Fig 1.
(A) Growth curves and (B) intracellular Ap4A levels of the wild type strain P. aeruginosa PAO1 and the isogenic deletion mutant ΔapaH, carrying or not the pMEapaH plasmid, cultured in flasks at 37°C in LB, supplemented with 100 μM IPTG in the case of ΔapaH pMEapaH. The control strains with the empty plasmid pME6032 are shown in S1 Fig. (C) Biofilm formation of PAO1 and ΔapaH in LB in 96-well polystyrene microtiter plates after 24-h incubation at 37°C. (D) Intracellular c-di-GMP levels of PAO1 and ΔapaH cultured in LB at 37°C. Cells for Ap4A and c-di-GMP quantification were collected after 12 h of growth. Values are the mean (± standard deviation) of at least three independent experiments. Asterisks indicate a statistically significant difference (P < 0.001) with respect to PAO1 (ANOVA).
Fig 2.
(A) Inhibition halos of streptomycin (Sm), gentamicin (Gm), tobramycin (NN), ciprofloxacin (Cip), imipenem (Imp), erythromycin (Ery), novobiocin (NB), or rifampicin (Rif) for P. aeruginosa PAO1 and the ΔapaH mutant in the Kirby-Bauer disc diffusion assay. (B) Time-kill curves of PAO1 and ΔapaH in the presence of kanamycin (Km) at 32 and 64 μg/mL or Gm at 0.5 and 1 μg/mL, corresponding to 1× and 2×MIC for both strains. Values are the mean (± standard deviation) of at least three independent assays.
Fig 3.
Functional analysis of differentially expressed genes (DEGs). Number (left panel) and relative percentage (right panel) of downregulated and upregulated genes (red and green bars, respectively) in the apaH mutant compared to PAO1 in the PseudoCAP functional categories listed on the left. Only DEGs with a fold change ≥ ± 4 were considered (S1 Table).
Fig 4.
(A) Protease activity, (B) elastase activity, and (C) PQS/HHQ levels, normalized to cell density (OD600), in the supernatants of P. aeruginosa PAO1 and the apaH mutant, carrying or not the pMEapaH plasmid, cultured at 37°C in LB, supplemented with 100 μM IPTG in the case of ΔapaH pMEapaH. (D) Growth curves and (E) pyoverdine levels, normalized to cell density (OD600), of the same strains cultured at 37°C in the iron-poor medium CAA, supplemented with 50 μM FeCl3 when indicated (+ Fe), and 100 μM IPTG in the case of ΔapaH pMEapaH. Values are the mean (± standard deviation) of at least three independent assays. Asterisks indicate a statistically significant difference (P < 0.001) with respect to PAO1 (ANOVA). The control strains with the empty plasmid pME6032 are shown in S6 Fig.
Fig 5.
(A) Pathogenicity of P. aeruginosa PAO1 and the apaH mutant in the lettuce leaf virulence assay. Bacterial viable cells (CFUs) per mg of lettuce midribs at three days post injection are shown. Two independent experiments were performed, each with five biological replicates. A representative picture of the infected midribs is shown for each strain. (B) Dose-dependent survival curves of G. mellonella larvae infected with different doses of PAO1 or ΔapaH cells. Lethal dose 90% (LD90) and R2 values are shown in the figure. (C) Survival of mice (n = 5 per group) challenged with intratracheal injection of 107 CFUs of PAO1 or ΔapaH. (D) Total bacterial CFU in lungs or spleen from mice (n = 5 per group) intra-tracheally infected with 107 CFU of PAO1 or ΔapaH and sacrificed within 24 h of infection (see Materials and methods for details). (E) Count of total leukocytes, polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes in BALF from the mice described in panel D. Asterisks indicate statistically significant differences (*P < 0.05, *** P < 0.001) with respect to PAO1 (Mann-Whitney test for data in panels A, D, and E; Mantel-Cox test for data in panel C).
Fig 6.
(A) Protease and (B) elastase activity, normalized to cell density (OD600), in the supernatants of P. aeruginosa PA14 and the indicated P. aeruginosa clinical isolates (WT) or the cognate apaH deletion mutants (ΔapaH) cultured at 37°C in LB. (C) Maximum growth yields and (D) pyoverdine levels normalized to cell density (OD600), of the same strains cultured at 37°C in the iron-poor medium CAA. Pyoverdine levels were not measured for BG80 ΔapaH as this mutant did not grow in CAA (see panel C). Values are the mean (± standard deviation) of three independent assays. Asterisks indicate statistically significant differences between each ΔapaH mutant and the corresponding parental strain (* P < 0.05, ** P < 0.01, *** P < 0.001; unpaired t test).
Fig 7.
(A) Pathogenicity of P. aeruginosa PA14 and the indicated P. aeruginosa clinical isolates (WT) or the cognate apaH deletion mutants (ΔapaH) in the lettuce leaf virulence assay. Bacterial viable cells (CFUs) per mg of lettuce midribs at three days post injection are shown. Five biological replicates for each strain were analyzed. A representative picture of the infected midribs is shown for each strain. (B) Reduction in infectivity, calculated as the ratio between the lethal dose 90% (LD90) of the ΔapaH mutant and the LD90 of the corresponding wild type strain, observed for apaH mutants of P. aeruginosa PA14 and the indicated P. aeruginosa clinical isolates in G. mellonella larvae. The dose-dependent survival curves used to calculate the LD90 values and the corresponding R2 values are shown in S10 Fig.