Targeting the Pseudomonas aeruginosa Virulence Factor Phospholipase C With Engineered Liposomes

Abstract

Engineered liposomes composed of the naturally occurring lipids sphingomyelin (Sm) and cholesterol (Ch) have been demonstrated to efficiently neutralize toxins secreted by Gram-positive bacteria such as Streptococcus pneumoniae and Staphylococcus aureus. Here, we hypothesized that liposomes are capable of neutralizing cytolytic virulence factors secreted by the Gram-negative pathogen Pseudomonas aeruginosa. We used the highly virulent cystic fibrosis P. aeruginosa Liverpool Epidemic Strain LESB58 and showed that sphingomyelin (Sm) and a combination of sphingomyelin with cholesterol (Ch:Sm; 66 mol/% Ch and 34 mol/% Sm) liposomes reduced lysis of human bronchial and red blood cells upon challenge with the Pseudomonas secretome. Mass spectrometry of liposome-sequestered Pseudomonas proteins identified the virulence-promoting hemolytic phospholipase C (PlcH) as having been neutralized. Pseudomonas aeruginosa supernatants incubated with liposomes demonstrated reduced PlcH activity as assessed by the p-nitrophenylphosphorylcholine (NPPC) assay. Testing the in vivo efficacy of the liposomes in a murine cutaneous abscess model revealed that Sm and Ch:Sm, as single dose treatments, attenuated abscesses by >30%, demonstrating a similar effect to that of a mutant lacking plcH in this infection model. Thus, sphingomyelin-containing liposome therapy offers an interesting approach to treat and reduce virulence of complex infections caused by P. aeruginosa and potentially other Gram-negative pathogens expressing PlcH.

Keywords: abscess; anti-virulence; cholesterol; dermonecrosis; plcH; sphingomyelin.

Figure 1

Engineered liposomes reduced the lysis of human cells induced by Pseudomonas aeruginosa bacterial supernatant grown in modified cystic fibrosis medium. (A) Human red blood cells were incubated with LESB58 (100 μl) bacterial supernatant and cholesterol-containing (Ch:Sm) or sphingomyelin only (Sm) liposomes for 1 h (n = 11, each treatment). (B) 16HBE14o- cells after challenge with Pseudomonas aeruginosa LESB58 stationary-grown supernatants (100 μl), normalized to Triton X-100. Both types of liposomes (300 μg/ml) decreased the release of lactate dehydrogenase (n = 17). Error bars, mean ± standard error. A one-way ANOVA with post-hoc Dunn’s multiple comparison test was performed between treated and non-treated exposure, value of p (^*** < 0.001) adjusted for multiple comparisons.

Figure 2

Pseudomonas aeruginosa supernatant-induced hemolysis is attenuated by the removal of phospholipase C and supernatants pre-incubated with liposomes have reduced hemolytic phospholipase C activity. (A) RBCs were treated with 100 μl of filter-sterilized supernatant from 24-h cultures of bacterial strains grown in MEM + 1% FBS incubated at 37°C. OD₄₅₀ was measured for each strain and normalized to control untreated RBCs and fully lysed RBCs (treated with 2% v/v Triton X-100). One-way ANOVA with post-hoc Dunnett test, ^**Bonferroni correct value of p < 0.01, and ^***Bonferroni corrected value of p < 0.001, n = 5, error bars represent ±SE. (B) The hemolytic phospholipase C driven hydrolysis of colorless p-nitrophenylphosphorylcholine to yellow p-nitrophenol was assessed for supernatants pre-treated with 300 μg/ml cholesterol-containing (Ch:Sm) and sphingomyelin only (Sm) liposomes or vehicle control (LESB58 wild type, LESB58.ΔplcH; six independently prepared wild-type supernatants for LESB58, Sm, Ch:Sm; three independently prepared LESB58.ΔplcH bacterial supernatants). Error bars are mean ± SE; SE not shown for mutant and media control due to very small variation. A two-way ANCOVA with post-hoc Bonferroni value of p adjustment was carried out using the wild-type (LESB58) phospholipase C activity as a control. All conditions were significantly different, adjusted value of p of 0.031 for Ch:Sm, and p < 0.0001 for all other conditions.

Figure 3

Effects of liposome treatment and phospholipase knockout mutant in a Pseudomonas aeruginosa LESB skin abscess mouse model. CD-1 mice were subcutaneously infected with a high bacterial density (5 × 10⁷ CFU) of LESB58 wild type (A,B), LESB58 carrying an empty vector pBBR5 or overexpression vector pBBR5.plcH⁺, and phospholipid mutant LESB58.ΔplcH carrying an empty vector pBBR5 or overexpression vector pBBR5.plcH⁺ (C,D). Lesion sizes (Box and whiskers plot) and CFU counts (with geometric mean) were determined 3 day post-infection. Liposome treatment (top) and mutant studies (bottom)—(A,C) abscess size measurements, and (B,D) bacterial counts per abscess. All experiments were done at least three times independently with 2–4 mice/group. Statistical analysis was performed using one-way ANOVA, Kruskal-Wallis test with Dunn’s correction. The asterisk indicates a significant difference to the wild type (^*p < 0.05; ^**p < 0.01).