Arrangement of the <i>enterotoxin gene cluster (egc)</i> in <i>S</i>. <i>aureus</i>.

<p>The relative positions of the 6 SE and SE<i>l</i> genes, in blue, are located but not drawn precisely to scale. The putative promoter and transcriptional terminator are shown.</p

Individual <i>egc</i> proteins have the capacity to induce IE in RN450.

<p>(A) SE<i>l</i>O; (B) SE<i>l</i>M; (C) SE<i>l</i>U; (D) SEG; (E) SEI; (F) SE<i>l</i>N; (G) Vector control.</p

Primers used to generate gene deletion constructs and in qRT-PCR to characterize expression of the <i>egc</i> in <i>Staphylococcus aureus</i> MN8.

<p>Primers used to generate gene deletion constructs and in qRT-PCR to characterize expression of the <i>egc</i> in <i>Staphylococcus aureus</i> MN8.</p

Fusions of 5′ <i>selu</i> and <i>5</i>′ <i>seg</i> regions of the <i>egc</i> induce GFP expression in RN4220.

<p>GFP expression as measured by relative fluorescence units (RFU) indicates 5′ <i>selu</i> and 5′ <i>seg</i> induce expression of GFP in RN4220 indicating potential promoter elements within the <i>egc</i>. Data displayed are means ± SEM, **<i>P</i> ≤ 0.01, Mann-Whitney test. <i>P</i> ≤ 0.05 is considered statistically significant.</p

The <i>egc</i> SAgs and TSST-1 are involved in vegetation formation during IE.

<p>(<i>A</i>) Total weight of vegetations dissected from aortic valves after intravenous inoculation of 3–4 x 10⁸ CFU of wild-type MN8, MN8Δ<i>tstH</i>Δ<i>egc</i>, MN8Δ<i>tstH</i>, or MN8Δ<i>egc</i>. (<i>B</i>) Bacterial counts recovered from vegetations shown in panel A. *<i>P</i> = 0.01, **<i>P</i> = 0.03, one-way ANOVA and non-parametric, Kruskal-Wallis test. Horizontal lines represent the median. <i>P</i> ≤ 0.05 is considered statistically significant.</p

Deletion of the <i>egc</i> and <i>tstH</i> from MN8 leads to decreased vegetation formation.

<p>Representative images of vegetative lesions from (<i>A</i>) MN8 wild-type, (<i>B</i>) MN8Δ<i>tstH</i>Δ<i>egc</i>, (<i>C</i>) MN8Δ<i>tst</i>, and (<i>D</i>) MN8Δ<i>egc</i>.</p

<i>egc</i> proteins differ in their ability to induce vegetations.

<p>Expression of SE<i>l</i>O, SE<i>l</i>M, SE<i>l</i>U, and SEI leads to vegetations. Expression of SEG and SE<i>l</i>N does not lead to development of vegetations. Horizontal lines represent the median. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, Mann-Whitney test. <i>P</i> ≤ 0.05 is considered statistically significant.</p

Model of <i>S</i>. <i>aureus</i> IE development on aortic valves.

<p>(<i>A</i>) Top down view of an aortic valve, with the represented boxed area used to provide an expanded model for the remaining panels. (<i>B</i>) Valve damage induces release of cytokines, chemokines, and clotting factors, leading to aggregation of host factors, including fibrin, erythrocytes, and platelets. (<i>C</i>) SAgs and exotoxins produced after valve colonization cause cytotoxicity, inhibiting healing of the damaged site and allowing bacterial growth (<i>D</i>) Bacterial persistence promotes further accumulation of host factors and vegetation expansion, inducing localized inflammation and increasing permeability of the aortic endothelium which allows SAgs access to the endocardium while recruiting immune cells. Superantigenicity within tissues is expected to promote immune dysfunction, chronic inflammation, and to support disease progression (<i>E</i>) Cross section of aortic valve. In large vegetations, pieces can detach leading to septic emboli that result in strokes and metastatic abscesses.</p

Vancomycin and IVIG treatment protects rabbits from lethality, vegetation formation, and bacteremia.

<p>Percent survival of rabbits infected intravenously with 2–4 x 10⁸ CFU/rabbit of <i>S</i>. <i>aureus</i> MN8 and treated with vancomycin alone, vancomycin plus IVIG or PBS 24 hrs post infection. <i>P</i> value determined with log-rank, Mantel-Cox test. (B) Rabbit temperatures over time. Red arrows indicate time points when temperature of some rabbits drops as they succumb to toxic shock. (C) Total weight of vegetations dissected from aortic valves and (D) bacterial counts recovered from vegetations; significance only between PBS and vancomycin plus IVIG treatment. (E) Bacterial counts per milliliter of blood recovered from rabbits <i>post mortem</i> and (E) splenomegaly resulting from <i>S</i>. <i>aureus</i> bacteremia. <i>P</i> value determined with one-way ANOVA and Dunnett’s multiple comparison test. (C-F) Horizontal lines represent mean values. <i>*P</i> ≤ 0.05, <i>**P</i> ≤ 0.01, <i>***P</i> ≤ 0.001. <i>P</i> ≤ 0.05 is considered statistically significant. No <i>P</i> value means no statistical significance.</p

The <i>egc</i> SAg genes are expressed in <i>S</i>. <i>aureus</i> strain MN8.

<p>Expression of the <i>egc</i> transcript is represented in relation to cell density (OD 600 nm wavelength). Data are represented as the relative expression of each gene normalized to <i>gyrB</i> expression ± standard error of the mean.</p