Fighting malaria
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This document discusses using paratransgenesis to combat malaria. It describes engineering the symbiotic bacteria Pantoea agglomerans from mosquito midguts to secrete proteins that kill Plasmodium parasites. Experiments show the engineered bacteria proliferate in mosquitoes, efficiently secrete anti-Plasmodium proteins, and reduce parasite levels in mosquitoes by 68-84% without impacting mosquito lifespan. The document concludes that using P. agglomerans to deliver multiple antimalarial proteins is an effective and resistance-proof strategy to block malaria transmission.
Fighting malaria
- 2. Introduction Malaria is a mosquito-borne infectious disease affecting humans and other animals . In 2016, there were 216 million cases of malaria worldwide resulting in an estimated 731,000 deaths. Transmission: Anopheles mosquito Symptoms usually begin ten to fifteen days after being bitten.
- 5. Life cycle of Plasmodium falciparum
- 6. Control of malaria Artemisinin (ACT) DNA vaccine
- 7. Big Question How can we control and eradicate malaria from the globe? Specific question What is the novel tool to fight against drug resistant malarial parasite? Or Can we implement “paratransgenesis” as a new weapon to combat malaria?
- 8. Malaria still remains as a major problem Continuous emergence of mosquito due to insecticide resistance, parasite drug resistance and the lack of an effective malaria vaccine Need for new weapons Background Genetically modified mosquitoes Paratransgenesis
- 9. Paratransgenesis Eliminate a pathogen from vector populations through transgenesis of a symbiont of the vector.
- 10. General approach • Engineering symbiotic bacteria (Pantoea agglomerans) from the mosquito midgut microbiome to produce the interfering proteins/effector molecules against Plasmodium (paratransgenesis) • Chagas disease caused by Trypanosoma cruzi triatomid bug commensal Gram-positive bacterium secrete cecropin A, a peptide that kills T. cruzi • Delivery of effector molecules: Use of E. coli hemolysin (Hly)A system
- 12. Q1. What is the colonization and survival fitness of recombinant P. agglomerans in the mosquito midgut? • Approach: Transformation of P. agglomerans with a highly stable plasmid pPnptII:gfp Result: Transgenic P. agglomerans rapidly proliferate in the midgut after a blood meal. Interpretation: Bacteria numbers increased dramatically by more than 200-fold during the first 2 d after a blood meal
- 13. Q2: Do the engineered P. agglomerans strains be efficiently secrete anti- Plasmodium effector proteins? • Approach: The target proteins were cloned to HlyA in a high-copy expression vector and transformed into P. agglomerans with plasmid coding for HlyB and HlyD. • Western blot analysis for culture supernatants and in-vivo immunofluorescence Results: 1.The smaller sized were more abundant in the culture supernatant than the larger sized proteins. 2.The recombinant P. agglomerans expressing (EPIP)4 had the highest secretion level and more efficiency. Interpretation: Recombinant P. agglomerans efficiently secrete anti-plasmodium effector molecules.
- 14. • Approach: Recombinant P. agglomerans were administered to mosquitoes in sucrose solution followed by a P. falciparum–infected blood meal. Q3:What is the effectiveness of recombinant bacteria in interfering the development of the human parasite P. falciparum in mosquitoes? Result: Infection prevalence (mosquitoes having oocyst)was 90% in Bact (control) mosquitoes and was reduced to 14% in mosquitoes carrying scorpine-secreting bacteria. Interpretation: Mosquitoes carrying scorpine-secreting bacteria have 84% transmission-blocking potential.
- 15. Q4: What is the effectiveness of recombinant P. agglomerans to inhibit development of the rodent parasite P. berghei in mosquitoes? • Approach: Mosquitoes harboring recombinant bacterial strains were allowed to feed on the same P. berghei–infected mouse, followed by determination of oocyst numbers on day 14 post infection. Result: The recombinant P. agglomerans expressing (SM1)2 reduced mean oocyst counts by 68%, whereas an equal mixture of effector molecules reduced count by 83%. Interpretation: Recombinant P. Agglomerans strains efficiently impair P. Berghei development in mosquitoes.
- 16. Q5:What is the impact of recombinant P. agglomerans strains expressing anti-Plasmodium molecules on mosquito lifespan? • Approach: Mosquitoes were fed on sugar alone or on various recombinant P. agglomerans and, 32 h later, were allowed to feed on a noninfected mouse and mortality was monitored. Result: No significant differences in mosquito lifespan was detected among any of the mosquito groups Interpretation: These recombinant anti-Plasmodium products pose no obvious negative impact on mosquito fitness in laboratory conditions.
- 17. Summary • Mosquito midgut is prime target for blocking Plasmodium transmission • The alternative strategy to deliver antimalarial effector molecules: paratransgenesis • Mosquitoes are getting benefits from increased number of bacteria • P. agglomerans an excellent candidate for delivery of antimalarial • The availability of multiple effector proteins, acting by different mechanism, greatly reduces the probability of parasite resistance. • Effectiveness is 85 to 90% - “Universal approach”
Editor's Notes
- #6: (Top) Life cycle of Plasmodium falciparum and gametocyte development. Malaria parasites enter the human bloodstream in the form of sporozoites that are injected by infected female Anopheles mosquitoes taking a blood meal. The majority of sporozoites migrate to the liver, where they invade hepatocytes and multiply. Merozoites are formed that are released into the bloodstream, where they invade red blood cells, initiating the asexual multiplication cycle. A fraction of merozoites that are released from infected red blood cells form gametocytes, the transmissible parasite form. The formation and maturation of gametocytes take place in five morphologically recognizable stages. Early-stage gametocytes are sequestered, and only mature stage V gametocytes circulate in the peripheral blood, where they can be taken up by mosquitoes. Once ingested by mosquitoes, each individual gametocyte forms 1 female macrogamete or up to 8 male microgametes. In the mosquito midgut, the fusion of gametes results in the formation of a zygote that develops into a motile ookinete that can penetrate the midgut wall to form oocysts. The oocysts enlarge over time and burst to release sporozoites that migrate to the mosquito salivary gland, rendering the mosquito infectious to human beings. (Bottom) The five developmental stages of P. falciparum gametocytes and mature P. vivax gametocytes. (The P. falciparum gametocyte photographs are reprinted from reference 410 with permission; the P. vivax gametocyte photographs are courtesy of Debbie Nolder, Malaria Reference Laboratory, London School of Hygiene and Tropical Medicine, United Kingdom, reproduced with permission.)