Hemolytic phospholipase C inhibition protects lung function during Pseudomonas aeruginosa infection - PubMed (original) (raw)

Hemolytic phospholipase C inhibition protects lung function during Pseudomonas aeruginosa infection

Matthew J Wargo et al. Am J Respir Crit Care Med. 2011.

Abstract

Rationale: The opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic lung infections and is particularly problematic in patients with cystic fibrosis and those undergoing mechanical ventilation. Decreased lung function contributes significantly to morbidity and mortality during P. aeruginosa infection, and damage inflicted by P. aeruginosa virulence factors contributes to lung function decline.

Objectives: We sought to describe direct contribution of a bacterial phospholipase C/sphingomyelinase, PlcHR, to alteration of host lung physiology and characterize a potential therapeutic for protection of lung function.

Methods: We infected C57Bl/6 mice with P. aeruginosa wild-type or isogenic plcHR deletion strains and measured lung function using computer-controlled ventilators. For in vivo testing, miltefosine was delivered intraperitoneally 1 hour after infection. Infection and respiratory endpoints were at 24 hours after infection.

Measurements and main results: P. aeruginosa wild-type infection caused significant lung function impairment, whereas the effects of a ΔplcHR strain infection were much less severe. Surfactometry analysis of bronchoalveolar lavage fluid indicated that PlcHR decreased pulmonary surfactant function. Miltefosine has structural similarity to the PC and sphingomyelin substrates of PlcHR, and we found that it inhibits the cleavage of these choline-containing lipids in vitro. Miltefosine administration after P. aeruginosa infection limited the negative effects of PlcHR activity on lung function.

Conclusions: We have directly linked production of a single virulence factor in P. aeruginosa with effects on lung function, and demonstrated that the inhibitor miltefosine protects lung function from PlcHR-dependent surfactant dysfunction.

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Figures

Figure 1.

Real-time quantification of relative plcH mRNA levels in Pseudomonas aeruginosa RNA isolated from cystic fibrosis sputum. The plcH/ppiD mRNA ratios from uninduced (pyruvate-grown) and induced (surfactant-grown) P. aeruginosa were determined in 4-hour cultures from four independent experiments and averaged together. Ratios of plcH to ppiD in sputum samples represent the average from nine patients. Error bars represent the SD. *P < 0.05.

Figure 2.

Intratracheal instillation of purified PlcHR resulted in a loss of lung function. (A) Pressure–volume (P-V) loops were measured in vehicle-treated (phosphate-buffered saline [PBS], white diamonds), PlcHR-treated (black circles), and catalytically inactive PlcHR T178A-treated (gray triangles) animals. Both PlcHR additions were 50 ng of protein in 50 μl of PBS. The quasistatic pressure is plotted against volume and the symbols represent mean ± SEM. Y-error bars have been removed for clarity. (B) Quasistatic elastance (E) calculated from the inspiratory limb of the P-V curves in (A). (C) Lung stiffness (H) calculated from respiratory impedance measurements based on the constant phase model. Means in (B) and (C) are shown ± 1 SD from a representative experiment of five mice per group. Two independent experiments with five animals per infected group showed similar results. Tests of significance conducted using a one-way analysis of variance with a Bonferroni multiple comparison test.

Figure 3.

Deletion of plcHR did not alter bacterial load or inflammation at high infective dose. (A) Bacterial burden from whole homogenized lung and spleen. CFU counts from individual mice plotted with mean noted by the horizontal lines. Means are not significantly different. (B) WBC infiltration into the BALF as measured by automated counter (Advia). Mean ± SEM plotted for 7 mice/group, and were not significantly different. (C) Hematoxylin and eosin stained lung sections from control and infected animals. Scale bar = 100 μm. Histology is from a representative experiment with 4 mice per group and is representative of two independent experiments. Statistical significance for bacterial burden calculated using a two-tailed t test.

Figure 4.

Deletion of plcHR alters respiratory mechanics during infection. (A) Pressure–volume (P-V) loops measured in sham-treated (white diamonds) and Pseudomonas aeruginosa_–infected mice (black circles for wild-type PAO1 infected; gray triangles for Δ_plcHR infected). The quasistatic pressure is plotted against volume and the symbols represent mean ± SEM. Y-error bars have been removed for clarity. (B) Quasistatic elastance (E) calculated from the inspiratory limb of the P-V curves in (D). (C) Lung stiffness (H) calculated from respiratory impedance measurements based on the constant phase model. (D) Capillary surfactometry was used to assess the surfactant function of the large aggregate fraction of mouse bronchoalveolar lavage fluid. Higher values of percent open indicate better surfactant function. The black bars and gray bars represent surfactant function from mice infected by wild-type and Δ_plcHR P. aeruginosa_, respectively. Data for A_–_C are from the same representative experiment with seven mice per group in the infected groups and four mice per group in the control (phosphate-buffered saline [PBS]) group. Data are representative of two independent experiments. Data for D are from one representative experiment, where three independent experiments with at least five animals per infected group show similar results. Mean plotted ± SEM for a minimum of six mice as described in the M

ethods

section. Tests of significance conducted using a one-way analysis of variance with a Bonferroni multiple comparison post-test.

Figure 5.

Miltefosine inhibited PlcHR hemolysis, enzymatic activity, and surfactant dysfunction in vitro. (A) Miltefosine inhibited nitrophenolphosporylcholine hydrolysis by purified PlcHR, measured by nitrophenol release followed as a change in absorbance at 410 nm. The legend indicates the concentration of miltefosine in the reaction (micromolar). Means for parts A and B are from three replicates and correspond to at least three independent experiments conducted on different days. Error bars indicate SD. (B) Miltefosine inhibited PlcHR-dependent hemolysis of sheep erythrocytes in a concentration-dependent manner. (C) Capillary surfactometry was used to assess the surfactant function of purified bovine surfactant (Survanta) after treatment with 10 mg/ml PlcHR with or without the labeled doses of miltefosine. Higher values of percent open indicate better surfactant function. Mean plotted ± SEM for six samples as described in the M

ethods

section. Data are representative of two independent experiments and significance conducted using a one-way analysis of variance with a Bonferroni multiple comparison test. *P < 0.05.

Figure 6.

Miltefosine protects against PlcHR-dependent alteration in lung physiology during Pseudomonas aeruginosa infection. (A) Pressure–volume (P-V) loops measured in mice infected for 24 hours with PAO1 that received injection 1 hour after infection of phosphate-buffered saline (PBS) (vehicle, gray diamonds) or 10 mg/kg miltefosine (black triangles). The quasistatic pressure is plotted against volume and the symbols represent mean ± SEM for six mice per group. Y-error bars have been removed for clarity. (B) Quasistatic elastance (E) calculated from the inspiratory limb of the P-V curves in (A). (C) Lung stiffness (H) calculated from respiratory impedance measurements based on the constant phase model. Means in (B) and (C) are shown ± 1 SD from a representative experiment of six mice per group for PAO1-infected mice, and five mice per group for Δ_plcHR_ infected mice. (D) Capillary surfactometry was used to assess the surfactant function of the large aggregate fraction of mouse bronchoalveolar lavage fluid. Higher values of percent open indicate better surfactant function. Bars represent surfactant function from mice infected with wild-type (WT) P. aeruginosa that received injection of PBS (black) or 10 mg/kg miltefosine (gray). Mean plotted ± SEM for a minimum of six mice as described in the M

ethods

section. Data are representative of two independent experiments. For all figure parts, three independent experiments with at least five animals per infected group show similar results. Test for significance done using one-way analysis of variance with a Bonferroni multiple comparison post-test (B_–_D). *P < 0.05, **P < 0.01, ***P < 0.001.

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Hemolytic phospholipase C inhibition protects lung function during Pseudomonas aeruginosa infection - PubMed (original) (raw)