Self-limiting paratransgenesis

Abstract

Presently, the principal tools to combat malaria are restricted to killing the parasite in infected people and killing the mosquito vector to thwart transmission. While successful, these approaches are losing effectiveness in view of parasite resistance to drugs and mosquito resistance to insecticides. Clearly, new approaches to fight this deadly disease need to be developed. Recently, one such approach-engineering mosquito resident bacteria to secrete anti-parasite compounds-has proven in the laboratory to be highly effective. However, implementation of this strategy requires approval from regulators as it involves introduction of recombinant bacteria into the field. A frequent argument by regulators is that if something unexpectedly goes wrong after release, there must be a recall mechanism. This report addresses this concern. Previously we have shown that a Serratia bacterium isolated from a mosquito ovary is able to spread through mosquito populations and is amenable to be engineered to secrete anti-plasmodial compounds. We have introduced a plasmid into this bacterium that carries a fluorescent protein gene and show that when cultured in the laboratory, the plasmid is completely lost in about 130 bacterial generations. Importantly, when these bacteria were introduced into mosquitoes, the bacteria were transmitted from one generation to the next, but the plasmid was lost after three mosquito generations, rendering the bacteria non-recombinant (wild type). Furthermore, no evidence was obtained for horizontal transfer of the plasmid to other bacteria either in culture or in the mosquito. Prior to release, it is imperative to demonstrate that the genes that thwart parasite development in the mosquito are safe to the environment. This report describes a methodology to safely achieve this goal, utilizing transient expression from a plasmid that is gradually lost, returning the bacterium to wild type status.

Overview of paratransgenesis as a strategy to control pathogen transmission by insect vectors

“…stephensi Serratia AS1 -mCherry and AS1 -gfp Semi-field simulation of transgenic bacteria spread [ 120 ] R. prolixus R. rhodnii and Gordona rubropertinctus Model showing negligible risk of horizontal transfer of transgenic bacteria [ 133 ] An. stephensi Serratia ASI -gfp + mCheery and kanR genes/+ microbiome in vivo No horizontal transfer of transgenic bacteria genetic material in vivo [ 134 , 162 ] Anopheles spp. P. agglomerans Modelling paratransgensisi b [ 130 ] An.…”

“… P. agglomerans Modelling paratransgensisi b [ 130 ] An. stephensi Serratia ASI -gfp + mCheery and kanR genes/+ microbiome in vivo Transiently expressed plasmids for checking environmental safety of released genes [ 134 ] An. stephensi Asaia + scorpine Transgene only expressed after blood meal, thus reducing fitness costs [ 126 ] R. prolixus R. rhodnii and Escherichia coli expressing dsRNA RNAi and knockdown of vector genes c [ 74 , 111 ] Anopheles spp.…”

“…stephensis Semi-field transfection study only [ 120 ] Serratia ASI -gfp + mCherry and kanR genes + microbiome in vivo An. stephensi No horizontal transfer of transgenic bacteria and transient plasmid expression [ 134 , 162 ] Serratia ASI −mCherry An. stephensi , Culex pipiens , Cx.…”

Genetic Technologies for Sustainable Management of Insect Pests and Disease Vectors

“…Paratransgenesis is compatible with both traditional and other genetic control methods [37], while having the potential for self-sustained or self-limiting effect according to the stability of the recombinant DNA and transmission rate of the symbiont. Examples of well-characterized paratransgenic systems include: the bacteria Rhodococcus rhodnii engineered to express Cecropin A to interfere with Trypanosoma cruzi in the triatomine bug Rhodnius prolixus, vector of Chagas disease [23]; Sodalis glossinidius, a natural symbiont of Glossina spp., vector of African trypanosomiasis [22]; Anopheles stephensi and Anopheles gambiae recolonized by engineered strains of Escherichia coli [24,29], Pantoea agglomerans [25,30], Asaia [26][27][28]38] and Serratia [39,40]. Confined semi-field studies employing Asaia in the two major malaria vectors A. stephensi and A. gambiae [41] have outlined key aspects regarding the behavioral ecology of paratransgenic mosquitoes and the transmission dynamics of the engineered symbiont in the vector population, through both horizontal (co-feeding and/or mating) and vertical (maternal and/or paternal inheritance) routes [42,43].…”

“…To mitigate the environmental impact (i.e., any undesired outcome such as the spread of recombinant microorganisms to non-targeted organisms or horizontal gene transfer), several technologies are being investigated. These include self-limiting paratransgenic systems in which the engineered symbiont reverts to a wild-type status at a predetermined rate [39] and microcapsule-based delivery strategies, which implies the release of the microorganism into the targeted organ of the insect [44]. An advantage of paratransgenesis over other control strategies is that the microorganisms used are relatively easy to isolate, engineer, and reintroduce into the vector and they can be produced at scale at an exceptionally low cost [36].…”