Forward Genetic Analysis of Mosquito/Parasite Interactions

Pratima Chennuri, Ph.D. is a Postdoctoral Research Associate in the Department of Entomology at Texas A&M University and is interested in Gene Drives, DNA Repair, small RNA pathways (piRNAs) and transposons. MORE ABOUT THE AUTHOR

Dong et al. (2018) in a recent paper in PLOS Pathogens describe the results of the genetic inactivation of a broad-spectrum agonist of the malaria parasite, FREP1 (fibrinogen-related protein 1, member of an immune and pattern recognition gene family called FREP) in Anopheles gambiae on:

  1. mosquito susceptibility to two species of Plasmodium, falciparum that infects human hosts and P. berghei that infects rodents and
  2. mosquito fitness.

Anopheles gambiae

Malaria is one of the most devastating infectious diseases that kills over half a million people every year. The causative agents of malaria are members of the genus Plasmodium that are transmitted to the host organisms by mosquito vectors belonging to the genus Anopheles. The life cycle of the Plasmodium species in their mosquito vectors involves intimate interactions with the mosquito’s midgut, hemolymph and salivary glands where the parasite interacts with both antagonists that prevent its replication and transmission and host factors or agonists that allow for the parasite’s successful replication and transmission to a host, usually a mammal.

Dong et al. generated FREP1 A. gambiae mutants by employing CRISPR/Cas9 genome engineering where transgenic mosquitoes carrying a single Golden Gate cloned construct containing three guide RNAs targeting the FREP1 gene were created by docking the construct in to an attP site-containing A. gambiae line, X1 (chromosome 2L).

Plasmodium life-cycle within the mosquito. Ingestion of Plasmodium infected blood by the mosquitoes is followed by reductional division in male and female gametocytes resulting in formation and fusion of male and female gametes within mosquito midgut, to form a zygote. The zygote then develops into an ookinete. The ookinete invades midgut epithelia to reach basal lamina, where it matures into oocyst. The oocysts upon maturation, release hundreds of sporozoites into haemocoel. Sporozoites migrate from midgut to salivary gland through haemolymph and then invade salivary glands. Once inside the salivary gland the sporozoites undergo further modifications and are injected into the host blood stream during a blood meal.

Phases of Plasmodium development in Anopheles mosquitoes and some of the proteins implicated in critical mosquito/parasite interactions to which FREP1 can now be added. Image Credit. Sreenivasamurthy et al (2013) A compendium of molecules involved in vector-pathogen interactions pertaining to malaria. Malaria Journal201312:216 https://doi.org/10.1186/1475-2875-12-216

Homozygous mosquitoes carrying this triple guide RNA expressing transgene were crossed to transgenic mosquitoes expressing Cas9 under the control of the germline-specific vasa regulatory elements, thereby generating FREP1 knock out mosquitoes.

Upon infection with either P. falciparum or P. berghei, the midguts of the A. gambiae FREP1 mutants showed strong suppression of the Plasmodium oocyte load when compared to the control mosquitoes. There was also a marked reduction in P. falciparum sporozoite infection of the mosquito salivary glands.

Cas9 + sgRNA

Knocking out FREP1 did not affect adult mosquito size but affected mosquito fitness where significant reduction in blood-feeding propensity, longevity after a blood meal, fecundity and egg hatching rates were observed, however, when fed on 10% sugar solution the adverse effects on longevity were absent.

These experiments provide strong evidence that mosquito-encoded Plasmodium agonists such as FREP1 can be targeted for significant epidemiological control of malaria vectors.

Therefore, studies like these are essential for developing safe and effective ways of using CRISPR/Cas9-mediated and other genome engineering technologies to create and rapidly spread (gene drive) such altered characteristics that suppress/prevent the spread of vector borne diseases such as malaria.

Dong Y, Simões ML, Marois E, Dimopoulos G (2018) CRISPR/Cas9 –mediated gene knockout of Anopheles gambiae FREP1 suppresses malaria parasite infection. PLoS Pathog 14(3): e1006898.

 

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