Staphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors

<div><p><i>Staphylococcus aureus</i> is an opportunistic pathogen that produces many virulence factors. Two major families of which are the staphylococcal superantigens (SAgs) and the Staphylococcal Superantigen-Like (SSL) exoproteins. The former are immunomodulatory toxins that induce a Vβ-specific activation of T cells, while the latter are immune evasion molecules that interfere with a wide range of innate immune defences. The superantigenic properties of Staphylococcal enterotoxin-like X (SElX) have recently been established. We now reveal that SElX also possesses functional characteristics of the SSLs. A region of SElX displays high homology to the sialyl-lactosamine (sLacNac)-specific binding site present in a sub-family of SSLs. By analysing the interaction of SElX with sLacNac-containing glycans we show that SElX has an equivalent specificity and host cell binding range to the SSLs. Mutation of key amino acids in this conserved region affects the ability of SElX to bind to cells of myeloid origin and significantly reduces its ability to protect <i>S</i>. <i>aureus</i> from destruction in a whole blood killing (WBK) assay. Like the SSLs, SElX is up-regulated early during infection and is under the control of the <i>S</i>. <i>aureus</i> exotoxin expression (Sae) two component gene regulatory system. Additionally, the structure of SElX in complex with the sLacNac-containing tetrasaccharide sialyl Lewis X (sLeX) reveals that SElX is a unique single-domain SAg. In summary, SElX is an ‘SSL-like’ SAg.</p></div

Analysis of host binding by affinity precipitation.

<p>(A) SDS-PAGE (12.5%) run under reducing and denaturing conditions of proteins from the various indicated human and mouse sources isolated by affinity with SElX2, SElX2-T130A, SSL6, SSL6-R181A, SSL11, or SSL11-T168A coupled to sepharose. Sepharose alone (control) was used as a control for non-specific binding. * indicates SELX/SSL that has dissociated from the sepharose. Marker is BenchMark Protein Ladder (Life Technologies). (B) The 40 top scoring leukocyte proteins identified by SElX-sepharose affinity binding are shown by descending rank in the bar graph and listed in order in the accompanying table. The ranking is based on the Unused Score (taken from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006549#ppat.1006549.s001" target="_blank">S1 Table</a>) given to each uniquely identified protein as calculated by the mass spectrometry analysis software ProteinPilot 5.0 (AB Sciex Pte. Ltd). The Unused Score indicates how much of the Total Score is unique to the particular protein hit. The Total Score is the sum of the Contrib values (contrib = the highest scoring peptide match for a peptide sequence) and determines the overall confidence for the protein identification. A and B are the Unused Scores of additional proteins identified in both the SElX-T130A/R141A and sepharose control samples with their corresponding Unused Scores from the SElX sample.</p

Data collection and refinement statistics parameters.

<p>Data collection and refinement statistics parameters.</p

Analysis of host binding by flow cytometry.

<p>(A) Median fluorescence intensity (MFI) of a two-fold dilution series of SElX-488 (green line) or SElX-T130A/R141A (red line) binding to human leukocytes with cell populations gated as granulocytes, monocytes, and lymphocytes based on size and granularity. Each data point represents the mean ± SD of three separate experiments using three individual donors. Comparison of the two proteins binding each cell population was performed in Graphpad Prism using two way analysis of variance (ANOVA) (p<0.0001) with Sidak’s multiple comparisons test: * p = 0.0336; ** p = 0.0037; ***p = 0.0005; **** p < 0.0001. (B) Binding of 100nM SElX-488 to human granulocytes (black bar) and in the presence of increasing concentrations of SElX (Green bars) or SElX-T130A/R141A (red bars). The MFI is the mean ± SD of three experiments performed using three separate human donors. The column data were compared by two-tailed paired t-tests: *p = 0.0333; ** p = 0.0038. (C) Comparison of the binding of 100nM SElX-488 to human and mouse leukocyte populations. The MFI is the mean ± SD of three experiments performed using three separate human donors or two experiments on n = 1 mouse per experiment and is the MFI (SElX-488 stained cells) minus MFI (unstained control population). Data compared by one way ANOVA (p<0.0001) with Tukey’s multiple comparisons test: * p = 0.0189; *** p = 0.0005.</p

Ten strongest SElX binding glycans from the glycomics consortium array.

<p>Ten strongest SElX binding glycans from the glycomics consortium array.</p

Primers used in this study.

<p>Primers used in this study.</p

Characterization of a Mouse-Adapted <i>Staphylococcus aureus</i> Strain

<div><p>More effective antibiotics and a protective vaccine are desperately needed to combat the ‘superbug’ <i>Staphylococcus aureus.</i> While in vivo pathogenicity studies routinely involve infection of mice with human <i>S. aureus</i> isolates, recent genetic studies have demonstrated that <i>S. aureus</i> lineages are largely host-specific. The use of such animal-adapted <i>S. aureus</i> strains may therefore be a promising approach for developing more clinically relevant animal infection models. We have isolated a mouse-adapted <i>S. aureus</i> strain (JSNZ) which caused a severe outbreak of preputial gland abscesses among male C57BL/6J mice. We aimed to extensively characterize this strain on a genomic level and determine its virulence potential in murine colonization and infection models. JSNZ belongs to the MLST type ST88, rare among human isolates, and lacks an <i>hlb</i>-converting phage encoding human-specific immune evasion factors. Naive mice were found to be more susceptible to nasal and gastrointestinal colonization with JSNZ than with the human-derived Newman strain. Furthermore, naïve mice required antibiotic pre-treatment to become colonized with Newman. In contrast, JSNZ was able to colonize mice in the absence of antibiotic treatment suggesting that this strain can compete with the natural flora for space and nutrients. In a renal abscess model, JSNZ caused more severe disease than Newman with greater weight loss and bacterial burden. In contrast to most other clinical isolates, JSNZ can also be readily genetically modified by phage transduction and electroporation. In conclusion, the mouse-adapted strain JSNZ may represent a valuable tool for studying aspects of mucosal colonization and for screening novel vaccines and therapies directed at preventing colonization.</p></div

The structural analysis of SElX.

<p>(A) crystal structure of SElX8 (cyan) in complex with sLeX (green) shown from the left of the glycan binding site (left panel) and from the right of the glycan binding site (right panel). (B) The sialylated glycan-binding site of SElX8 (blue) showing the residues that hydrogen-bond (yellow dotted lines) with sLeX (green). The side chains that interact with sLeX are labelled and shown in bold. The components of sLeX are labelled as follows: N-Acetylneuraminic Acid (S); galactose (G); fucose (Fuc); and N-Acetylglucosamine (N). (C) Structural overlay of SElX8 (blue) with the SAg TSST-1 (silver) and SSL5 (sage). (D) Comparison of sialyl Lewis X (sLeX) (in green) bound in the glycan binding sites of SElX8 (blue), SSL4 (silver), SSL5 (sage), and SSL11 (orange). The side chains of residues that hydrogen-bond with sLeX are shown in bold. An overlay of these binding sites (centre) shows the conservation of residues that interact with sLeX.</p

Determination of SElX2 and SElX8 binding to sLeX and sLacNac by surface plasmon resonance.

<p>(A) Quantitative measure of SElX2 and SElX8 binding to sLeX and sLacNac. Binding responses at equilibrium (Req) are shown against the concentration and fitted to a steady-state affinity binding model to calculate an equilibrium affinity constant (KD). (i) sLeX sensor chip binding and equilibrium binding analysis of 0.25 to 50 μM SElX2 in duplicate. (ii) sLacNac sensor chip binding and equilibrium binding analysis of 0.25 to 50 μM SElX2 in duplicate. (iii) sLeX sensor chip binding and equilibrium binding analysis of 0.25 to 50 μM SElX8 in duplicate. (iv) sLacNac sensor chip binding and equilibrium binding analysis of 0.25 to 50 μM SElX8 in duplicate. (B) Comparison of SElX and its glycan-binding mutants to sLeX and sLacNac. (i) sLeX sensor chip binding SElX2 (red), SElX2-T130A (pink), SElX2-R141A (green), and SElX2-T130A/R141A (blue) at 20 μM. (ii) sLacNac sensor chip binding SElX2 (red), SElX2-T130A (pink), SElX2-R141A (green), and SElX2-T130A/R141A (blue) at 20 μM. (iii) sLeX sensor chip binding SElX8 (red), SElX8-T130A (pink), SElX8-R141A (green), and SElX8-T130A/R141A (blue) at 20 μM. (iv) sLacNac sensor chip binding SElX8 (red), SElX8-T130A (pink), SElX8-R141A (green), and SElX8-T130A/R141A (blue) at 20 μM. The plots shown are representative of three independent experiments where each experiment was performed in duplicate. The affinity (K_<i>D</i>) values are expressed as mean ± SD of the repeats.</p

Comparison of SElX with SAgs and SSLs.

<p>(A) Phylogenetic analysis of the SAgs and SSLs of <i>S</i>. <i>aureus</i>. The Phylogenetic tree created using FigTree (v1.4.2) from an amino acid alignment of the staphylococcal SAgs and SSLs generated using Clustal Omega (EMBL-EBI). SAgs are shown in black text and the SSLs in grey text. SElX is shown in red. (B) Amino acid sequence alignment of the two SElX variants used in this study, SElX2 and SElX8 (in bold), with the SSLs in the region of the sialylated glycan-dependent binding site. The glycan binding SSL subfamily is highlighted by the horizontal grey box and the region of the 17 amino acid glycan binding site is highlighted by the vertical grey box. Residues that have been experimentally determined to interact with the sialylated glycan are shown in bold type with those that hydrogen bond to the glycan underlined. Residues with homology to these amino acids are highlighted in dark grey. The conserved Threonine (T) and Arginine (R) residues mutated to affect sialylated glycan binding are indicated by the red asterisks.</p