<i>Emm</i>3 STSS isolates show <i>emm</i>-type specific differences in overall mitogenicity.

<p><i>A.</i>Human MNC proliferation response to culture supernatants from 63 <i>Streptococcus pyogenes</i> streptococcal toxic shock syndrome (STSS) isolates grouped by <i>emm</i>-type (numbers per group: <i>emm</i>1, n = 11; <i>emm</i>3, n = 12; <i>emm1</i>2 and <i>emm</i>87, n = 11; <i>emm</i>18, n = 5; <i>emm</i>28, n = 7; <i>emm</i>89, n = 6). Negative; tissue culture media (RMPI) alone. Outliers are represented as individual circles. Representative of 2 experiments performed on different donors. <i>B.</i> Human MNC proliferation response to sera obtained from CD1 mice 24 hours after being infected with one of three <i>S. pyogenes</i> strains representing each <i>emm</i>-type; two mice were infected per strain. Negative; uninfected mouse serum. Proliferative response is measured as counts per minute (cpm) of tritiated-thymidine uptake. Median and 5th, 25th, 50th, 75th, and 95th centiles shown.</p

<i>emm</i>3 strains have low mitogenicity due to the mutation in <i>smeZ</i>.

<p><i>A.</i> MNC proliferative response to culture supernatants from an <i>emm</i>3 isolate, GAS-M3 carrying the typical M3-<i>smeZ</i> with 13 bp deletion, GAS-M3 over-expressing the functional M89 form of SMEZ (GAS-M3<i>_smeZ</i>_-M89) and GAS-M3 over-expressing the M3-SMEZ (GAS-M3<i>_smeZ</i>_-M3). <i>B.</i> Experimental <i>smeZ</i> mutation in <i>emm</i>1 <i>S. pyogenes</i> (GAS-M1) reduced MNC proliferation (GAS-M1Δ<i>smeZ</i>). Proliferative response was restored when GAS-M1Δ<i>smeZ</i> over-expressed the functional M89 form of SMEZ (GAS-M1<i>_smeZ</i>_-M89) but not with M3-type form of SMEZ (GAS-M1<i>_smeZ</i>_-M3). <i>C</i>. A similar result was obtained using an <i>emm</i>89 strain (GAS-M89) with an experimental mutation in <i>smeZ</i> (GAS-M89Δ<i>smeZ</i>). Proliferation was restored when GAS-M89ΔsmeZ over-expressed the functional M89 form of SMEZ (GAS-M89<i>_smeZ</i>_-M89) but not with M3-type form of SMEZ (GAS-M89<i>_smeZ</i>_-M3). Negative; media alone.Proliferation was measured as counts per minute (cpm) of tritiated-thymidine uptake and the percentage proliferation for each strain was calculated relative to the wild type strain (GAS-M3, GAS-M1, GAS-M89 respectively). Data are mean (+standard deviation) of three measurements. Representative of two experiments performed using different donor MNC.</p

<i>emm</i>3 isolates have a 13 base pair (bp) deletion within the <i>smeZ</i> locus.

<p>Representation of the <i>smeZ</i> locus from <i>emm</i>1 (M1) and <i>emm</i>3 (M3) strains. Nucleotides 1 to 3 encode the start codon (shown in bold). From 73 bp of the nucleotide sequence, the amino acid sequence of the mature SMEZ protein is shown. <i>Emm</i>3 strains have a 13 bp deletion at 316 bp (highlighted by a shaded box) that results in a frameshift and a predicted premature stop codon after 86 amino acids (shown as *). The forward primer and the reverse primer amplify the full length <i>smeZ</i> locus. The 13 bp deletion was detected using a forward (truncated) primer that anneals specifically to the region containing the deletion.</p

Functional SMEZ is required to stimulate production of cytokines from human tonsil cells.

<p>Human tonsil cell suspensions were cultured with bacterial cell-free culture supernatants from GAS-M3_control(white bars), GAS-M3<i>_smeZ</i>_-M89 (black bars) and GAS-M3<i>_smeZ</i>_-M3 (gray bars). After 2 and 5 days incubation cell-free media were obtained from cultures and production of TNFα, TNFβ, IL-10, IL-17 and IFNγ were measured by ELISA. Horizontal dotted lines represent the mean level of cytokines produced after co-culture with bacterial cell-free culture supernatants from GAS-M1_control on day 2 and day 5. Mean (+ standard deviation) of three replicates measured in duplicate. Representative of two experiments performed on different donors. Statistical analysis was performed using ANOVA with Bonferroni multiple comparison.</p

<i>S. pyogenes</i> strains used in this study.

<p><i>S. pyogenes</i> strains used in this study.</p