Absence of membrane phosphatidylcholine does not affect virulence and stress tolerance phenotypes in the opportunistic pathogen Pseudomonas aeruginosa

Abstract

During growth in presence of choline, both laboratory and clinical Pseudomonas aeruginosa strains synthesize phosphatidylcholine (PC), and PC makes up ∼4% of the total membrane phospholipid content. In all the strains tested, PC synthesis occurred only when choline is provided exogenously. Mutants defective in synthesis of PC were generated in the strain backgrounds PAO1 and PA14. Minimum inhibitory concentration studies testing sensitivity of PC-deficient strains towards various antibiotics and cationic antimicrobial peptides revealed no differences as compared to wild-type strains. Mutants incapable of synthesizing PC were also found to be unaffected in motility and biofilm formation on abiotic surfaces, colonization of biotic surfaces and virulence in a mouse infection model. A global phenotypic microarray was further used to identify conditions wherein membrane PC may play a role of in P. aeruginosa. No culture conditions were identified wherein wild-type and PC-deficient mutants showed phenotypic differences. Membrane PC may serve a highly specific role during P. aeruginosa interactions with its eukaryotic hosts based on all the clinical strains tested retaining the ability to synthesize it during availability of choline.

Figure 1. Phospholipid profiles of P. aeruginosa strains grown in rich and minimal media.

Separation of phospholipids by 1-D thin layer chromatography and detection by charring with sulphuric acid-methanol solution (1∶19, v/v) is shown. (A) Panel showing complementation of the PAO1 and PA14 Δpcs deletion mutants with pcs gene expressed in trans at a separate genomic location (att::pcs). Phospholipids were extracted from PAO1 and PA14 strains grown overnight in MOPS-20mM choline. (B) Phospholipid profiles of PAO1 and PA14 strains grown overnight in LB, MOPS-20mM glucose and MOPS-20mM choline. TLC spots corresponding to PC are shown as black arrowheads. The nature of the spot corresponding to white arrowheads is observed under all growth conditions just above the PC migration front remains unknown. This spot has been observed under all growth conditions, regardless of the presence of choline in the medium. Panels A and B are representative images of observations from three independent experiments. (Slight differences in the migration fronts of PL standards and the extracted phospholipids are likely a result of their acyl chains varying in lengths and saturation levels).

Figure 2. PC-deficient mutants are not affected for motility and biofilm formation on abiotic surfaces.

(A) Twitching motility assays of PA14 strains on LB and tryptone agar plates were performed as previously described . Motility was determined by measuring diameter (mm) of the zone of expansion from the point of inoculation. (B) Biofilm assays of PA14 strains grown in LB and MOPS-20mM choline media were performed using 96-well PVC microtiter plates as previously described . At 12 hours, the biofilms formed were stained with crystal violet, solubilized with 95% ethanol, and quantified by measuring absorbance at 595 nm. In panels A and B, values represent averages and error bars are SD (n = 3). Tests of significance were conducted using a one- way analysis of variance (ANOVA) with a Bonferroni multiple comparison test, ***P<0.001, ^ns P>0.05.

Figure 3. PC-deficient mutants are not affected for colonization of biotic surfaces.

Initial attachment and biofilm formation of PAO1 strains on epithelial cells was performed as previously described , . Seven-day-old confluent monolayers of CFBE epithelial cells were inoculated with bacterial strains and CFUs were estimated at (A) 1 hour post infection for enumerating initial attachment and (B) 6 hours post infection for enumerating biofilm formation. Values in A and B represent averages and SD from three independent replicates, and statistical significance of data was evaluated using a two-tailed unpaired t test, ***P<0.001, ^ns P>0.05. (C) Phase contrast micrographs show 48-hour biofilms formed by P. aeruginosa PA14 strains on the fungus Candida albicans. PA14 strains were co-cultured with constitutively-filamentous Candida albicans nrg1/nrg1 strain as previously described . The media used were MG (MOPS 30 mM glucose) and MGC (MOPS 20 mM glucose, 10 mM choline). Note: Presence of choline enhances colonization. The micrographs are representative images of from three independent experiments.

Figure 4. PC-deficient mutants are not defective in virulence.

(A) PA14 PlcH activity was assayed for by measuring NPPC hydrolysis activity in defined medium with (black bars) or without (white bars) choline. (B) PA14 T3SS-mediated cytotoxicity towards epithelial cells was assessed by monitoring LDH release 6 hours post infection. LDH released from control cells lysed with Triton-X 100 was set as 100% cytotoxicity. In A and B, error bars represent SD of the means of triplicate experiments (C) Bacterial burden 24 hours post infection from whole homogenized lungs of mice inoculated at a high infective dose. CFU counts from individual mice are plotted with means of each group denoted by horizontal lines. Means were not significantly different. Statistical significance of data in panels A, B and C were evaluated using a two-tailed unpaired t test, ***P<0.001, ^ns P>0.05.