Drosophila as a model host for Pseudomonas aeruginosa infection

Abstract

Using the fruit fly Drosophila melanogaster as model host, we have identified mutants of the bacterium Pseudomonas aeruginosa with reduced virulence. Strikingly, all strains strongly impaired in fly killing also lacked twitching motility; most such strains had a mutation in pilGHIJKL chpABCDE, a gene cluster known to be required for twitching motility and potentially encoding a signal transduction system. The pil chp genes appear to control the expression of additional virulence factors, however, since the wild-type fly-killing phenotype of a subset of mutants isolated on the basis of their compact colony morphology indicated that twitching motility itself was not required for full virulence in the fly.

FIG. 1

Time course of infection of wild-type flies with PAO1 (■), a pilD mutant (○), and a chpA mutant (▵). Bacteria were grown as described for PAO1 and introduced into flies by pricking with a syringe needle. The chpA37 mutant (see the legend to Fig. 2) was used for this and subsequent experiments. The values plotted are the averages of five replicate experiments, each with 10 flies, and the standard error of the mean is shown for each point.

FIG. 2

Chromosomal genes disrupted in PAO1 twitching-motility mutants. The seven depicted loci are dispersed along the PAO1 chromosome. Horizontal arrows denote the extent of individual genes and their direction of transcription (adapted from Fig. 2 in reference 1); the organization of the pil chp gene cluster is derived from nucleotide sequences with GenBank accession numbers L10831 (pilG), L22036 (pilHIJ), U11382 (pilK), and U79580 (pilL chpABCDE). The sites of insertion of the ISphoA/hah transposable element are indicated by vertical arrows. Black arrowheads denote mutations that impair fly killing (i.e., that delay 50% fly killing by 2 to 24 h relative to infection with wild-type bacteria), and white arrowheads denote mutations in strains that appear to have wild-type virulence in the fly. The strain with the chpA37 mutation, the second insertion from the 5′ end of chpA (at position 458351 in the PAO1 genome), was used for the experiment in Fig. 1 as well as subsequent experiments; the insertion in orf406 is at position 448168, the insertion in orf2982 is at position 3339667, and the insertion in the pilU mutant with a unique colony morphology is at position 438420 in the PAO1 genome. The first insertion from the 5′ end of pilR was identified in two independently generated mutants.

FIG. 3

Growth of PAO1 and the chpA37 mutant in wild-type flies. Homogenates of batches of five flies were made at various time points after infection and before flies began dying. Homogenates were plated on LB agar to determine viable bacterial cell counts. The bacterial cultures used in this experiment were also used for one of the replicates in the experiment in Fig. 1. Three subsequent experiments gave equivalent results. Symbols: ■, PAO1; ▵, chpA37 mutant.

FIG. 4

Time course of infection of Bc,imd/Bc,imd mutant flies with PAO1 and the chpA37 mutant. Bacteria were grown and introduced into flies as in the experiment in Fig. 1. The values plotted are the averages of five replicate experiments, each with 10 flies, and the standard error of the mean is shown for each point. Symbols: ■, PAO1; ▵, chpA37.

FIG. 5

Model for the role of the pil chp gene cluster in both twitching motility and virulence in the fruit fly. The P. aeruginosa pilGHIJKL chpABCDE gene cluster could encode a signal transduction system and is required for twitching motility mediated by type IV pili (1, 12) and possibly other adaptations for surface growth. Such adaptations appear to include the expression of as yet undetermined virulence factors, since pil chp mutants are impaired in fly killing even though twitching motility is not required for full virulence in the fly.