Identification of Drosophila mutants altering defense of and endurance to Listeria monocytogenes infection

Abstract

We extended the use of Drosophila beyond being a model for signaling pathways required for pattern recognition immune signaling and show that the fly can be used to identify genes required for pathogenesis and host-pathogen interactions. We performed a forward genetic screen to identify Drosophila mutations altering sensitivity to the intracellular pathogen Listeria monocytogenes. We recovered 18 mutants with increased susceptibility to infection, none of which were previously shown to function in a Drosophila immune response. Using secondary screens, we divided these mutants into two groups: In the first group, mutants have reduced endurance to infections but show no change in bacterial growth. This is a new fly immunity phenotype that is not commonly studied. In the second group, mutants have a typical defense defect in which bacterial growth is increased and survival is decreased. By further challenging mutant flies with L. monocytogenes mutants, we identified subgroups of fly mutants that affect specific stages of the L. monocytogenes life cycle, exit from the vacuole, or actin-based movement. There is no overlap between our genes and the hundreds of genes identified in Drosophila S2 cells fighting L. monocytogenes infection, using genomewide RNAi screens in vitro. By using a whole-animal model and screening for host survival, we revealed genes involved in physiologies different from those that were found in previous screens, which all had defects in defensive immune signaling.

Figure 1.—

L. monocytogenes monocytogenes infection in Drosophila melanogaster. Wild-type flies were injected with 10³ CFUs of either wild-type or mutant L. monocytogenes. Survival and growth (growth of wild-type bacteria only) were monitored over the course of the infection. (A) L. monocytogenes strain 10403S (wild type) infection. Wild-type flies exhibit a median time to death (MTD) of ∼5 days postinfection. (B) Growth of 10403S in flies. Bacteria reach levels >10⁴ CFUs by 48 hr postinfection. **P < 0.005 (t-test). (C) Δhly infection in flies. Wild-type flies exhibit a MTD of ∼20 days postinfection. (D) ΔactA infection in flies. Infected flies exhibit a MTD of ∼10 days postinfection. Statistical analysis on survival curves was done using log-rank analysis and lines with P < 0.05 were considered statistically significant.