Response variables per treatment -Van Cauwenberghe et al. 2016 Oikos

Response variables per treatment -Van Cauwenberghe et al. 2016 Oiko

Data_Sheet_1_The Putative De-N-acetylase DnpA Contributes to Intracellular and Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to Fluoroquinolones.pdf

<p>Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in E_max: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1–3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5–7 fold) and mexX (3.6–5.4 fold) in the parental strain but to a lower extent (3–4-fold for gyrA and 1.8–2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target.</p

Revealing the Excited-State Dynamics of the Fluorescent Protein Dendra2

Green-to-red photoconversion is a reaction that occurs in a limited number of fluorescent proteins and that is currently mechanistically debated. In this contribution, we report on our investigation of the photoconvertible fluorescent protein Dendra2 by employing a combination of pump–probe, up-conversion and single photon timing spectroscopic techniques. Our findings indicate that upon excitation of the neutral green state an excited state proton transfer proceeds with a time constant of 3.4 ps between the neutral green and the anionic green states. In concentrated solution we detected resonance energy transfer (25 ps time constant) between green and red monomers. The time-resolved emission spectra suggest also the formation of a super-red species, first observed for DsRed (a red fluorescent protein from the corallimorph species Discosoma) and consistent with peculiar structural details present in both proteins

All primary data for Stepanyan et al (2015) Mol Ecol

Zip archive containing all primary data for Stepanyan et al (2015) Mol Ecol. Relates to Figure 1, Figure 2, Figure 3, Figure 4, Figure S1, Figure S2 and Figure S5

Structure of compound SPI031.

<p>Structure of compound SPI031.</p

Integrity of <i>S</i>. <i>aureus</i> liposomes after treatment with SPI031 at 1x MIC and 4x MIC.

<p>SDS (5%) and tetracycline (TET) (4x MIC) were used as positive and negative controls, respectively. Means and standard deviation (SD) of 3 independent experiments are shown (*p < 0.05; **p < 0.01; ***p < 0.001 compared to treatment with tetracycline).</p

Time-kill kinetics of SPI031 against <i>S</i>. <i>aureus</i> and <i>P</i>. <i>aeruginosa</i>.

<p>(A) Concentration-dependent killing of <i>S</i>. <i>aureus</i> by SPI031 and vancomycin (VAN). (B) Concentration-dependent killing of <i>P</i>. <i>aeruginosa</i> by SPI031 and polymyxin B (PMB). All data represent means ± standard error of the mean (SEM) from 3 independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). The black dotted lines indicate the lower limit of detection.</p

Network analysis of differential expression data.

<p>Resulting subnetwork from network analysis of RNAseq data using PheNetic [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155139#pone.0155139.ref027" target="_blank">27</a>]. This subnetwork shows molecular mechanisms which are differentially active when comparing <i>P</i>. <i>aeruginosa</i> cells treated with 0.2x MIC of SPI031 to <i>P</i>. <i>aeruginosa</i> cells treated with DMSO. Nodes and connecting lines represent genes and interactions between these genes, respectively. Red dots represent genes overexpressed in the SPI031-treated organisms with respect to the DMSO-treated organism and vice versa for green dots. The color of the connecting lines represents the type of interaction in between genes. Yellow lines represent metabolic interactions, red lines represent regulatory interactions and green lines represent protein-protein interactions.</p

Effect of SPI031 on membrane permeability.

<p>(A) Effect of increasing concentrations of SPI031 on the membrane permeability of <i>S</i>. <i>aureus</i>, monitored by the uptake of SYTOX green. Cells treated with melittin (MEL) (1x MIC) served as a positive control. (B) Inner membrane permeabilization of <i>P</i>. <i>aeruginosa</i> after treatment with different concentrations of SPI031, determined by measuring SYTOX green uptake. Melittin (MEL) (1x MIC) was used as a positive control. (C) Outer membrane permeabilization of <i>P</i>. <i>aeruginosa</i> after treatment with different concentrations of SPI031, assessed by quantifying NPN uptake. Cells treated with polymyxin B (PMB) (1x MIC) were used as a positive control. Data represent the means of three independent replicates ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001 compared to untreated control).</p

Microscopic analysis of the cell membrane after treatment with SPI031.

<p>Fluorescent images of <i>S</i>. <i>aureus</i> cells (upper row) and <i>P</i>. <i>aeruginosa</i> cells (lower row) stained with FM 4–64 in the absence or presence of 1x MIC of SPI031. Scale bar corresponds to 2 μm. Images were processed with unsharp mask of Zen 2.0.</p