Calcium ion entry into human PMNs by combinations of S and F components from LukGH, PVL, LukDE and γ-hemolysin.

<p>Calcium flux was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089308#pone-0089308-g001" target="_blank">Figure 1</a>. Each panel shows a particular S component (header) as mixed with various F components (LukG: red, LukF-PV: green, LukD: brown, HlgB: blue). The concentration of all proteins was 500 nM. The results shown are the mean of three independent experiments.</p

LukG and LukH induction of IL-8 production by PMNs pretreated with an inhibitor of NFκB.

<p>PMNs (2×10⁶/mL) were pretreated with Bay11-7082 (50 µM) for 1 h and then mixed with 500 nM LukG or LukH for 2–8 h. IL-8 levels in the supernatants were measured by ELISA.</p

Characteristics of heterologous toxin combinations.

<p>Characteristics of heterologous toxin combinations.</p

IL-8 production by PMNs treated with LukGH or PVL.

<p>PMNs (2×10⁶/mL) were incubated in the presence or absence of (A) indicated concentrations of LukGH, PVL, LPS (50 µg/mL) and (B) single leukotoxin components, protease-digested single components (LukG dig and LukH dig), LukGH, PVL (all 500 nM) or LPS (50 µg/mL) for 2–8 h. IL-8 levels in the supernatants were measured by ELISA using recombinant IL-8 as a standard. The results represent the mean of three independent experiments ± SEM. In (A), <b>*</b> indicates statistical significance compared to PVL. In (B), * indicates significance compared to both LukF-PV and LukS-PV and <b>°</b> indicates significance to LukF-PV only.</p

Calcium ion entry into human PMNs in presence of PVL (A) and LukGH (B) at the indicated concentrations.

<p>Leukotoxins were mixed with 2×10⁶/mL PMNs loaded with 4 µM Fluo-4 in presence of 1.1 mM CaCl₂. Fluorescence intensity was recorded immediately and then every minute. Percent Fluo-4 fluorescence was calculated according to the formula described in the methods section by using 1% Triton X-100 to estimate maximal fluorescence and 1 mM EGTA to determine minimal fluorescence. The results represent the mean of four independent experiments.</p

Induction of IL-8 mRNA by individual components of LukGH or PVL.

<p>PMNs were incubated for 1(cells) or with 500 nM LukG, LukH, LukF-PV, LukS-PV, protease-digested LukG (LukG dig) or LukH (LukH dig), or 50 µg/mL LPS, and total mRNA was isolated from cell lysate. cDNA was synthesized and the DNA was amplified using IL-8 and β-actin specific primers and PCR. The ratio of IL-8 transcripts to β-actin transcripts is shown below the gel image.</p

The effect of calcium channel blockade on calcium ion entry into human PMNs in presence of 20(A) or 500 nM LukGH (B).

<p>PMNs (2×10⁶/mL) were loaded with 4 µM Fluo-4 in presence of 1.1 mM CaCl₂ and then exposed to the indicated concentration of methoxyverapamil for 60 min prior to adding PVL or LukGH. Calcium flux was then determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089308#pone-0089308-g001" target="_blank">Figure 1</a>. The results represent the mean of three independent experiments.</p

Cytolytic activities of LukGH and PVL in human PMNs.

<p>PMNs (1×10⁶/mL) were incubated with indicated concentrations of LukGH or PVL for 20–80 min and LDH concentration in the supernatant was measured using the Roche Cytotoxicity Detection Kit. Percent LDH release was calculated according to the formula described in the methods section by using 1% Triton X-100 to estimate maximal LDH release. The results represent the mean of three independent experiments ± SEM. * indicates statistical significance from PVL at the same concentration and time.</p

Effects of the staphylococcal bicomponent leukotoxins [10], [30], [42], [43].

1<p>HlgA has also been called Hlg2, HlgB has been variously called Hlg1 or LukF and HlgC is also known as LukS.</p>2<p>ND no data available.</p

Primers used for plasmid construction and reverse-transcription PCR.

<p>Primers used for plasmid construction and reverse-transcription PCR.</p