A novel core genome-encoded superantigen contributes to lethality of community-associated MRSA necrotizing pneumonia

Abstract

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone.

Figure 1. Phylogenetic analysis of selx and its species-wide distribution.

a) Neighbor-joining tree based on the sequences of all known staphylococcal SAgs and the SAg-like protein, SSL7 b) Neighbor-joining tree of concatenated MLST sequences of representative S. aureus STs representing the breadth of species diversity. Coloured circles denote the presence of specific selx alleles.

Figure 2. SElX is expressed by clinical isolates in vitro and during infection.

a) Growth-phase dependent transcription of selx determined by qRT-PCR. b). Representative Western blot analysis of rSElX proteins with human infective endocarditis (IE) or ovine mastitis sera samples.

Figure 3. SElX is predicted to have a truncated B-domain.

Schematic diagram of the solved structures of TSST-1, SSL7 and modelled 2-domain structures of human, bovine and ovine variants of SElX. SElX composite structure was obtained by superimposition of protein variants in PyMol.

Figure 4. SElX stimulates proliferation of human and rabbit lymphocytes.

a) Proliferation of human PBMC cultures with recombinant SElX2, SElXbov1, and SElXov variants measured by [3H]-thymidine incorporation. Proliferation of human (b) or rabbit (c) PBMC cultures with recombinant SElX2 (red line) or TSST-1 (black line) measured by [3H]-thymidine incorporation.

Figure 5. SElX activates Vβ-specific human and bovine Tcells.

(ii) Relative fold-change in Vβ expression (mean ± S.E.M.) for human (A) or bovine (B) T cells after stimulation with (i) SElX2_, (ii) SElXbov1, and (iii) SElXov. *indicates statistical significance (p<0.05).

Figure 6. SElX causes TSS and contributes to the severity and lethality of necrotizing pneumonia caused by CA-MRSA USA300.

A) The ability of rSElX2, administered at a dose of 200 µg/kg, to cause TSS was measured using a standard miniosmotic pump model of TSS. TSST-1 administration was used as the positive control and PBS the negative control. B) Fever in response to intravenous injection of rSElX2 at concentrations of 10 µg/kg (□), 1 µg/kg (▪), 0.1 µg/kg (Δ), of body weight per ml, or PBS (▴), was evaluated over a 4 h test period, followed by the ability to enhance the lethality of LPS over 48 h. C) Kaplan-Meier Curves of % survival of rabbits infected with S. aureus LAC, S. aureus LACΔselx, and LACΔselx rep. D). Increase in rabbit core temperature (ΔT °C) 2 d after pulmonary infection with LAC wt, LACΔselx, and LACΔselx rep. E). Gross pathology of lungs from uninfected rabbit (i) or from rabbits infected with LAC wt (ii), LACΔselx (iii), and LACΔselx rep (iv). F) Haematoxylin Eosin-stained tissue sections from (i) uninfected rabbit lung, or from rabbit lungs infected with (ii) LAC wt, (iii) LACΔselx and (iv) LACΔselx rep isogenic strains.