<i>C. albicans</i> directly detoxifies ROS, but cannot block PMA-induced neutrophil ROS.

<p><u>ROS scavenging</u>: Different masses of <i>C. albicans</i> yeasts and hyphae, RPMI medium and unstimulated neutrophils were added to hydrogen peroxide. Each column represents the corresponding AUC from RLUs measured by luminol (A). A representative experiment out of three independent experiments from three different donors is shown. Data are presented as means of three technical replicates ±SD (NS: P>0.05 and ***P≤0.001). <u>Spiking ROS assay II</u>: ROS production over time is shown for a total of 3 h as relative light units (RLUs). For each sample, 10⁵ neutrophils were infected with 6 µg <i>C. albicans</i> yeasts (closed circles, B) or hyphae (closed squares, C) and after 30 min PMA was added. Additionally, neutrophils were stimulated with 100 nM PMA only (stars, D). A representative experiment out of three independent experiments from three different donors is shown. Data are presented as means of three technical replicates ±SD. Masses to MOI conversion for 10⁵ neutrophils: (Yeasts) 4 µg≙1.6, 6 µg≙2.5, 8 µg≙3.3, 16 µg≙6.6, 32 µg≙13.1; (hyphae) 4 µg≙0.7, 6 µg≙1, 8 µg≙1.4, 16 µg≙2.7, 32 µg≙5.4.</p

DM, cell surface area per <i>C. albicans</i> yeast or hypha and their ratios.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077993#pone-0077993-t001" target="_blank">Table 1:</a> To determine the DM per yeast cell at the indicated time points the correlation coefficient factors of DM versus time and cell number versus time were used. With these values the average yeast concentration (DM per ml) was divided by the total number of yeast cells per ml. Similarly, calculations were performed for hyphae. Cell numbers of the initial inoculum at OD 0.1 were used assuming that under hypha-inducing conditions each yeast cell germinates and grows as hypha. To calculate the cell surface area and volume of yeasts and hyphae at different time points, the dimensions (length, width and depth) of approximately 50 immune-stained cells were measured by confocal microscopy as described in materials and methods. The ratio of cell surface areas per µg DM and cell number per µg DM from yeasts and hyphae were calculated for different time points. Data are presented as average of three biological replicates ± SD.</p

Not heat-killed, but thimerosal-killed <i>C. albicans</i> and live <i>S. cerevisiae</i> detoxify neutrophil ROS.

<p><u>Spiking ROS assay IV</u>: ROS production over time is shown for a total of 3 h as relative light units (RLUs). For each sample, 10⁵ neutrophils were infected with a first infection dose of 3 µg of <i>C. albicans</i> wild-type hyphae (closed squares, A-D); 30 min thereafter, the cells were re-infected with 10 µg of <i>C. albicans</i> heat-killed yeasts (open circles, A) or of heat-killed hyphae (open squares, B), with 10 µg of thimerosal-killed yeasts (open triangles, C) or of thimerosal-killed hyphae (open rhombuses, D). In (E), neutrophils were infected with a first infection dose of 6 µg of <i>C. albicans</i> wild-type hyphae (closed squares) and 30 min thereafter re-infected with <i>S. cerevisiae</i> at MOI 12 (gray squares) or MOI 6 (open squares). In (F), neutrophils were stimulated with PMA (100 nM, star), different MOIs of <i>S. cerevisiae</i> (rhombuses and triangles) or remained unstimulated (cross). A representative experiment out of three independent experiments from three different donors is shown. Data are presented as means of three technical replicates ±SD. Masses to MOI conversion for 10⁵ neutrophils: (Yeasts) 10 µg≙4.1; (hyphae) 3 µg≙0.5, 6 µg≙1, 10 µg≙1.7.</p

Neutrophil ROS response pattern depends on amount, morphotype and viability of <i>C. albicans</i>.

<p>Neutrophil ROS generation was measured in a luminol-based assay. Different DM amounts of <i>C. albicans</i> were used to infect 10⁵ neutrophils. Each data point represents AUC corresponding to the total ROS in the course of 3 h. Neutrophils were infected either with wild-type yeasts (closed circles) or <i>Δefg1</i> mutant (open triangles) (A) or wild-type hyphae (closed squares) or <i>Δtup1</i> mutant (open inverted triangles) (B). Neutrophils were infected with heat-killed (open triangles) or thimerosal-killed (open rhombuses) yeasts (C) or hyphae (D). The dashed line represents the best-fit curve of three individual assays from different donors. Masses to MOI conversion for 10⁵ neutrophils: (Yeasts) 1 µg≙0.4, 5 µg≙2.1, 10 µg≙4.1, 20 µg≙8.2, 30 µg≙12.3; (hyphae) 1 µg≙0.2, 5 µg≙0.9, 10 µg≙1.7, 20 µg≙3.4, 30 µg≙5.1.</p

Neutrophil IL-8 response pattern depends on amount, morphotype and viability of <i>C. albicans</i>.

<p>Neutrophil IL-8 secretion was measured by ELISA. For each sample, 10⁵ neutrophils were infected with <i>C. albicans</i>. Each data point represents the total amount of IL-8 secreted by neutrophils in the course of 6 h. To compare C. <i>albicans</i> wild-type to morphotype-locked mutants neutrophils were infected with wild-type yeasts (closed circles, A), the <i>Δefg1</i> mutant (open triangles, A), wild-type hyphae (closed squares, B) or the <i>Δtup1</i> mutant (open inverted triangles, B). Neutrophils were infected with C. <i>albicans</i> wild-type yeasts which were alive (closed circles, C), thimerosal-killed (half-closed circles, C) or heat-killed (open circles, C) or were infected with C. <i>albicans</i> wild-type hyphae which were alive (closed squares, D), thimerosal-killed (half-closed squares, D) or heat-killed (open squares, D). The dashed line indicates the best-fit which was obtained from three individual assays from three different donors. Masses to MOI conversion for 10⁵ neutrophils: (Yeasts) 1 µg≙0.4, 5 µg≙2.1, 10 µg≙4.1, 20 µg≙8.2, 30 µg≙12.3; (hyphae) 1 µg≙0.2, 5 µg≙0.9, 10 µg≙1.7, 20 µg≙3.4, 30 µg≙5.1.</p

Neutrophil ROS and IL-8 response triggered with different amounts and morphotypes of wild-type <i>C. albicans</i>.

<p>Neutrophil ROS generation was measured in a luminol-based assay. Different DM amounts of <i>C. albicans</i> were used to infect 10⁵ neutrophils. Each data point represents AUC corresponding to the total ROS in the course of 3 h (A). IL-8 secretion was measured from supernatants of 10⁵ neutrophils after 6 h by ELISA (B). Neutrophils were infected with <i>C. albicans</i> yeasts (closed circles) or hyphae (open squares). The dashed line represents the best-fit curve of three individual assays from different donors. Masses to MOI conversion for 10⁵ neutrophils: (Yeasts) 1 µg≙0.4, 5 µg ≙2.1, 10 µg≙4.1, 20 µg≙8.2, 30 µg≙12.3; (hyphae) 1 µg≙0.2, 5 µg≙0.9, 10 µg≙1.7, 20 µg≙3.4, 30 µg≙5.1.</p

<i>C. albicans</i> morphotype and strain-specific correlation factors.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077993#pone-0077993-t002" target="_blank">Table 2:</a> Basic linear equations (y = mx±b) are presented to calculate DM. For yeast all three types of correlations: DM/MA, DM/OD and MA/OD are used. For the hyphal growth form the DM/MA correlation factor is proposed. The valid range of OD and MA measurements per 1 ml are indicated. The equations and R-square (R²) values were obtained from plotted graphs.</p

a-b: Bar graphs with element weight fractions within entire neutrophils throughout PMA stimulation for the elements P, S, K, Cl, K, Ca, Mn, Fe, Co (upper graph, Fig 3a) and for Ni, Cu, Zn, Se, Br, Sr and Pb (lower graph, Fig 3b).

<p>Neutrophils from control culture are indicated in green, 1 <i>h</i> PMA-stimulated neutrophils in blue and 2 <i>h</i> PMA-stimulated neutrophils in red. Weight fractions are expressed in <i>%</i>, <i>ppm</i> or <i>ppb</i> in a (different) logarithmic scale. Weight fraction values are normalized to the Compton intensity of neutrophil “0h_donorA_ cell1”. Error bars are based upon Poisson counting statistics and the certified uncertainty values of NIST SRM1577C “Bovine liver”.</p

a-d: Bar graphs with element weight fractions within neutrophil nuclei (left column, Fig 4a-b) and cytoplasms (right column, Fig 4c-d) throughout PMA stimulation for P, S, K, Cl, K, Ca, Mn, Fe, Co (upper row) and for Ni, Cu, Zn, Se, Br, Sr and Pb (lower row).

<p>Neutrophils from control culture are indicated in green, 1 <i>h</i> PMA-stimulated neutrophils in blue and 2 <i>h</i> PMA-stimulated neutrophils in red. Weight fractions are expressed in <i>%</i>, <i>ppm</i> or <i>ppb</i> in a (different) logarithmic scale. Weight fraction values are normalized to the Compton intensity of neutrophil “0h_donorA_ cell1”. Error bars are based upon Poisson counting statistics and the certified uncertainty values of NIST SRM1577C “Bovine liver”.</p

RGB composite images of the trace level element distribution of Ca, Zn and Fe (represented by the red, green and blue color channel respectively) of two single, high pressure frozen and cryosubstituted human neutrophils (white blood cells) before and after stimulation with phorbol myristate acetate (PMA), inducing the formation of so-called Neutrophil Extracellular Traps (or NETs).

<p>NETs are newly discovered structures which are believed to act as a defense mechanism against microbes via chelating proteins, removing crucial trace elements from the pathogen. As all intensities in the trichromatic maps are normalized to a single upper value, increases in color brightness also represent increases in concentration. A clear increase in cellular Ca concentration is revealed via appearance of a yellow nucleus and reddish cytoplasm. Intracellular Fe and Fe/Ca-rich structures are also emerging after 2 <i>h</i> stimulation with PMA, visible as blue and pinkish hot-spots. The absent green color in the PMA-stimulated neutrophil indicates the strong association of Zn to Ca and/or Fe. Results were obtained using synchrotron radiation based X-ray fluorescence at the ID22NI beamline (European Synchrotron Radiation Facility, Grenoble) operating at a spatial resolution of 50 <i>nm</i>.</p