Recombinant Tuf of <i>A</i>. <i>baumannii</i> binds plasminogen.

<p>(A) Purity of the recombinant, hexahistidine-tagged proteins was assessed by silver staining (left panel) and Western blotting using a monospecific antibody raised against the hexahistidine-tag (anti-His₆, middle panel). Western blot experiments using a polyclonal antiserum raised against Tuf of <i>S</i>. <i>pneumonia</i> (anti-Tuf_Sp, right panel) revealed that this antiserum also reacts with Tuf proteins from <i>A</i>. <i>baumannii</i> (Tuf_Ab) and <i>L</i>. <i>pneumophila</i> (Tuf_Lp), making it suitable for detection of these proteins in subsequent experiments. (B) Binding of plasminogen (20 μg/ml) to purified Tuf proteins. Far Western blotting shows that recombinant Tuf_Ab and Tuf_Lp bound plasminogen. Tuf_Sp served as a positive control, BSA as a negative control for unspecific binding.</p

Localization of Tuf_Ab on the outer surface of <i>A</i>. <i>baumannii</i> ATCC 19606.

<p>Late log-phase <i>A</i>. <i>baumannii</i> cells (5 x 10⁸) were harvested and resuspended in PBS with 1% (w/v) BSA to block unspecific binding sites. Cells were then incubated with a cross-reacting, polyclonal Tuf_Sp antiserum (1:10). Following several wash steps, cells were incubated with an Alexa Fluor 488 anti-rabbit conjugate (1:25). After incubation, cells were washed again and fixated with 3.75% PFA. Surface exposure of Tuf_Ab was then assayed using flow cytometry. 50,000 events were counted and approximately 40% (± 4.9%) of <i>A</i>. <i>baumannii</i> cells stained positive for Tuf_Ab. Shown are representative results of three separate experiments.</p

<i>A</i>. <i>baumannii</i> Tuf-bound plasminogen is converted to active plasmin by uPA.

<p>Microtiter plates were coated with 5 μg/ml of recombinant Tuf_Ab (A), Tuf_Lp (B), Tuf_Sp (C) or BSA as a negative control for unspecific binding (D) and incubated with plasminogen (10 μg/ml). Following several wash steps, a reaction mixture containing the plasminogen activator uPA (final concentration of 0.1 μg/ml) and the chromogenic substrate D-Val-Leu-Lys-<i>p</i>-nitroanilide dihydrochloride (S-2251) was added (■). Control reactions included 50 mM of the lysine analog tranexamic acid (♦) or omitted plasminogen (▼) or uPA (▲), respectively. Microtiter plates were incubated at RT for 18 h and absorbance at 405 nm was measured at 30 min intervals. At least three independent experiments were conducted, each in triplicate. Data shown are from a representative experiment. For clarity, graphs of negative controls are shaded gray.</p

Tuf-bound plasmin degrades the complement opsonin C3b.

<p>Microtiter plates were coated with recombinant Tuf proteins (10 μg/ml), BBA70 or gelatin as a negative control for unspecific binding. Following incubation with plasminogen (20 μg/ml) and several wash steps, a reaction mixture containing the plasminogen activator uPA (0.16 μg/ml) and C3b (20 μg/ml) was added and microtiter plates were incubated at 37°C. Control reactions included the lysine analog tranexamic acid (+T) or omitted the incubation step with plasminogen (-Plg). Samples were taken at the indicated time intervals and separated by SDS-PAGE. C3b and its degradation products were detected by Western blot analysis probing the membranes with a polyclonal C3 antiserum. Degradation products with apparent molecular masses of approximately 43 kDa, 37 kDa, and 27 kDa are marked by asterisks. Results shown are representative of several independent experiments.</p

<i>A</i>. <i>baumannii</i> ATCC 19606 binds plasminogen.

<p>(A) Binding of plasminogen (10 μg/ml) to increasing numbers of <i>A</i>. <i>baumannii</i> cells was analyzed by whole cell ELISA. Bound plasminogen was detected using a polyclonal plasminogen antiserum. BSA was used as a control for nonspecific binding. Black bars represent plasminogen binding, gray bars represent background signals in the absence of plasminogen. Data represent mean values from at least three independent experiments, each performed in triplicate. Error bars represent standard deviation. *, p ≤ 0.05 and ***, p ≤ 0.001, one-way ANOVA with Bonferroni post hoc test. (B) Binding of plasminogen (20 μg/ml) to viable <i>A</i>. <i>baumannii</i> cells. 2 x 10⁹ cells were incubated with plasminogen. Following incubation, cells were washed thoroughly and bound proteins were eluted. The last wash fraction and eluate fraction were separated via SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and probed with a polyclonal plasminogen antiserum. Purified plasminogen (500 ng) and NHS (2 μl of a 1:10 dilution) served as controls. Arrowhead indicates plasminogen with a molecular mass of 92 kDa.</p

Degradation of fibrinogen by Tuf-bound plasmin.

<p>Tuf proteins, BBA70 and gelatin (5 μg/ml) were immobilized on microtiter plates, blocked and incubated with plasminogen (10 μg/ml). Following several wash steps, a reaction mixture containing the plasminogen activator uPA (0.16 μg/ml) and fibrinogen (20 μg/ml) was added and plates were incubated at 37°C. Samples were taken at the indicated time intervals and separated via SDS-PAGE. Upon transfer to nitrocellulose membranes, fibrinogen or its degradation products were detected in a Western blot analysis using a polyclonal fibrinogen antiserum. Controls included the lysine analog tranexamic acid (+T) and omission of plasminogen (-Plg). Fg, fibrinogen. Shown are representative results from several independent experiments.</p

Further characterization of Tuf-Plasminogen interaction.

<p>(A) Binding of plasminogen (10 μg/ml) to immobilized recombinant Tuf proteins (5 μg/ml) derived from various species was assessed by ELISA. Tuf_Sp was used as a positive control, BSA as a negative control for nonspecific binding. Bound plasminogen was detected using a polyclonal plasminogen antiserum. (B) Binding of plasminogen to immobilized Tuf_Ab and Tuf_Lp occurred in a dose-dependent manner. Tuf proteins (5 μg/ml) were immobilized and incubated with increasing amounts of plasminogen. Binding of plasminogen was analyzed by ELISA using a polyclonal plasminogen antiserum. (C) Role of lysine residues in the Tuf_Ab-plasminogen interaction. Binding of plasminogen (10 μg/ml) to immobilized Tuf_Ab was assayed by ELISA, using a polyclonal plasminogen antiserum, in the presence of increasing concentrations of the lysine analog tranexamic acid. (D) Impact of ionic strength on plasminogen binding to Tuf_Ab. Tuf_Ab was immobilized and incubated with plasminogen (10 μg/ml) and increasing concentrations of NaBr. Plasminogen binding was analyzed by ELISA using a polyclonal plasminogen antiserum. Data represent means and standard deviation of at least three different experiments, each conducted in triplicate. **, p ≤ 0.01 and ***, p ≤ 0.001, one-way ANOVA with Bonferroni post hoc test.</p

Conserved lysine residues of elongation factor Tuf and charge distribution.

<p>3D-structure of Tuf of <i>E</i>. <i>coli</i> in its GDP-bound state. (A) Lysine residues conserved among the organisms analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134418#pone.0134418.s003" target="_blank">S3 Fig</a> are highlighted in blue. Note that residues K3 and K5 from sequence alignment are missing, as the PDB file did not include those amino acids. (B) Predicted charge distribution across the Tuf protein. Fig was created using PyMOL, Version 1.3 and is based on PDB file 2FX3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134418#pone.0134418.ref045" target="_blank">45</a>].</p

Gpm1 and Pra1 expression levels vary among the <i>C</i>. <i>albicans</i> isolates.

<p>Gpm1 (<b>A</b>) and Pra1 (<b>B</b>) surface expression of the thirteen clinical <i>C</i>. <i>albicans</i> isolates. Each <i>C</i>. <i>albicans</i> isolate as well as the reference strain SC5314 was incubated in Gpm1- or Pra1 specific anti-serum and after washing bound antibodies were detected by flow cytometry using secondary Alexa Fluor-488, or Alexa Fluor-647 labeled goat-anti rabbit serum. Control means that SC5314 was incubated with pre-immune serum instead of anti Pra1 or anti Gpm1 sera. Each panel shows representative results of three independent experiments. (<b>C</b>) Median fluorescence intensity of Gpm1 and Pra1 from three independent experiments. The data shown represent mean values ± SD of three separate experiments. (<b>D</b>) Correlation of Gpm1- and Pra1 surface levels for the in thirteen clinical isolates and SC5314. When further analyze the expression level of Candida Gpm1 and Pra1 within these thirteen clinical isolates, we found that Gpm1 and Pra1 surface levels correlated with each other. Most of the high Gpm1 expressing Candida isolates had relatively high Pra1 surface levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113192#pone.0113192.g002" target="_blank">Fig. 2D</a>). Similarly the medium or low Gpm1 expressing strains had medium/low Pra1 surface levels, respectively. The exceptions were isolates J#5 and J#9, of which both had low or high Gpm1, but medium Pra1 levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113192#pone.0113192.t002" target="_blank">Table 2</a>). Thus Gpm1 and Pra1 surface levels are variable among the clinical isolate and surface expression levels of the two fungal immune evasion proteins correlate with each other to a great extent.</p

<i>Candida albicans</i> Gpm1 binds to human cells.

<p>(A) Candida Gpm1 binds to HUVEC and HaCaT. Recombinant Gpm1 was added to human endothelial cells (HUVEC), keratinocytes (HaCaT) or monocytic U937 cells. After incubation and subsequent washing, bound Gpm1 was detected in flow cytometry using rabbit Gpm1 antiserum followed by Alexa Fluor 647 goat anti-rabbit IgG. Human cells without Gpm1 were used as control. Histograms are representative of three independent experiments. (B) Binding of Gpm1 to HUVEC and HaCaT was confirmed by confocal microscopy. HUVEC (top panels) or HaCaT cells (bottom panels) were incubated with Gpm1, fixed with parafolmaldehyde and after washing, bound Gpm1 was detected with rabbit Gpm1 antiserum followed by Alexa Fluor 488 goat anti-rabbit IgG (green). The cellular DNA was stained with DAPI (blue). Scale bar = 10 µm.</p