Combining transgenesis with paratransgenesis to fight malaria
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Evaluation Summary:
This study tests the potential of using a combination of mosquito-based approaches, transgenesis and paratransgenesis, for malaria control relative to the use of the individual technologies. The results show that a combination of approaches can be more powerful at preventing the transmission of malaria parasites, opening the possibility of using similar combination approaches to reduce the malaria burden. The findings will be interesting for a broad audience of mosquito biologists and malaria researchers, but as they are limited to a specific transgenic-paratransgenic combination, more work will be needed to determine the true potential of this strategy for disease control.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Malaria is among the deadliest infectious diseases, and Plasmodium , the causative agent, needs to complete a complex development cycle in its vector mosquito for transmission to occur. Two promising strategies to curb transmission are transgenesis, consisting of genetically engineering mosquitoes to express antimalarial effector molecules, and paratransgenesis, consisting of introducing into the mosquito commensal bacteria engineered to express antimalarial effector molecules. Although both approaches restrict parasite development in the mosquito, it is not known how their effectiveness compares. Here we provide an in-depth assessment of transgenesis and paratransgenesis and evaluate the combination of the two approaches. Using the Q-system to drive gene expression, we engineered mosquitoes to produce and secrete two effectors – scorpine and the MP2 peptide – into the mosquito gut and salivary glands. We also engineered Serratia , a commensal bacterium capable of spreading through mosquito populations to secrete effectors into the mosquito gut. Whereas both mosquito-based and bacteria-based approaches strongly reduced the oocyst and sporozoite intensity, a substantially stronger reduction of Plasmodium falciparum development was achieved when transgenesis and paratransgenesis were combined. Most importantly, transmission of Plasmodium berghei from infected to naïve mice was maximally inhibited by the combination of the two approaches. Combining these two strategies promises to become a powerful approach to combat malaria.
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Evaluation Summary:
This study tests the potential of using a combination of mosquito-based approaches, transgenesis and paratransgenesis, for malaria control relative to the use of the individual technologies. The results show that a combination of approaches can be more powerful at preventing the transmission of malaria parasites, opening the possibility of using similar combination approaches to reduce the malaria burden. The findings will be interesting for a broad audience of mosquito biologists and malaria researchers, but as they are limited to a specific transgenic-paratransgenic combination, more work will be needed to determine the true potential of this strategy for disease control.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
Evaluation Summary:
This study tests the potential of using a combination of mosquito-based approaches, transgenesis and paratransgenesis, for malaria control relative to the use of the individual technologies. The results show that a combination of approaches can be more powerful at preventing the transmission of malaria parasites, opening the possibility of using similar combination approaches to reduce the malaria burden. The findings will be interesting for a broad audience of mosquito biologists and malaria researchers, but as they are limited to a specific transgenic-paratransgenic combination, more work will be needed to determine the true potential of this strategy for disease control.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The key question that Huang et al. are addressing is which approach, paratransgenesis, transgenesis, or the combination of both, is the most promising to combat malaria, killing parasites without affecting the mosquito host. They explored this question by generating a transgenic mosquito line secreting two effector molecules in the midgut and salivary glands, and infecting mosquitoes with Serratia bacteria expressing effector molecules. Their major finding is that a combination of both strategies has the highest inhibition of parasite development compared to transgenesis or paratransgenesis alone. This is further confirmed by mouse infections with a rodent malaria model showing that a combination of both strategies inhibits transmission to naïve mice.
This study is comprehensive and provides significant …
Reviewer #1 (Public Review):
The key question that Huang et al. are addressing is which approach, paratransgenesis, transgenesis, or the combination of both, is the most promising to combat malaria, killing parasites without affecting the mosquito host. They explored this question by generating a transgenic mosquito line secreting two effector molecules in the midgut and salivary glands, and infecting mosquitoes with Serratia bacteria expressing effector molecules. Their major finding is that a combination of both strategies has the highest inhibition of parasite development compared to transgenesis or paratransgenesis alone. This is further confirmed by mouse infections with a rodent malaria model showing that a combination of both strategies inhibits transmission to naïve mice.
This study is comprehensive and provides significant information on the possible use of these approaches for malaria control. The effects on parasite development are clear and convincingly confirm that these strategies have the potential for reducing malaria transmission. It cannot be ruled out, however, that the more pronounced effects on parasite development of the combined approaches may be due to differences in the fitness of these mosquitoes rather than a true additive or synergistic action between transgenesis and paratransgenesis. Another limitation is that the authors do not show when parasites are killed and do not provide direct evidence of the role of the bacterial-expressed factors in the killing mechanism.
The authors show very convincingly that transgenic mosquitoes (all possible combinations) have comparable fitness to wild types. However, these fitness studies are lacking in Serratia-infected mosquitoes, and in the transgenic-paratransgenic combination. Are those mosquitoes as fit as WT? Fitness costs could negatively affect parasite development indirectly, rendering the comparison between the treatments impossible (and negatively impacting this possible strategy). These are key controls that need to be added to the manuscript in order to support the finding that the combination is the best approach.
It is surprising that the Sg/E line inhibits oocyst development given it uses a salivary gland promoter. The authors hypothesize that this is most likely explained by mosquitoes ingesting saliva with the blood meal. This hypothesis is interesting but needs to be tested by determining the presence of Scorpine and MP2 protein in the blood bolus. Also, at what stage are parasites killed?
While the authors test the expression levels of Scorpine and MP2 by qRT-PCR and western blot in transgenic mosquitoes, they did not test levels in paratransgenic ones. In which tissues are these factors produced in Serratia-infected mosquitoes? Are Scorpine and MP2 produced in the midguts and/or salivary glands? And at what level? A quantitative comparison of scorpine and MP2 protein levels in transgenic and paratransgenic mosquitoes is important to determine whether levels are correlated to the effects on parasite development.
Related to this, the engineered Serratia bacteria appear to express 5 effector molecules rather than just MP2 and Scorpine. This obviously can affect the results and also makes a direct comparison less meaningful, but we couldn't find any information on the other effectors, or on whether they are expressed and potentially responsible for the observed anti-parasitic activity.
More information about the experimental setup is needed. The authors used a piggybac approach that has led to multiple insertions in some of the mosquito lines. Which lines did they use for the experiments? This is not clear in the manuscript. If multiple insertions were used, this should be stated and the feasibility of maintaining them (and efficacy) over different generations should be discussed.
Oocyst and sporozoite data are not normally distributed, and therefore presenting the median instead of the mean is more informative. Furthermore, the statistical analyses done do not appear to be appropriate for this data. The authors need to either FDR-correct for multiple comparisons or do a Kruskal-Wallis test with post hoc testing. It would also be important to do statistical analyses on the prevalence.
When discussing the ethical consequences of this approach, the authors should also discuss the possible effects of QF2, scorpine, and MP2 secretions in humans upon a blood feed.
The authors show Serratia vertical transmission over three generations, but as the CFUs decrease over multiple generations, they should discuss whether low levels of Serratia can still block parasite development. In general, the manuscript lacks a thorough discussion of the limitations of this study.
The discussion around line 280 should be more nuanced. I don't think the word 'protected' can be used as mice were not immunized but were simply not infected.
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Reviewer #2 (Public Review):
The authors look at two relatively novel ways of mosquito control: one working by genetically altering symbiotic bacteria of the mosquito (paratransgenesis) and the other working by genetically altering the mosquito genome (transgenesis). They do this by expressing combinations of effector molecules designed to act directly against the malaria parasite as it completes its life cycle in the mosquito, either by killing it directly or by affecting its ability to invade key mosquito cells. There have been several attempts to show the feasibility of each approach recently, and the effector genes in question have each been previously validated, but the novelty proposed with this approach is the combination of the two approaches.
The experiments performed are well described and the data is quite convincing in …
Reviewer #2 (Public Review):
The authors look at two relatively novel ways of mosquito control: one working by genetically altering symbiotic bacteria of the mosquito (paratransgenesis) and the other working by genetically altering the mosquito genome (transgenesis). They do this by expressing combinations of effector molecules designed to act directly against the malaria parasite as it completes its life cycle in the mosquito, either by killing it directly or by affecting its ability to invade key mosquito cells. There have been several attempts to show the feasibility of each approach recently, and the effector genes in question have each been previously validated, but the novelty proposed with this approach is the combination of the two approaches.
The experiments performed are well described and the data is quite convincing in showing that there is an additive effect when combining anti-parasite effectors from a transgenic source as well as a paratransgenic source. My initial reaction is to suggest that it is not surprising that the two effects are additive, it would be more interesting and surprising if they were not. Given that there are no general recommendations, nor avenues for synergy investigated, I have some reservations about the impact on the field since the findings are unlikely to be generalizable.
The thoroughness of the experiments is to be commended and I do think the experiments to check the transmissibility of the parasite, in the face of these interventions, are valuable.
The recommendations for how the overall knowledge provided by these findings could change or impact vector control approaches, other than saying they could be complementary, seem a bit vague. The starting point for the Discussion seems to be that removing a vector population is not a stable strategy as there will always be vectors that resist, or re-fill the empty niche, but why do the same considerations not apply to the parasite in the face of effector genes designed to act against it?
A common concern about any paratransgenesis intervention is how stable the association of the symbiont and the mosquito is likely to be and, in turn, how stable the presence of any transgene within the symbiont is likely to be. The authors show here that at least for 3 generations there is continuous inter-generational transmission, though there does seem to be a decrease in the total load with each successive generation. Undoubtedly there are strategies to strengthen these associations and combat any transgene loss, and I would like to see more discussion of these points.
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