Photopolymerization of Polydiacetylene in Hybrid Liposomes: Effect of Polymerization on Stability and Response to Pathogenic Bacterial Toxins

Liposomes containing lipids and polydiacetylene (PDA) are hybrid systems encompassing both a fluid phospholipid membrane and a polymer scaffold (PDA). However, the biophysical role of PDA in such liposomes is not well understood. In this report, we studied the effects of photopolymerization of PDA on the stability of lipid–PDA liposomes, and their sensitivity to selected purified toxins and bacterial supernatants, using a fluorescence assay. Of the three different types of liposomes with variable lipid chain lengths that were chosen, the degree of polymerization had a significant impact on the long-term stability, and response, to external microbial exotoxins secreted by pathogenic bacteria, namely, <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i>. The degree of polymerization of TCDA played an important role in lipid-chain-length-dependent stabilization of lipid–PDA liposomes, as well as in their response to bacterial toxins of <i>S. aureus</i> and <i>P. aeruginosa</i>

Analysis of surface behaviors to determine efficacy of Varpa_4680 (<i>wbu</i>B) and Varpa_5900 (<i>pil</i>Y1) complementation.

<p>Biofilm levels after 24 h incubation and swarm diameters at 48 h for wt and mutant strains expressing either <i>wbu</i>B (A, B) or <i>pil</i>Y1 (C, D) <i>in trans</i> compared to vector controls. 86 = Varpa_4680::Tn5, 223 = Varpa_5900::Tn5. Error was computed as +/−SEM. All p-values were calculated using the student's unpaired T-test. For all panels a = p<0.01 compared to wt+vec, b = p<0.01 compared to 86+vec (A,B) or 223+vec (C,D).</p

Expression of <i>wbu</i>B (grey bars) and <i>pil</i>Y1(black bars) assessed directly by qPCR.

<p>Planktonic cells, biofilm cells, and plate cultures from 0.5% agarose solidified YE plates or FW swarming plates were harvested at 48 h of growth and compared to aerated liquid culture in log phase (19 h) or stationary phase (26 h). RNA levels were determined in comparison to a luciferase spike added to each sample, and fold expression relative to log phase was assessed using the Pfaffl method.</p

Mutants from Colony Morphology Screens.

<p>Mutants from Colony Morphology Screens.</p

Phylogenetic and nucleotide composition suggests Varpa_4680 entered the <i>V. paradoxus</i> EPS genome by horizontal transfer.

<p>A) Phylogenetic tree based on WbuB amino acid sequences created using ClustalW multiple sequence alignment and subsequent Bayesian inference of phylogeny(Mr. Bayes). B) Phylogenetic tree using the 16s rDNA sequences from the same set of organisms as in (A), using Bayesian inference of phylogeny based on the HKY85 nucleotide substitution model. For both A) and B) Vp EPS = <i>Variovorax paradoxus</i> EPS, Pn = <i>Polaromonas naphthlenivorans</i> CJ2, Bp = <i>Bordatella pertussis</i> Tohama 1, Pao1 = <i>Pseudomonas aeruginosa</i> PAO1, Cv = <i>Chromobacterium violaceum</i> ATCC 12472, Sd = <i>Sulfurimonas denitrificans</i> DSM 1251 C) G/C analysis of 35 kb region of the <i>V. paradoxus</i> EPS genome including <i>wbu</i>B. Total region spans orfs Varpa_4661-4691. Low G/C region from 18.4 kb to 27.1 kb spans orfs Varpa_4679-4684.</p

Swarm edges at 24 h on 0.5% FW plates+25 µg/ml Km were photographed using a 10× phase contrast lens (scale bar = 10 microns).

<p>A) wt+vec, B) wt+<i>wbu</i>B, C) mut86+vec, D) mut86+<i>wbu</i>B.</p

Analysis of surface behaviors to examine complementation of Varpa_4664 (<i>shk</i>S).

<p>Biofilm levels after 24 h incubation (A) and swarm diameters (B) at 48 h for wt and mut99 expressing either <i>shk</i>S or <i>shk</i>R <i>in trans</i> compared to vector controls. Error was computed as +/−SEM. P-values were calculated using the student's unpaired T-test. For both panels, a = p<0.01 compared to wt+vec, b = p<0.01 compared to mut99+vec.</p

Strains, Vectors, Primers.

<p>Strains, Vectors, Primers.</p

Swarms of <i>V. paradoxus</i> EPS imaged using a stereomicroscope.

<p>(A) wt+vec, (B) wt+<i>pil</i>Y1, (C) mut223+vec, (D) mut223+<i>pil</i>Y1.</p