SElX protein sequence encodes a conserved sialic acid-binding motif.

<p>(i) A conserved glycan binding motif (colored red) is present in the amino acid sequences of characterised SSl-proteins and SElX. (ii) The sialic acid-binding motif is conserved in all 17 alleles of SElX. (iii) Amino acid conservation across the sialic acid-binding region of 7 staphylococcal neutrophil binding proteins (SSl2, SSl3, SSl4 SSl5, SSl6, SSl11 and SElX). The probability of residues at each position is proportional to the size of the letters. Image generated using the weblogo 3.4 program (<a href="http://weblogo.threeplusone.com/" target="_blank">http://weblogo.threeplusone.com/</a>). Chemical property color scheme: green is polar, blue is basic, red is acidic, purple is neutral and black is hydrophobic. Sequences were obtained from previously published work [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006461#ppat.1006461.ref006" target="_blank">6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006461#ppat.1006461.ref023" target="_blank">23</a>].</p

Neutrophil binding-deficient mutants of SElX retain mitogenic activity.

<p>Isolated human PBMC were stimulated with recombinant SElX and sialic acid-binding mutants. After 72 h incubation, proliferation was determined by analysing the incorporation of [³H] thymidine. Results shown are the means of triplicate measurement from 3 human donors ± standard deviation of the mean.</p

List of primers used in this study.

<p>List of primers used in this study.</p

SElX binds to multiple neutrophil surface glycoproteins in a sialic acid-dependent manner.

<p>(i) Neutrophil lysates were analysed for peptide enrichment following incubation with wild-type SElX (WT) and SElX EKQD-A. Data shown indicates the mean number of peptides detected from each enriched protein (± SD from three donors). * denotes significance difference (p-value <0.05) in peptide enrichment between the wild-type and mutant protein as determined by multiple t-test comparisons (one unpaired, two tailed test per protein), without assuming similar standard deviation. (ii) ELISAs were performed comparing the interaction between staphylococcal proteins and recombinant CD50. Two-way ANOVA statistical analyses were performed. **** denotes a significant difference between SElX and SElX EKQD-A.</p

SElX binds to human monocytes and neutrophils from multiple mammalian species.

<p>Flow cytometry analysis of recombinant staphylococcal proteins binding to isolated human cells. Cell type was determined by forward (FSC-H) and sideways (SSC-H) scatter (i). SElX binding to human neutrophils (ii) monocytes (iii) and lymphocytes (iv) in addition to neutrophils isolated from mice (v), cattle (vi) and rabbits (vii), was examined. For all graphs binding was detected using mouse anti-HIS-FITC IgG binding to the 6 x HIS-tag on the recombinant proteins. Mean median fluorescence of three donors is shown ± standard error of the mean (SEM). SSl5 and SSl7 were used as positive and negative controls, respectively. The same legend is used for all graphs.</p

SElX does not contribute to <i>S. aureus</i> virulence in a mouse skin abscess infection model.

<p>(i) Lesion size of each mouse (n = 10) was measured every 24 h post inoculation (p.i.) for 6 d. Mean lesion size ± SEM is plotted for each of the 4 USA300 LAC mutant groups. (ii) Bacteria were recovered from excised skin lesions and enumerated by serial dilutions. CFU were normalised to the weight of tissue homogenised to give the bacterial load per mg of tissue. CFU per mg are displayed for each infected animal; the horizontal line indicates the mean CFU/mg of tissue and vertical bars show the SEM for each group (n = 10) (iii) Representative images from histological examinations of skin lesions 72 h and 144 h post inoculation. Mounted sections were stained with haematoxylin and eosin. Black arrows on each image indicate the surface of the epidermis.</p