Use of the Galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis

Abstract

Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using Delta exoU Delta exoY, Delta exoT Delta exoY, and Delta exoT Delta exoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a Delta exoT Delta exoU Delta exoY triple mutant and a Delta pscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.

FIG. 1.

LD₅₀s of P. aeruginosa strains in G. mellonella larvae. G. mellonella larvae were infected with P. aeruginosa strains as described in Materials and Method and incubated for 1 day at 37°C. LD₅₀s were determined by using the SYSTAT program as previously described (31). Bars represent the means and standard deviations of at least three experiments.

FIG. 2.

Extracellular protein profiles of P. aeruginosa strains. (A) Coomassie brilliant blue-stained polyacrylamide gel (10%) of concentrated culture supernatants from strains grown in the presence of the calcium chelator NTA (27). Wild-type PA14 was also grown in the presence of Ca²⁺ as a negative control. Secreted proteins were precipitated with ammonium sulfate as described in Materials and Methods. Relative mobilities of the known proteins from PA103 are indicated by arrows. PA14 that was not induced (PA14-U), PA14 that was induced (PA14-I), and PA103 that was induced (PA103-I) by the calcium chelator NTA are shown in lanes 1, 2, and 9, respectively. (B) Immunoblot of polyacrylamide gel probed with antisera reactive to ExoT. (C) Immunoblot of polyacrylamide gel probed with antisera reactive to ExoU. (D) Immunoblot of Coomassie brilliant blue-stained polyacrylamide gel probed with antisera reactive to ExoY and PcrV.

FIG. 3.

Effects of secreted effectors ExoT and ExoU in a mammalian tissue culture system. (A) Internalization of PA14 mutants. HeLa cells were infected with the ΔpscD, ΔexoU, ΔexoT ΔexoU, and ΔexoU ΔexoY isogenic mutants of P. aeruginosa strain PA14 and incubated for 2 h at 37°C. Internalized bacteria were released as described previously (23). Bars represent the means and standard deviations of three independent experiments performed in triplicate. (B) Cytotoxicity of PA14 mutants. HeLa cells were infected with wild-type PA14 and with the ΔpscD, ΔexoT, ΔexoU, and ΔexoY mutants as described in Materials and Methods. At 3 h postinfection, HeLa cells were stained with trypan blue, and the percentage of dead cells was scored with a hemocytometer. Bars represent the means and standard deviations of at least three independent experiments that showed similar results.

FIG. 4.

Killing of G. mellonella larvae by PA14 type III secretion mutants. LD₅₀s of P. aeruginosa strain PA14 and ΔpscD, ΔexoT, ΔexoU, ΔexoY, ΔexoU ΔexoY, ΔexoT ΔexoY, ΔexoT ΔexoU, and ΔexoT ΔexoU ΔexoY mutants are shown. G. mellonella larvae were inoculated with P. aeruginosa strains as described in Materials and Methods and incubated for 1 day at 37°C. LD₅₀s were determined by using the SYSTAT program as previously described (31). Bars represent the means and standard deviations of bacterial counts from at least three experiments.

FIG. 5.

Time course experiment for strain PA14 and ΔpscD, ΔexoU, ΔexoT ΔexoU, and ΔexoT ΔexoU ΔexoY mutants. Infection of G. mellonella larvae and determination of LD₅₀s were performed as described in Materials and Methods. Wax moths were incubated for 1 to 4 days at 37°C. Data represent the means and standard deviations of at least three independent experiments that showed similar results.

FIG. 6.

C. elegans and Arabidopsis pathogenicity assays. (A) Assay of fast killing of C. elegans feeding on wild-type PA14 and ΔpscD, ΔexoT, and ΔexoU mutants. The assay was carried out as described in Materials and Methods. (B) Assay of slow killing of C. elegans feeding on wild-type PA14 and ΔpscD, ΔexoT, and ΔexoU mutants. E. coli OP50 was used as a negative control in each assay and showed killing close to zero. For both experiments, data represent the means and standard deviations of three replicates. The experiments were repeated at least three times, with similar results. (C) Arabidopsis ecotype Columbia leaves were infiltrated with wild-type PA14 and ΔpscD, ΔexoT, and ΔexoU mutants. Bacterial growth within the leaves was monitored for 6 days. Data represent the means and standard deviations of bacterial colony counts from six different leaves, except for time zero (at which point only four leaves were sampled). The experiment was repeated at least three times, with similar results.