Fig 1.
C. elegans predation induces Pf4 replication and enhances P. aeruginosa virulence.
(A-D) Bacterial CFUs and Pf4 PFUs were enumerated after three days in the absence (A-B) or presence (C-D) of C. elegans. nd, not detected (below detection limit of 333 PFU/mL indicated by dashed line). Results are the mean ±SD of three experiments, **P<0.01, Student’s t-test. (E) Wild-type N2 C. elegans were maintained on lawns of 1) E. coli OP50 (non-pathogenic nematode food) or 2) OP50 supplemented with 109 Pf4 virions labeled with Alexa-fluor 488 (green). Representative brightfield and fluorescent images after 24 hours are shown. (F) Kaplan-Meier survival curve analysis of C. elegans exposed to P. aeruginosa. N = 90 worms per condition (three replicate experiments of 30 worms each). The mean survival of C. elegans maintained on lawns of PAO1 was four days compared to seven days for nematodes maintained on lawns of ΔPf4 (dashed gray lines). Note that worms that may have escaped the dish rather than died were withdrawn from the study, explaining why the black PAO1 line does not reach zero percent survival.
Fig 2.
Pyocyanin production is enhanced in ΔPf4 compared to PAO1.
(A) The structure of pyocyanin, a redox-active green pigment produced by P. aeruginosa. (B) Representative images of PAO1 and ΔPf4 growing on NNGM agar plates after 24 hours at 37°C. (C) Pyocyanin was chloroform-acid extracted from NNGM agar plates, absorbance measured (520 nm), and values converted to μg/mL. Data are the mean ±SEM of six replicate experiments. ***P<0.003, Student’s t-test.
Fig 3.
PQS quorum sensing is upregulated in P. aeruginosa ΔPf4.
GFP fluorescence from the transcriptional reporters (A) PrsaLI-gfp, (B) PrhlA-gfp, (C) PpqsA-gfp and (D) Pempty-gfp was measured in PAO1 (black) or ΔPf4 (green) at 18 hours in cultures growing in lysogeny broth. For each measurement, GFP fluorescence was corrected for bacterial growth (OD600). Data are the mean ±SEM of six replicates. **P<0.001, Student’s t-test.
Fig 4.
Pf4 modulates expression of C. elegans proteins associated with respiration, the extracellular matrix, and motility.
(A) Volcano plot showing differentially expressed proteins in C. elegans maintained on lawns of ΔPf4 compared to C. elegans maintained on lawns of PAO1 for three days. The dashed lines indicate proteins with expression levels greater than ±1.5-fold and a false discovery rate (FDR) <0.05. Results are representative of quadruplicate experiments. (B-D) Enrichment analysis of significant upregulated proteins shown in (A). Fold enrichment of observed proteins associated with specific Gene Ontology (GO) terms each had an FDR of <0.002.
Fig 5.
PAO1 compromises C. elegans cuticle integrity compared to ΔPf4.
Synchronized young adult N2 worms were collected from lawns of PAO1, ΔPf4, or E. coli OP50 after 48 hours and stained with the nucleic acid stain Hoechst. Cuticle permeability was assessed by visualization of stained nuclei in live nematodes exposed to (A) PAO1 or (B) ΔPf4. Representative images are shown. (C) The percent C. elegans with stained nuclei were scored as permeable and plotted. **P<0.01, Student’s t-test. N = 3 replicates of 25–50 animals per replicate, 92–137 total worms per group.
Fig 6.
Inactivation of AhR signaling in C. elegans enhances ΔPf4 virulence.
(A, C, and E) Kaplan-Meier survival curve analysis (Log-rank) of wild-type N2 or isogenic ahr-1(ia3) C. elegans maintained on lawns of P. aeruginosa PAO1, ΔpqsA, or ΔPf4 for the indicated times. N = 3 groups of 90 animals per condition (270 animals total per condition). Error bars represent standard error of the mean. P-values of pairwise log-rank survival curve analyses are shown. (B, D, and F) The median survival of C. elegans in days was plotted for each group.
Fig 7.
Pf4 suppresses the production of quorum-regulated pigments by P. aeruginosa allowing bacteria to evade AhR-mediated immune responses in C. elegans.
Table 1.
Bacterial strains, phage, and plasmids used in this study.