Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Total Synthesis of Alloviroidin

Alloviroidin is a cyclic heptapeptide, produced by several species of Amanita mushrooms, that demonstrates high affinity for F-actin as is characteristic of virotoxins and phallotoxins. Alloviroidin was synthesized via a [3 + 4] fragment condensation of Fmoc-d-Thr(OTBS)-d-Ser(OTBS)-(2 S,3 R,4 R)-DHPro(OTBS)-OH and H-Ala-Trp(2-SOMe)-(2 S,4 S)-DHLeu(5-OTBS)-Val-OMe to form bond A. The linear heptapeptide favored a turn conformation, facilitating cyclization between Val and d-Thr (position B). Global deprotection and HPLC purification afforded alloviroidin with NMR spectra in excellent agreement with the natural product

Pyocyanin facilitates extracellular DNA binding to Pseudomonas aeruginosa influencing cell surface properties and aggregation.

Pyocyanin is an electrochemically active metabolite produced by the human pathogen Pseudomonas aeruginosa. It is a recognized virulence factor and is involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognized as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. eDNA has also been demonstrated to be a major component in establishing P. aeruginosa biofilms. In this study we discovered that production of pyocyanin influences the binding of eDNA to P. aeruginosa PA14 cells, mediated through intercalation of pyocyanin with eDNA. P. aeruginosa cell surface properties including cell size (hydrodynamic diameter), hydrophobicity and attractive surface energies were influenced by eDNA in the presence of pyocyanin, affecting physico-chemical interactions and promoting aggregation. A ΔphzA-G PA14 mutant, deficient in pyocynain production, could not bind with eDNA resulting in a reduction in hydrodynamic diameter, a decrease in hydrophobicity, repulsive physico-chemical interactions and reduction in aggregation in comparison to the wildtype strain. Removal of eDNA by DNase I treatment on the PA14 wildtype strain resulted in significant reduction in aggregation, cell surface hydrophobicity and size and an increase in repulsive physico-chemical interactions, similar to the level of the ΔphzA-G mutant. The cell surface properties of the ΔphzA-G mutant were not affected by DNase I treatment. Based on these findings we propose that pyocyanin intercalation with eDNA promotes cell-to-cell interactions in P. aeruginosa cells by influencing their cell surface properties and physico-chemical interactions

Size analysis of <i>P. aeruginosa</i> PA14 strains.

<p>(A to C) Hydrodynamic diameter of PA14 wildtype and Δ<i>phzA-G</i> strains, grown over 1 to 3 days respectively and effect of DNase I treatment on it. (D) Represents the change in reduction in hydrodynamic diameter of wildtype and Δ<i>phzA-G</i> after DNase I treatment. Error bars represents standard deviations from the mean (n = 5). Asterisks and hash indicate statistically significant (p<0.05) differences in hydrodynamic diameter in comparison to DNase I treated wildtype and Δ<i>phzA-G</i> strain (regardless of DNase I treatment) respectively. Dollar indicates statistically significant (p<0.05) differences in reduction in hydrodynamic diameter between wildtype (before and after DNase I treatment) and Δ<i>phzA-G</i> (before and after DNase I treatment) respectively.</p

Difference in <i>P. aeruginosa</i> cell surface.

<p>(A and B) Schematic showing changes in PA14 wildtype and Δ<i>phzA-G</i> cell surface respectively before and after DNase I treatment. (A) Natural production of pyocyanin by wildtype strain facilitates EPS (mostly constitute of eDNA) binding to <i>P. aeruginosa</i> cell wall and removal of eDNA by DNase I treatment subsequently removes EPS from its cell wall. (B) The mutant Δ<i>phzA-G</i> deficient in pyocyanin production could not facilitate eDNA/EPS binding with <i>P. aeruginosa</i> cell wall, therewith no effect of DNase I treatment on its cell wall.</p

Effect of DNA addition in aggregation of <i>P. aeruginosa</i> PA14.

<p>The % reduction in optical density after 60 min due to aggregation of PA14 wildtype and Δ<i>phzA-G</i> strains before and after addition of 1 µg/ml of final concentration of exogenous DNA isolated from <i>P. aeruginosa</i> cultures. Error bars represents standard deviations from the mean (n = 3). Asterisks indicate statistically significant (p<0.05) differences in % of aggregation in comparison to DNase I treated wildtype strain.</p

Pyocyanin production in <i>P. aeruginosa</i> PA14 strains and effect of DNase I treatment in PA14 aggregation.

<p>(A) Shows production of pyocyanin in planktonic culture of PA14 DKN370 and wildtype (indicated by green colour) and lack of pyocyanin production in Δ<i>phzA-G</i> strain even over 3 days growth period. (B) Represents an example of raw data showing decrease in absorbance at 600 nm OD due to aggregation of PA14 DKN370, wildtype and Δ<i>phzA-G</i> over 60 minutes. DKN370: before DNase I treatment (closed square), after DNase I treatment (open square), wildtype: before DNase I treatment (closed circle), after DNase I treatment (open circle), Δ<i>phzA-G</i>: before DNase I treatment (closed triangle), after DNase I treatment (open triangle).</p

Pyocyanin intercalation with DNA.

<p>(A) Fluorescence emission spectra of ethidium bromide before and after DNA addition. (B) Decrease in intensity of emission spectra of ethidium bromide-DNA in presence and absence of pyocyanin. (C) Showing structure and colour of pyocyanin at acidic and neutral pH. (D and E) Showing colour change in DNA pellet from white to greenish blue in neutral pH and pink under acidic pH, after treated with pyocyanin and incubated at room temperature for overnight, indicates pyocyanin binded with DNA and the reappearance of white colour of DNA by simple washing of coloured DNA pellet with water resulted in removal of pyocyanin stain (Fig. 6D and E).</p

Interfacial free energy of aggregation of <i>P. aeruginosa</i> PA14 strains.

<p>(A to C) Components: Lifshitz-Van der Waals (LW ΔG) and acid-base (AB ΔG) and total interfacial free energy (Total ΔG) of aggregation of PA14 strains before and after DNase I treatment. Error bars represents standard deviations from the mean (n = 3). Asterisks and hash indicate statistically significant (p<0.05) differences in the free energy of aggregation in comparison to DNase I treated wildtype and Δ<i>phzA-G</i> strain regardless of DNase I treatment respectively.</p