Mouse neutrophils do not require transcription or translation to release NETs.

<p>Inhibitors were used at the following concentrations: Flavopiridol (0.05 μM), CAS 577784-91-9 (10 μM), CHX (1 μg/ ml). (A) Flavopiridol and CHX, but not CAS 577784-91-9 block Mip-1α production in response to LPS. (B) PMA-induced NET formation is not inhibited by flavopiridol, CAS 577784-91-9 or CHX. Neutrophils isolated from Nox2 -/-mice are used as negative control. (C) NET formation in response to heat-killed <i>C</i>. <i>albicans</i> is not blocked by inhibitors. Graphs show mean ± SEM of experiments with neutrophils isolated from 3 mice. (D) Representative immunofluorescence pictures of murine neutrophils stained with PL2-3 (chromatin, red) and DAPI (blue) after infection with <i>C</i>. <i>albicans</i> (MOI 3) and treatment with transcription/ translation inhibitors. (B, C) Statistical analysis revealed no significant changes after inhibitor treatment.</p

Human neutrophils do not require transcription or translation to release NETs in response to PMA.

<p>(A-G). Inhibitors were used at the following concentrations: Actinomycin D (1 μg/ ml), flavopiridol (0.05 μM), CAS 577784-91-9 (10 μM), CHX (1 μg/ ml). (A) Actinomycin D, flavopiridol and CHX, but not CAS 577784-91-9 inhibit <i>de novo</i> production of Mip-1α induced by LPS. (B) Actinomycin D and flavopiridol, but not CAS 577784-91-9or CHX block mRNA transcription of PMA-treated human neutrophils. (C.) Actinomycin D, flavopiridol and CHX, but not CAS 577784-91-9 inhibit <i>de novo</i> production of IL-8 induced by PMA. (D-G) Actinomycin D, flavopiridol, CAS 577784-91-9 or CHX do not block NET formation in response to PMA (100 nM). (D, E). NET formation was quantified by adding the DNA dye SYTO green (total cells) and the cell impermeable DNA dye SYTOX orange (NETs). (F) NET formation was quantified by immunofluorescence staining of chromatin and Hoechst as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157454#pone.0157454.ref010" target="_blank">10</a>]. Graphs show mean ± SEM from independent experiments with at least 3 different donors. (G) Representative images of human neutrophils stained with PL2-3 (chromatin, red), NE (Neutrophil Elastase, green) and Hoechst (blue) after treatment with transcription or translation inhibitors and PMA induction. (D-F) Statistical analysis revealed no significant changes after inhibitor treatment.</p

Human neutrophils do not require transcription or translation to release NETs in response to <i>C</i>.<i>albicans</i>.

<p>(A, B) Inhibitors were used at the following concentrations: Actinomycin D (1 μg/ ml), flavopiridol (0.05 μM), CAS 577784-91-9 (10 μM), CHX (1 μg/ ml). Cells were infected with opsonized <i>C</i>.<i>albicans</i> at MOI 5 and subsequently fixed for immunofluorescence staining. (A) NET formation was quantified at 2h after infection by immunofluorescence staining of chromatin and Hoechst. Graphs show mean ± SEM from independent experiments with 3 different donors. (B) Representative images of an early time point (2h) of human neutrophils infected with <i>C</i>.<i>albicans</i> and treated with transcription or translation inhibitors. Cells were stained with PL2-3 (chromatin, red), NE (Neutrophil elastase, green) and Hoechst (blue). (C) Representative images of a late time point (4h) of <i>C</i>.<i>albicans</i>-infected neutrophils showing spread NETs that are unaffected by inhibitors of gene expression. (A) Statistical analysis revealed no significant changes after inhibitor treatment.</p

Involvement of the selected genes in the colanic acid pathway and cloning under arabinose promoter.

<p>(A) The genes screened for histone resistance (bold pink) are represented in the scheme of the colanic acid pathway. The first steps of the colanic acid pathway consist on the production of activated sugar in the cytoplasm of the bacterium. Mannose-6-phosphate (Man-6-P) leads to the production to GDP-fucose by CpsG and CpsB. Others activated sugars, UDP-galactose (UDP-Gal), -glucosamine (-Glc) and –glucuronic acid (-GlcA), are produced also for other cellular components. The assembly of the first sugar unit, colanic acid (CA) unit, is assembled on the lipid carrier, called undecaprenyl pyrophosphate, by Wca proteins, such as WcaJ, WcaI and WcaA. Then the CA unit is exported and polymerized by the Wzc machinery, for the production of the exopolysaccharide. Another possibility is the addition of CA unit on the short form of the lipopolysaccharide (LPS) by an unknown mechanism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071097#pone.0071097-Meredith1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071097#pone.0071097-Stevenson1" target="_blank">[22]</a>. (B) The selected genes belonging to the colanic acid cluster were cloned under arabinose promoter on pBAD33 and pBAD18 vectors. Different constructs were obtained to address different gene combinations in the histone resistance and to refine the essential genes for the resistance. (C) The toxicity of the arabinose-induced genes was tested by following the bacterial population (CFU/ml). After 2 h of culture (indicated by an arrow and considered as time 0), L-arabinose was added at 0.4 (open triangle, full line), 0.04 (light gray triangle, dash line) and 0.004% (dark gray triangle, dot line) final concentration or not (black diamond, full line), and the culture was prolonged during 6 h.</p

Contribution of capsule and LPS in histone resistance in <i>Klebsiella pneumoniae</i> and <i>Shigella flexneri</i>.

<p>MBC90 and MBC99 have been determined for (A–B) <i>K. pneumoniae</i> wild type (Kp52), acapsulated mutant (52K10) and O-antigen mutant (52O21), and (C–D) in <i>S. flexneri</i> background: wild type (M90T) and O-antigen mutant (<i>waaL-</i>). Each dot represents the result of one experiment and at least 4 independent experiments have been done. The differences were considered significant by the Mann-Whitney test with p≤0.05 (*).</p

Primers designed for the different clonings of the <i>wcaI/J</i> and <i>wzc/wcaA</i> fragments in pBAD33 and pBAD18 (see Fig. 2).

<p>On the sequence of the primers is represented: the restriction site (underlined); the Shine Dalgarno (lower case letter); start or stop codon (bold) for the forward (F symbol in the primer name) or reverse (R symbol) primers, respectively.</p

O-Antigen Protects Gram-Negative Bacteria from Histone Killing

<div><p>Beyond their traditional role of wrapping DNA, histones display antibacterial activity to Gram-negative and -positive bacteria. To identify bacterial components that allow survival to a histone challenge, we selected resistant bacteria from homologous <i>Escherichia coli</i> libraries that harbor plasmids carrying pieces of the chromosome in different sizes. We identified genes required for exopolysaccharide production and for the synthesis of the polysaccharide domain of the lipopolysaccharide, called O-antigen. Indeed, O-antigen and exopolysaccharide conferred further resistance to histones. Notably, O-antigen also conferred resistance to histones in the pathogens <i>Shigella flexneri</i> and <i>Klebsiella pneumoniae</i>.</p></div

bacterial strains and plasmids.

<p>bacterial strains and plasmids.</p

Characterization of exopolysaccharide and lipopolysaccharide production in <i>E. coli</i> MACH1 expressing <i>wcaI, cpsB</i> and <i>cpsG</i> genes.

<p>(A) We purified the exopolysaccharide from supernatants of overnight liquid culture <i>E. coli</i> MACH1 pBAD18 empty vector or carrying <i>wcaI/cpsB</i>, <i>cpsB/cpsG</i>, <i>wcaI</i>, <i>cpsB</i> or <i>cpsG</i>, growing in presence of arabinose. The isolated exopolysaccharide was quantified by determining the carbohydrate content. Each dot represents the result of one measurement. The differences were considered significant by the Mann-Whitney test with p≤0.001 (***). (B) From the same liquid cultures, an aliquot was taken to analyze the LPS. The preparations of proteinase K-treated whole cell lysates were run on a Tris-tricine polyacrylamide gel and silver stained after periodate treatment. The gel represents an example of 3 independent experiments.</p

Impact of over-expression of colanic acid related genes identified in the screen on the histone resistance.

<p>We determined the minimal bactericidal concentration of histones to kill 90% (MBC90, left panels) or 99% (MBC99, right panels) of the initial bacterial inoculum after 1 h of incubation. (A–B) The simultaneous over-expression of <i>wcaI</i>, <i>cpsB</i>, <i>cpsG</i> genes in MACH1 carrying the plasmids pBAD33<i>cpsG</i> pBAD18<i>wcaI/cpsB</i> or pBAD33<i>wcaI</i> pBAD18<i>cpsB/cpsG</i> led to a high histone resistance in comparison with the strain carrying the empty vectors. (C–D) To refine the genes contributing the resistance, we tested the over-expression of <i>wcaI/cpsB</i>, <i>cpsB/cpsG,</i> or single genes, <i>wcaI</i>, <i>cpsB</i>, <i>and cpsG,</i> carried by pBAD18. (E–F) The other set of genes related to the colanic acid, <i>wzc/wcaA</i>, did not show any significant histone resistance in term of MBC90 and MBC99 in comparison with the bacteria carrying the empty vector, while the genes were over-expressed together (MACH1 pBAD18<i>wzc/wcaA</i>) or individually (MACH1 pBAD18<i>wzc</i> and MACH1 pBAD18<i>wcaA</i>). (G) The <i>E. coli</i> over-expressing this different combination of <i>wzc</i> and <i>wcaA</i> genes showed a higher number of survivors after 1 h treatment with histones at 100 µg/ml. To be able to compare the different clones, we standardized the values by dividing the CFU/ml after 1 h treatment by the CFU/ml of the inoculum. Each dot represent the result of one experiment and at least three independent experiments have been done. The differences were considered significant by the Mann-Whitney test with p≤0.05 (*) and p≤0.01 (**).</p