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.
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
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
(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.)