Positions selected for mutations in LukS-PV.

<p>A. Sequence alignment of the three stretches of residues constituting the rim domain of class S components of leukotoxins. Numbering corresponds to the mature LukS-PV protein. Red asterisks indicate positions selected for mutation. Strictly conserved residues are indicated on a red background, while similar residues in group 1 (LukS-PV and HlgC) or in group 2 (all others) are indicated with red letters. The secondary structure of LukS-PV is indicated above the alignment, colored according to the corresponding structural domain: β-sandwich (cyan) and rim (purple). GenBank accession numbers are LukS-PV: CAA51251.1, HlgC: AAA26638.1, HlgA: AAA26637.1, LukE: CAA73667.1, LukE-V: BAB47174.1, LukS-I: CAA55782.1, LukM: BAA97866.1. B. Schematic representation of the three dimensional structure of LukS-PV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092094#pone.0092094-Guillet1" target="_blank">[35]</a>, PDB entry 1T5R, highlighting the three structural domains: β-sandwich (cyan), stem (orange) and rim (purple). C. Stereo view of the Cα trace of wild-type LukS-PV. Residues selected for mutations are displayed as sticks and labeled.</p

Binding properties of LukS-PV* and LukS-PV mutants to hPMNs and U937-C5aR cells.

<p>A. Flow cytometry measurement of LukS-PV* and fluorescein-labeled LukS-PV G10C binding to human PMNs and U937-C5aR cells (<i>n</i> = 3). B. Graphic representation of the <i>K</i>_i values obtained for wild-type or mutant LukS-PV. The dotted line corresponds to the value of wild-type LukS-PV. Error bars represent the 95% confidence interval. Statistical analysis: **: <i>p</i><0.01, ***: <i>p</i><0.001 (<i>n</i> = 3).</p

Crystallographic data and refinement statistics of the LukS-PV mutants¹.

1<p>Numbers in parentheses report statistics for the highest resolution shell.</p

Molecular surface of the rim domain of LukS-PV.

<p>Residues identified in this study as important for the binding of LukS-PV on the C5a receptor (<i>K_i</i> increased more than 50 fold upon mutation to Ala) are depicted in red. Mutated residues affecting binding to a lesser extent (i.e. increase in <i>K_i</i> by a factor between 5 and 50) are depicted in pink whereas residues for which no effect on binding was found upon mutation (increase in <i>K_i</i> less than 3 fold) are depicted in blue (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092094#pone-0092094-t001" target="_blank">Table 1</a>). Two orthogonal views around a vertical axis are presented, with the orientation on the left being the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092094#pone-0092094-g005" target="_blank">figure 5</a>.</p

Binding properties as well as cellular and pore-forming activities of wild-type and LukS-PV mutants¹.

1<p><i>K_i</i> values, calcium entry slope and ethidium entry at 30 min were obtained for wild-type LukS-PV and all mutants with hPMNs. <i>K_i</i> values with U937-C5aR cells were obtained for wild-type LukS-PV and for the most affected mutants (values given in parenthesis).</p>2<p>Mutations causing a significant decrease in LukS-PV affinity for hPMNs (<i>p</i><0.001, one-way ANOVA with Dunnett's post test).</p

Biological activity of LukS-PV and corresponding mutants: rise of cytoplasmic calcium concentration due to human neutrophil activation.

<p>Values represent the calcium entry slope expressed in percent of maximum calcium fluorescence (after neutrophil lysis with Triton X-100 0.05% v/v) per second. Statistical analysis: ns, non-significant; *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001 (one-way ANOVA with Dunnett's post test, <i>n</i> = 6).</p

Primers used for PCR detection of genes encoding virulence factors.

<p>Other virulence factors have been detected by multiplex PCR onto purified total DNA (Qiagen). Presence of genes encoding enterotoxins E, G, H, K, L, T, epidermolysin D (Etd), genes encoding Edin A and EDIN B and the genes encoding seven adhsesion factors (Cna, FnbA, FnbB Bbp, Clfb, Fib, Ebp and Lbp) was checked in 8 set in function of base size.</p

Production of toxins and identification of genes encoding toxins in <i>Staphylococcus aureus</i>.

<p>The majority of <i>SA</i> strains isolated from HIV patient-derived furuncles significantly produced PVL (<i>p</i><0.05), whereas only 10% of <i>SA</i> strains produced this toxin in secondary infected dermatosis. A high prevalence of LukE-LukD-producing isolates (56 to 78%) was recorded in patient groups.</p

Pulsed field gel electrophoresis (PFGE) dependent dendogram of isolated <i>Staphylococcus aureus</i>.

<p>Pulsed field gel electrophoresis (PFGE) proves no specific clonal relationship between PVL-producing isolates issued from furuncles or secondary dermatosis. The similarity of the different pulsotypes was established by using Molecular Analyst™ software. Twenty four pulsotypes corresponded to the 55 isolates and their distribution is given according to the groups of isolates issued from secondary infected dermatosis (1), furuncles from HIV (−)(2) or HIV (+)(3) patients.</p

The proteolytic cleavage sites induced after treatment of bCAT, hCAT and CTL with different <i>S. aureus</i> strains (ATCC25923, ATCC49775, S1 and S2).

<p>The experimental molecular masses obtained after MALDI-TOF analysis, are indicated.</p