Injection of ExoS-Bla into differentiated HL-60 cells is sensitive to pharmacological agents.

<p><b>A/</b> VD3-differentiated HL-60 cells were exposed to 10 µM of cytochalasin D (CytoD), 10 µM of latrunculin B (LtrB) or 50 nM of Wortmannin for 30 min prior and during infection, or to 2 µM of TAT-C3 toxin, 100 µM of LY-294002, 12 µM of genistein, 10 µM of PP2, and 10 µM of PF-573-228 for 120 min prior and during a 3 h infection period at MOI 10 with the PAO1ΔSTY-SBlaR146A strain. The percentage of injection-positive cells was evaluated by flow cytometry. <b>B/</b> Eukaryotic plasma membranes were purified by fractionation on sucrose gradient, after infection of cells pre-treated with inhibitors as above. Proteins were analysed by immunoblotting using anti-PopB. Ni : non-infected VD3-differentiated HL-60 cells, CTRL: infected VD3-differentiated HL-60 cells without inhibitor. <b>C/</b> The ability of PAO1ΔSTY-SBlaR146A strain to secrete ExoS-Bla <i>in vitro</i> was assayed for each inhibitor modifying injection by immunoblotting of total secreted ExoS-Bla protein using the anti-ß-lactamase antibody. Pa: supernatant of T3SS non-induced <i>P.aeruginosa</i>, IPa: supernatant of T3SS induced <i>P. aeruginosa</i> without inhibitor.</p

Impact of the CFTR-Potentiator Ivacaftor on Airway Microbiota in Cystic Fibrosis Patients Carrying A G551D Mutation

<div><p>Background</p><p>Airway microbiota composition has been clearly correlated with many pulmonary diseases, and notably with cystic fibrosis (CF), an autosomal genetic disorder caused by mutation in the CF transmembrane conductance regulator (CFTR). Recently, a new molecule, ivacaftor, has been shown to re-establish the functionality of the G551D-mutated CFTR, allowing significant improvement in lung function.</p><p>Objective and Methods</p><p>The purpose of this study was to follow the evolution of the airway microbiota in CF patients treated with ivacaftor, using quantitative PCR and pyrosequencing of 16S rRNA amplicons, in order to identify quantitative and qualitative changes in bacterial communities. Three G551D children were followed up longitudinally over a mean period of more than one year covering several months before and after initiation of ivacaftor treatment.</p><p>Results</p><p>129 operational taxonomy units (OTUs), representing 64 genera, were identified. There was no significant difference in total bacterial load before and after treatment. Comparison of global community composition found no significant changes in microbiota. Two OTUs, however, showed contrasting dynamics: after initiation of ivacaftor, the relative abundance of the anaerobe <i>Porphyromonas</i> 1 increased (p<0.01) and that of <i>Streptococcus</i> 1 (<i>S</i>. <i>mitis</i> group) decreased (p<0.05), possibly in relation to the anti-Gram-positive properties of ivacaftor. The anaerobe <i>Prevotella</i> 2 correlated positively with the pulmonary function test FEV-1 (r=0.73, p<0.05). The study confirmed the presumed positive role of anaerobes in lung function.</p><p>Conclusion</p><p>Several airway microbiota components, notably anaerobes (obligate or facultative anaerobes), could be valuable biomarkers of lung function improvement under ivacaftor, and could shed light on the pathophysiology of lung disease in CF patients.</p></div

Efficiency of ExoS-Bla and ExoY-Bla translocation in different cell lines.

<p><b>A/</b> A549 epithelial cells, BJAB, Jurkat, non-differentiated HL-60 cells (HL-60), or HL-60 differentiated into neutrophils, macrophages and monocytes by DMSO, PMA or Vitamin D3 (VD3), respectively, were infected at MOI of 10, for 3 h, with CHAΔS expressing ExoS-BlaR146A and analyzed by flow cytometry. <b>B/</b> HL-60 and U937 were differentiated in monocytes with VD3 (black bars) or not (white bars) and infected at MOI 10 with PAO1ΔSTY strains expressing either ExoS-BlaR146A or ExoY-Bla as described above. The error bars indicate standard deviation (n = 3). <b>C/</b> Non-differentiated HL-60 (thin lane) or HL-60 differentiated in neutrophils (dot line), monocytes (dash line) or macrophage (thick line) were labelled with a FITC conjugated antibody specific for CD11b and analysed by flow cytometry.</p

Switching T3SS resistant HL-60 cells to T3SS permissive cells by serum starvation or panning to anti-CD43.

<p>Non-differentiated HL-60 cells were treated as follows: maintained during 15 min in medium supplemented (+ FCS) or not (− FCS) (a), incubated during 5 min in either naive (− anti CD43) or coated wells with a monoclonal anti CD43 (+ anti CD43), first panned in a anti CD43-coated well during 5 min and then transferred to a naïve well, (transferred) (b) and cells were next incubated at MOI 10 with the CHAΔS-SblaR146A strain and analysed by flow cytometry. Non-differentiated HL-60 were incubated during 5 min in a well coated with anti CD43 in the absence (− CytoD) or the presence (+ CytoD) of 10 µM of cytochalasine D for 30 min prior and during infection at MOI 10 with the CHAΔS-SblaR146A strain (c). The error bars indicate standard deviation (n = 3). Insert: schematic drawing of the experiments presented in the histogram (part <b>b</b>).</p

Characterization of ExoS-Bla and ExoY-Bla reporters.

<p><b>A/</b> Secretion profiles of <i>P. aeruginosa</i> strains carrying the reporter fusion ExoS-Bla. Fifteen µL of culture supernatants of either wild-type CHA strain or mutant CHA strains expressing ExoS-Bla grown under T3SS-inducing conditions were analyzed by immunoblotting with antibodies directed against ExoS, PopB, PopD and PcrV. <b>B/</b> Co-cultures of A549 cells with <i>P. aeruginosa</i> strains for 3 h at MOI 10. Injection of either ExoS-Bla or ExoY-Bla fusions by strains CHA and PAO1ΔSTY was detected after incubating cells with β-lactamase substrate CCF2-AM either by fluorescence microscopy using a 20× objective (upper panel) or by flow cytometry (lower panel). The horizontal bar in flow-cytometry histograms indicates the gating used to determine the percentage of β-lactamase positive cells revealing ExoS-Bla injection. Mean Fluorescence Intensity is indicated in each panel. Scale bar, 100 µm.</p

Relative abundance (RA) of OTUs belonging to the major core microbiota.

<p>A) RA of OTUs for the three patients (GM, PM, and RM) highlighted that each individual harbored his or her own microbiota, even if several genera were shared. B) RA of OTUs before ivacaftor treatment (BT) and after the beginning of ivacaftor treatment (AT) for each patient. RA of <i>Streptococcus</i> 1 showed a tendency to decrease from BT to AT samples, whereas <i>Porphyromonas</i> 1 increased. C) Grouping all BT samples (on the left of the graph) and all AT samples (on the right of the graph) confirmed the tendency observed per patient: after ivacaftor treatment, the RA of <i>Streptococcus</i> 1 decreased while that of <i>Porphyromonas</i> 1 increased.</p

Association between relative abundance of OTUs belonging to the major core microbiota and FEV-1 (n = 15 sputum samples).

<p>SCC: Spearman's correlation coefficient (r).</p><p>FDR: false discovery rate.</p><p>Significant correlations after FDR adjustment are in bold (threshold = 0.1).</p><p>Association between relative abundance of OTUs belonging to the major core microbiota and FEV-1 (n = 15 sputum samples).</p

Patient characteristics associated with each sputum sample.

<p>^aDates in bold correspond to the beginning of ivacaftor treatment; sputum samples corresponding to the first day of ivacaftor treatment were collected before its administration.</p><p>FEV-1: forced expiratory volume in 1 second.</p><p>Hi: <i>Haemophilus influenzae</i>, MSSA: methicillin sensitive <i>Staphylococcus aureus</i>, <i>Pa</i>: <i>Pseudomonas aeruginosa</i>.</p><p>Patient characteristics associated with each sputum sample.</p

Dynamics and interrelations of 7 key-role OTUs throughout ivacaftor treatment, and their correlations with lung function.

<p><i>Streptococcus</i> 1 (<i>S</i>. <i>mitis</i> group) and <i>Porphyromonas</i> 1 were the two OTUs for which a significant association with ivacaftor treatment period emerged (Colin-White test; See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124124#pone.0124124.s007" target="_blank">S2 Table</a>): <i>Streptococcus</i> 1 (<i>S</i>. <i>mitis</i> group) was associated with sputum samples collected before (p<0.05) and <i>Porphyromonas</i> 1 with samples collected after initiation of treatment (p<0.01). The depicted correlations between OTUs were all statistically significant with the adjusted p-values (Spearman correlation test; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124124#pone.0124124.t003" target="_blank">Table 3</a>). Significant correlations between OTUs and lung function (on FEV-1 test) are shown by red arrow when negative (p<0.05) and green arrow (light green if p<0.1; dark green if p<0.05) when positive (adjusted p-values; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124124#pone.0124124.t002" target="_blank">Table 2</a>).</p

FDR adjusted p-values associated with Spearman correlations between relative abundance (RA) of OTUs belonging to the major core microbiota and qPCR results.

<p>Significant correlations after FDR adjustment are represented in bold (threshold: 0.05), or are indicated by a star (threshold: 0.1). Negative correlations are underlined (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124124#pone.0124124.s008" target="_blank">S3 Table</a>).</p><p>Ge: <i>Gemella</i>, Str2: <i>Streptococcus</i> 2 (<i>S</i>. <i>salivarius</i> group), Neis2: <i>Neisseria</i>, Ha1: <i>Haemophilus</i> 1, Neis1: <i>Neisseria</i> 1, Pre2: <i>Prevotella</i> 2, Sta: <i>Staphylococcus aureus</i>, Fus1: <i>Fusobacterium</i> 1, Rot2: <i>Rothia</i> 2, Pre1: <i>Prevotella</i> 1, Rot1: <i>Rothia</i> 1, Str3: <i>Streptococcus</i> 3 (<i>S</i>. <i>anginosus</i> group), Str1: <i>Streptococcus</i> 1 (<i>S</i>. <i>mitis</i> group), Vei1: <i>Veillonella</i> 1, Other: OTUs out of the major core microbiota, Pept: <i>Peptostreptococcus</i>, Por1: <i>Pophyromonas</i> 1.</p><p>FDR adjusted p-values associated with Spearman correlations between relative abundance (RA) of OTUs belonging to the major core microbiota and qPCR results.</p