Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors

Abstract

We reported recently that the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 kills Caenorhabditis elegans and that many P. aeruginosa virulence factors (genes) required for maximum virulence in mouse pathogenicity are also required for maximum killing of C. elegans. Here we report that among eight P. aeruginosa PA14 TnphoA mutants isolated that exhibited reduced killing of C. elegans, at least five also exhibited reduced virulence in mice. Three of the TnphoA mutants corresponded to the known virulence-related genes lasR, gacA, and lemA. Three of the mutants corresponded to known genes (aefA from Escherichia coli, pstP from Azotobacter vinelandii, and mtrR from Neisseria gonorrhoeae) that had not been shown previously to play a role in pathogenesis, and two of the mutants contained TnphoA inserted into novel sequences. These data indicate that the killing of C. elegans by P. aeruginosa can be exploited to identify novel P. aeruginosa virulence factors important for mammalian pathogenesis.

Figure 1

Pathogenicity phenotypes of representative P. aeruginosa PA14 TnphoA mutants on C. elegans and Arabidopsis relative to the wild-type strain. C. elegans mortality rates under slow-killing conditions mediated by P. aeruginosa PA14 TnphoA mutants are shown in A and C, and growth of the same mutants in Arabidopsis leaves are shown in B and D. (A and B) Mutants 35A9 (▴) and 44B1 (■) exhibited reduced pathogenicity in nematodes but grew the same as the parental strain, PA14 (○) in Arabidopsis. (C and D) Mutants 48D9 (▾) and 50E12 (+) exhibited reduced pathogenicity in both plants and nematodes compared with PA14 (○).

Figure 2

Complementation analysis of the ability of P. aeruginosa PA14 mutants to mediate slow killing of C. elegans. (A) Slow killing of 1-day-old adult C. elegans by wild-type PA14, 12A1, and 12A1 (pKDT17; expressing PAO1 lasR under the control of a constitutive E. coli lacZ promoter). (B) Slow killing of L4 larval-stage C. elegans by PA14 (pUCP18; vector control), pho15 (pUCP18; vector control), pho15 (pEcdsbA; expressing E. coli dsbA under the control of the constitutive lacZ promoter), and pho15 (pPAdsbA; expressing P. aeruginosa dsbA under the control of a constitutive E. coli lacZ promoter). (C) Slow killing of L4 larval-stage C. elegans by PA14 (pUCP18; vector control), 25F1 (pUCP18; vector control), 25F1 (pORF338; expressing orf338 from its native promoter), and 25F1 (p3-ORFs; expressing orf338-orf224-orf252 from their native promoters). (D) Slow killing of L4 larval-stage C. elegans by 50E12 (pUCP18; vector control), PA14 (pUCP18; vector control), 50E12 (p206-lac; expressing the putative orf159-ptsP_Pa operon under the control of a constitutive E. coli lacZ promoter), and 50E12 (p206-nat; expressing the putative orf159-ptsP_Pa operon under the control of its native promoter). ∗, Zero mortality was observed for worms feeding on 50E12(pUCP18). For all experiments shown, each data point represents means ± SD of three to four replicates. At least two independent experiments were performed for each analysis.