New Strategy to End Ancient War on Malaria

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Malaria is one of the most devastating infectious diseases in the world today. About 400 million people are infected each year and of those 1.2 million die. Efforts to control malaria have been held back by the lack of an effective vaccine, the alarming rapidity with which Plasmodium, the protozoan parasite, develops drug resistance, plus the failure to eradicate the Anopheles mosquito vector. Fresh approaches to fight malaria are urgently needed, to be used singly or perhaps in combination. One novel approach to a vaccine was recently discussed in this blog.

 

Figure1
Formation of Plasmodium berghei (a rodent parasite) oocysts in culture. A. Ookinetes. B. Transforming ookinete and C. young oocysts. F. Transformation begins with a small hump on the outer edge of the ookinete. Transforming ookinetes (“tooks”) then take on a snail-like appearance (v). The entire population of ookinetes transform in 12–36 h, depending on nutrient availability. Source.

 

Plasmodium undergoes an unusually large number of lifestyle changes in its trip from the female mosquito to a human and back. This is one of the most complex life cycles extant, with so many details that it taxes one’s memory. Each one of the dozen or so stages is labeled with a fancy name and ought be a target for intervention but, for a host of reasons, that has proven elusive. However, the parasite is especially vulnerable in one stage, the transition from a cell called the ookinete to one named the oocyst, which occurs in the mosquito’s midgut. Eventually, the oocysts cross the midgut epithelium into the insect’s circulatory space, the hemocoel. But before going across, the ookinetes are detected by recognition components of the mosquito’s immune system. Killing factors from both the midgut and nearby tissues are recruited and the ookinetes targeted for destruction. In some model systems, fewer that 5% of the ookinetes survive. This, then, is a bottleneck in the Plasmodium life cycle and an attractive place to try to intervene for extermination of the parasite.

 

Figure2

Transgenic P. agglomerans rapidly proliferate in the midgut after a blood meal. GFP-tagged P. agglomerans were administered to 2-d-old Anopheles gambiae via a sugar meal, and, 32 h later, the insects were fed on blood. A. Midgut with GFP-tagged bacteria 24 h after a blood meal. B. GFP-fluorescent bacteria recovered from a mosquito midgut. Source.

 

An innovative approach for killing the protozoa while they are in the mosquito’s midgut has recently been exploited by Wang and coworkers. This procedure involves a genetically modified bacterium, Pantoea (once Enterobacter) agglomerans, a symbiont that normally reside in the insect’s midgut. This organism was bioengineered to deliver destructive compounds to the developing parasites. Using a naturally occurring mosquito symbiont beats having to introduce a foreign strain, most likely a poorer colonizer. This strategy, termed paratransgenesis, has been previously attempted for the control of other insect-borne diseases.

 

The first step in this work was successful: introducing GFP-tagged bacteria into mosquitoes led to their becoming localized in the midgut. The bacteria increased in number more than 200-fold two days after ingestion of a spiked blood meal—a perfect scenario for exploiting bacterial conveyance of anti-malarial effectors. The next step was to clone the genes for eight anti-Plasmodium compounds into P. agglomerans. In order for this to be of value, the bacteria must be able to secrete the effector molecules into the midgut’s medium. For this purpose, the researchers used an E. coli hemolysin system consisting of three components, HlyA, HlyB, and HlyD. They cloned the genes for the effector molecules in frame with the HlyA signal and introduced them singly into P. agglomerans via transformation. The same recipients were transformed with a separate plasmid containing genes coding for HlyB and HlyD. Fusion proteins with predicted size for each recombinant strain were found in Western blots of culture supernatants, successfully confirming their secretion.

 

Figure3

 

The investigators then tested the ability of the recombinant P. agglomerans strains to inhibit the ookinete to oocyst transition of Plasmodium falciparum (the most deadly species for humans). The highest inhibition (about 98%) was noted in recombinant strains expressing scorpine (a scorpion derived broad-spectrum antimicrobialdefensin-like peptide) and an enolase-plasminogen interaction peptide (which interferes with the binding of plasminogen to the ookinite surface). Infection prevalence (the percentage of mosquitoes containing one or more oocysts) was 90% in controls and 14% in those containing the scorpine-secreting bacteria. The inhibitory effect of the bacteria lasted for 4 days following administration. A study of several recombinant P. agglomerans strains revealed that their presence had no effect on mosquito longevity. Inhibition of oocyst formation was equally effective in Asian and African strains of the Anopheles mosquito.

 

These studies point toward the possible efficacy of paratransgenesis in the war against malaria, but the experiments were carried out in the laboratory. A big hurdle is how to introduce recombinant P. agglomerans into mosquitoes in the field. The authors indicate that they have had some success in dealing with this crucial problem by placing baiting stations consisting of clay pots containing cotton balls soaked with sugar and recombinant bacteria surrounding villages where malaria is prevalent. But we don’t yet know the extent of control achieved with this approach, meaning that much more work is needed to establish if this strategy is effective under field conditions. Wouldn’t it be wonderful if the engineered bacteria were to spread across the mosquito population and also be transmitted to their offsprings?

 

Let us hope that such original and ingenious approaches will pay off in the long run. In the ancient war on malaria, it’s time we got the upper hand.

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