The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a gram-negative pathogen that infects immunocompromised and cystic fibrosis patients. The molecular basis of the host-P. aeruginosa interaction and the effect of specific P. aeruginosa virulence factors on various components of the innate immunity pathways are largely unknown. We examine interactions between P. aeruginosa virulence factors and components of innate immunity response in the Drosophila melanogaster model system to reveal the importance of the Toll signaling pathway in resistance to infection by the P. aeruginosa human isolate PA14. Using the two PA14-isogenic mutants plcS and dsbA, we show that Drosophila loss-of-function mutants of Spatzle, the extracellular ligand of Toll, and Dorsal and Dif, two NF-kappa B-like transcription factors, allow increased P. aeruginosa infectivity within fly tissues. In contrast, a constitutively active Toll mutant and a loss-of-function mutant of Cactus, an I kappa B-like factor that inhibits the Toll signaling, reduce infectivity. Our finding that Dorsal activity is required to restrict P. aeruginosa infectivity in Drosophila provides direct in vivo evidence for Dorsal function in adult fly immunity. Additionally, our results provide the basis for future studies into interactions between P. aeruginosa virulence factors and components of the Toll signaling pathway, which is functionally conserved between flies and humans.

FIG. 1.

P. aeruginosa proliferates within D. melanogaster tissues with a lethal outcome. (A) Percent survival of adult OR flies following injury and infection. Control flies were injured with a needle that had been dipped into 10 mM MgSO₄. Infected flies received 10 to 100 bacterial cells/fly. Approximately 40 flies were used for each experiment. The data reflect results from five independent experiments. (B) Proliferation of P. aeruginosa strain PA14 in OR flies. Means ± standard deviations (SD) of results for five flies per time point are shown. The data reflect results from four independent experiments.

FIG. 2.

P. aeruginosa invades and degrades D. melanogaster tissues. OR flies infected with PA14 were collected and sectioned at 12, 24, and 40 h postinoculation, stained with hematoxylin and eosin, and evaluated by light microscopy. (A) Intact striated flight muscle exhibited no sign of bacterial invasion at 12 h. (B) P. aeruginosa cells (arrowhead) associated with flight muscles at 24 h. (C) Flight muscle degradation with large numbers of P. aeruginosa cells (arrowheads) at 40 h. Original magnifications: ×100 for panels A and B and ×40 for panel C.

FIG. 3.

Mutations in genetic components of Toll signaling alter P. aeruginosa lethality and proliferation. (A) Comparative survival analysis of adult flies infected with P. aeruginosa strain PA14 or its isogenic mutant derivatives dsbA and plcS. Survival in wild-type flies (OR and ywDD1,cnbw) or Toll component-mutated flies (spz^−/−, dl^−/−, cact^−/−, dif^−/−, and Tl^10b/+) was assessed. A total of 100 to 150 flies were infected and monitored for survival at 48 h postinoculation. The results reflect data from three independent experiments. (B) Comparative analysis of bacterial proliferation in adult wild-type flies (OR and ywDD1,cnbw) or Toll component-mutated flies (spz^−/−, dl^−/−, cact^−/−, dif^−/−, and Tl^10b/+). Flies were infected with P. aeruginosa strain PA14 or its isogenic mutants dsbA and plcS. Five flies per time point were used for CFU determination. Means ± SD of results for five flies per time point are shown. The results reflect data from three independent experiments.

FIG. 3.

Mutations in genetic components of Toll signaling alter P. aeruginosa lethality and proliferation. (A) Comparative survival analysis of adult flies infected with P. aeruginosa strain PA14 or its isogenic mutant derivatives dsbA and plcS. Survival in wild-type flies (OR and ywDD1,cnbw) or Toll component-mutated flies (spz^−/−, dl^−/−, cact^−/−, dif^−/−, and Tl^10b/+) was assessed. A total of 100 to 150 flies were infected and monitored for survival at 48 h postinoculation. The results reflect data from three independent experiments. (B) Comparative analysis of bacterial proliferation in adult wild-type flies (OR and ywDD1,cnbw) or Toll component-mutated flies (spz^−/−, dl^−/−, cact^−/−, dif^−/−, and Tl^10b/+). Flies were infected with P. aeruginosa strain PA14 or its isogenic mutants dsbA and plcS. Five flies per time point were used for CFU determination. Means ± SD of results for five flies per time point are shown. The results reflect data from three independent experiments.

FIG. 4.

Mutations in genetic components of the Drosophila Imd signaling pathway increase P. aeruginosa lethality and proliferation. (A) Survival analysis of wild-type OR and loss-of-function imd^−/− and rel^−/− (homozygous) mutant flies following infection with strain PA14 and its isogenic derivatives dsbA and plcS. A total of 100 to 150 flies were infected, and survival was scored at 48 h postinoculation. (B) Comparative analysis of bacterial proliferation in adult wild-type OR flies and imd^−/− and rel^−/− mutant flies infected with P. aeruginosa strain PA14 or its isogenic mutants dsbA and plcS. Five flies per time point were used for CFU determination. Means ± SD for five flies per time point are shown. The results reflect data from three independent experiments.

FIG. 4.

Mutations in genetic components of the Drosophila Imd signaling pathway increase P. aeruginosa lethality and proliferation. (A) Survival analysis of wild-type OR and loss-of-function imd^−/− and rel^−/− (homozygous) mutant flies following infection with strain PA14 and its isogenic derivatives dsbA and plcS. A total of 100 to 150 flies were infected, and survival was scored at 48 h postinoculation. (B) Comparative analysis of bacterial proliferation in adult wild-type OR flies and imd^−/− and rel^−/− mutant flies infected with P. aeruginosa strain PA14 or its isogenic mutants dsbA and plcS. Five flies per time point were used for CFU determination. Means ± SD for five flies per time point are shown. The results reflect data from three independent experiments.