Characterization of <i>vacJ</i> and <i>yrb</i> mutants.

<p>(<b>A</b>) Effect of mutations in <i>vacJ</i> and genes of the <i>yrb</i> ABC transporter on serum resistance of strain R2866. Survival was determined over 60 min in 5% normal human serum and expressed relative to controls in which complement was inactivated. (<b>B</b>) Representative histogram comparing the binding, as measured by fluorescence intensity (x-axis), of total IgM purified from normal human serum to parent strain (WT) or <i>vacJ</i> by flow cytometry. Control performed without IgM (<b>C</b>) Percent IgM binding for each mutant was determined by calculating the percentage of 50,000 events with an increase in mean fluorescence intensity following incubation in 5% heat-inactivated normal human serum compared to no serum controls. (<b>D</b>) Survival of mutants in 10% normal human serum in the presence (black bars) or absence (white bars) of Mg-EGTA to inhibit the classical pathway of complement activation. Values represent two independent experiments in triplicate ± SD. ^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001.</p

Membrane stability and <i>vacJ</i> and <i>yrb</i> expression among clinical isolates.

<p>(<b>A</b>) Outer membrane stability of serum sensitive (n = 31) compared to serum resistant (n = 16) clinical isolates. Following overnight culture, viable counts were obtained after incubation at 37°C for 4 h with or without 25 mM EDTA. Values represent two independent experiments in triplicate ± SD. ^*<i>P</i><0.05, Serum sensitive (SS), Serum resistant (SR). (<b>B</b>) Relative expression of <i>vacJ</i> mRNA, (C) <i>yrbD</i> and (D) <i>yrbE</i> by qRT-PCR in serum sensitive (white bar, n = 7) and serum resistant isolates (black bar, n = 7). Error bars indicate SD, ^**<i>P</i><0.01.</p

Repeated serum treatment selects for serum resistance, increased <i>vacJ</i> expression, and increased outer membrane stability.

<p>(<b>A</b>) Serum sensitive clinical isolate H725 was treated three times in 2.5% NHS and survival quantified after the passage indicated. (<b>B</b>) Following each passage in the bactericidal assay survivors were tested for relative expression of <i>vacJ</i> mRNA by qRT-PCR and (<b>C</b>) survival in the presence of 25 mM EDTA. Values represent two independent experiments in triplicate ± SD.^*<i>P</i><0.05, ^***<i>P</i><0.001.</p

Characterization of clinical isolates.

<p>(<b>A</b>) Comparison of serum sensitivity between lower and upper respiratory tract isolates. Survival was determined over 60 min in 5% normal human serum and expressed relative to controls in which complement was inactivated. Groups included lower respiratory tract isolates from patients with chronic obstructive pulmonary disease (COPD) at the time of clinical exacerbation (black bars; n = 11); isolates from patients with COPD during clinically stable periods (grey bars; n = 11); and upper respiratory tract colonizing strains (white bars; n = 25). Values are the mean of three determinations in triplicate ± SEM. (<b>B</b>) Comparison of antibody binding in serum resistant (>50% survival in pooled NHS, black bars; n = 10) and serum sensitive (<50% survival in pooled NHS, white bars; n = 10) isolates. B1 and B2 show percent of IgG and IgM bound following incubation in 5% heat-inactivated NHS as determined by flow cytometry, respectively. (<b>C</b>) Serum IgM contributes to bactericidal killing of clinical isolates of NTHi. Strains were incubated with or without IgG (C1, 0.25 µg/ml) or IgM (C2, 0.07μg/ml) purified from NHS for 60 min in 2.5% baby rabbit serum as a complement source. Percent survival was calculated by viable counts with and without antibody. The mean values of two independent experiments in triplicate are shown ± SD, ^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001. Serum sensitive (SS), Serum resistant (SR).</p

Effect of mutations in <i>vacJ</i> and genes of the <i>yrb</i> ABC transporter on outer membrane characteristics.

<p>(<b>A</b>) To compare outer membrane stability, following overnight culture of the strain indicated, viable counts were obtained after incubation at 37°C for 4 h in the presence (white bars) or absence (black bars) of 25 mM EDTA. Values represent two independent experiments in triplicate ± SD. ^*<i>P</i><0.05. (<b>B</b>) To compare surface hydrophobicity, the rate of uptake of membrane permeant 1-<i>N</i>-phenylnaphthylamine (NPN) was monitored by fluorescence. NPN was added at the time indicated and a representative experiment shown. (<b>C</b>) To compare amounts of surface phospholipids, diacylglycerol (boxed area) released by phospholipase C treatment of whole bacteria was detected by thin layer chromatography. Lane 1; diacylglycerol (standard), Lane 2 and 3; R2866 (wild type), Lane 4 and 5; 32F2 (<i>vacJ</i> mutant), Lane 6 and 7; 69G3 (<i>yrbE</i> mutant).</p

List of sites with multiple transposon insertions affecting serum resistance in strain R2866.

<p>*NCBI Reference Sequence: NC_000907.1 (<i>Haemophilus influenzae</i> Rd KW20, complete genome).</p>#<p>NCBI Reference Sequence: NZ_AADP01000001 and NZ_AADP01000002 (<i>Haemophilus influenzae</i> R2866 whole genome).</p

Effect of <i>vacJ</i> and <i>yrb</i> mutants on antibody binding and bactericidal activity.

<p>Binding of (<b>A</b>) mAb 4C4 to the LOS structure Galα1-4Gal or (<b>B</b>) mAb TEPC-15 to the LOS structure phosphorylcholine was compared by flow cytometry for the mutants indicated. Percent mAb binding for each mutant was determined by calculating the percentage of 50,000 events with an increase in mean fluorescence intensity compared to no primary antibody controls. (<b>C</b>) Effect of mutations in <i>vacJ</i> and genes of the <i>yrb</i> ABC transporter on the bactericidal effect of mAb 4C4. Bactericidal assays were performed with (white bars) or without (black bars) mAb with 5.0% normal mouse serum as a complement source. Values represent two independent experiments in triplicate ± SD. ^*<i>P</i><0.05, ^***<i>P</i><0.001.</p

Effects of two known azole transporter inhibitors, carbonyl cyanide 3-chlorophenylhydrazone and milbemycin A4 oxime, on micafungin susceptibility in <i>C</i>. <i>glabrata</i> wild-type strain and <i>CDR1</i>, <i>CDR2</i>, and <i>PDR1</i> mutant strains.

<p>Spot dilution tests were performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g001" target="_blank">Fig 1B</a>. Final drug concentrations: carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 40 μM; milbemycin A4 oxime (Milbemycin), 4 μM; micafungin (MCFG), 0.03 μg/ml. <i>C</i>. <i>glabrata</i> strains: Wild type, CBS138; Δ<i>cdr1</i>, TG-C1; Δ<i>cdr2</i>, TG-C2; and Δ<i>pdr1</i>, TG-C3.</p

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in <i>Candida glabrata</i> and other pathogenic <i>Candida</i> species - Fig 4

<p>(A) Time-course analysis of <i>FMS1</i> expression in <i>C</i>. <i>glabrata</i> wild-type strain. Cell culture, RNA extraction, real-time qRT-PCR, and data presentation were performed as described in the Materials and Methods section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g003" target="_blank">Fig 3A</a> legend. (B) Effects of clorgyline on susceptibility to micafungin and amphotericin B in the <i>C</i>. <i>glabrata</i> wild-type and <i>FMS1</i> mutant strains. Spot dilution tests were performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g001" target="_blank">Fig 1B</a>. Final drug concentrations: clorgyline, 80 μg/ml; micafungin (MCFG), 0.03 μg/ml; and amphotericin B (AMPH-B), 1.25 μg/ml. <i>C</i>. <i>glabrata</i> strains: Wild type, CBS138; and Δ<i>fms1</i>, TG-C4.</p

Strains used in this study.

<p>Strains used in this study.</p