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Use of a Drosophila model to identify genes regulating Plasmodium growth in the mosquito - PubMed

Use of a Drosophila model to identify genes regulating Plasmodium growth in the mosquito

Stephanie M Brandt et al. Genetics. 2008 Nov.

Abstract

We performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. We identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element. None of the identified genes have been previously implicated in innate immune responses or interactions with Plasmodium. The functions of five Anopheles gambiae homologs were tested by using RNAi to knock down gene function followed by measuring the growth of the rodent parasite, Plasmodium berghei. Loss of function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito.

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Figures

F<sc>igure</sc> 1.—
Figure 1.—

Survival curves for P. gallinaceum-resistant lines that are susceptible to bacterial infection. Flies were injected with 50 nl S. typhimurium (OD600 = 0.1) or L. monocytogenes (OD600 = 0.01) and then incubated at 29°. Significance was determined by a log-rank test. n = 30 flies. (A) GC15 S. typhimurium challenge. (B) GC15 L. monocytogenes challenge. (C) iG1 S. typhimurium challenge. (D) iG1 L. monocytogenes challenge. (E) 11603 S. typhimurium challenge. (F) 11603 L. monocytogenes challenge. (G) 12516 S. typhimurium challenge. (H) 12516 L. monocytogenes challenge. (I) 13425 S. typhimurium challenge. (J) 13425 L. monocytogenes challenge. (K) 16497 S. typhimurium challenge. (L) 16497 L. monocytogenes challenge.

F<sc>igure</sc> 2.—
Figure 2.—

Gene knockdown in An. gambiae. (A) Validation of gene silencing. One-day-old female mosquitoes were injected with double-stranded LacZ (dsLacZ) or dsRNA of the target gene and whole-body mRNA was extracted 5 days later. Gene expression was determined by qRT–PCR. Each sample was normalized using ribosomal protein S7 expression as an internal control. Samples were analyzed in triplicate. Columns indicate averages and standard deviations. The silencing efficiency is expressed as a percentage reduction in mRNA levels relative to the dsLacZ control. (B) Effect of gene silencing on P. berghei infection. One-day-old female mosquitoes were injected with either dsLacZ or dsRNA of the target gene. Four days later, mosquitoes fed on anesthetized P. berghei-infected mice. Midguts were dissected and genomic DNA extracted 6 days post-infection. The intensity of P. berghei infection was established on the basis of the abundance of the parasite 28S RNA gene relative to the mosquito ribosomal protein S7 gene in genomic DNA extracted from individual infected midguts determined by qRT–PCR. Medians were compared using the Kolmogorov–Smirnov test and the P-values <0.05 were considered significantly different.

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