authors report their work in a paper titled, “Engineered Anopheles immunity to Plasmodium infection.”
The transmission of malaria parasites between human hosts depends on the Plasmodium parasite completing its life cycle in the Anopheles mosquito vector. The mosquito’s innate immune system does, however, exhibit defenses against Plasmodium at multiple stages of parasite infection, and the most potent of the anti-Plasmodium immune factors identified to date are all controlled by the IMD pathway, via the transcription factor Rel2.
Previous work by the team has demonstrated that knocking out a negative regulator of Rel2, known as Caspar, renders three major Anopheles species vectors resistant to the human P. falciparum parasite, and that overexpressing Rel2 in transgenic mosquitoes results in increased resistance to an avian malaria parasite. This work indicated that such modifications have no detrimental effect on the fitness of the engineered vector, suggesting the approach could be used for the development of malaria-control strategies based the spread of P. falciparum-resistant mosquitoes.
The latest work by the Johns Hopkins team led by Dr. Dimopoulos aimed to develop an immuno-enhanced, P. falciparum-resistant strain of Anopheles stephensi mosquito that expresses an IMD pathway-controlled NF-kB Rel2 transcription factor when it takes a blood meal, to enhance immune responses at the earliest stage of infection.
The researchers tested three transgenes that triggered overexpression of the active form of Rel2 either in the mosquito’s midgut or fat-body tissues, under the control of a blood meal-inducible carboxylpeptidase (Cp) or vitellogenin (Vg) promoter, respectively. Cross-breeding allowed the generation of third hybrid transgenic line (Hyb) demonstrating blood meal-inducible expression of Rel2 in both the midgut and fat-body tissue compartments.
Initial results indicated that blood meal-induced overexpression of Rel2 in the transgenic mosquito resulted in significantly increased transcript abundance of several IMD pathway-regulated antimicrobial and anti-Plasmodium genes. When the three immune-enhanced transgenic mosquito lines were allowed to feed on different P. falciparum gametocyte cultures, all were found to be significantly less susceptible to the highly virulent P. falciparum strain NF54 at the pre-oocyst stage, and exhibited marked reductions in infectious sporozoite-stage parasites in their salivary glands.
An insight into the mechanism behind this enhanced resistance was provided by experiments demonstrating that silencing the Rel2-regulated anti-Plasmodium factors, TEP1, APL1, and LRRD7 resulted in a greater susceptibility to P. falciparum infection in the transgenic mosquitoes. The data indicated that each of the anti-Plasmodium factors was at least partially responsible for the transgene Rel2-mediated resistance, because their silencing didn’t lead to in an increased infection level to the same degree as did silencing the same genes in wild-type mosquitoes. “This observation, taken together with the blood meal-inducible transcription of these genes, suggests that Rel2-dependent immune activation generates an anti-Plasmodium response through a combination of effector molecules that can each attack the parasite independently or synergistically,” the team states.
Interestingly, the immune-enhanced transgenic mosquitoes, and especially the hybrid strain that expressed Rel2 in both the midgut and fat-body tissues, demonstrated increased resistance to systemic challenge with both Gram-negative and Gram-positive bacteria. Because prior research had indicated that the mosquito’s midgut microbioal flora triggers basal activation of the IMD pathway-regulated immune genes—and therefore influences resistance to Plasmodium—the researchers carried out infection assays to see whether the presence or absence of midgut bacteria would influence resistance of the transgenic immune-enhanced mosquitoes to Plasmodium infection. Encouragingly, while removing the majority of midgut bacterial flora through antibiotic treatment increased the susceptibility of wild-type Anopheles to Plasmodium infection, the increased resistance of the immune-enhanced transgenic mosquitoes wasn’t affected, “suggesting that the recombinant Rel2 prevails over the activity of the bacteria-inducible endogenous Rel2,” the team writes.
If immune-enhanced mosquitoes are to represent a viable approach to preventing malaria spread in the field, it’s important that the vectors are not at a disadvantage in terms of lifespan or reproductive capacity, compared with wild-type mosquitoes. Initial studies by the Johns Hopkins team indicated that the transgenic Anopheles weren’t at a significant fitness disadvantage, and while the investigators admit more work will be needed to address a greater range of parameters, they suggest that “our fitness assays suggest that the fitness loss associated with expression of the transgene Rel2 is unlikely to impair the spread of the immune-enhancement trait in natural mosquito populations when an effective genetic drive system that can overcome fitness disadvantages is employed.”
The team concludes that its previous and newly reported work indicates that their strategy of engineering mosquitoes with a transgenic Rel2-mediated innate immune response fulfills several criteria required for an anti-Plasmodium effector system that could be used to control malaria.
Importantly, they point out, the approach doesn’t involve the use of a foreign recombinant gene, but rather boosts the mosquito’s innate immune system through overexpression of its own Rel2 transcription factor using its own carboxypeptide and vitellogenin promoters. “A plausible future scenario could involve the spread of a Rel2 transgene through a powerful genetic drive system that can overcome the fitness cost of transgene expression, thereby conferring enhanced-immune properties on existing natural malaria vector populations,” they conclude. “This approach has the advantage of being logistically simple and self-propagating as well as environmentally friendly, since it does not eliminate the mosquito from its ecologic niche or involve chemical insecticide or drug treatments.”