RopB and SpeB-negative bacteria show reduced virulence in systemic infection models.

<p>(A) Intraperitoneal infection of C57BL6/J mice with 5×10⁷ cfu of GAS strains with 5% mucin. (B) Intravenous challenge of C57BL6/J mice with 2×10⁸ cfu of GAS strains.</p

Mutation in <i>ropB</i> results in truncation of the RopB protein and abrogation of SpeB expression.

<p>(A) Schematic representation of the RopB protein expressed by GAS strains 5448 and 5628. In 5628, white region represent homology to 5448, shaded region indicates unique protein sequence in 5628. (B) Western blot for SpeB using overnight culture supernatants. (C) SpeB activity assay using azocasein substrate.</p

RopB mutation does not affect <i>in vitro</i> growth, hemolytic activity, extracellular DNase activity or resistance to neutrophil killing.

<p>(A) OD₆₀₀ of <i>in vitro</i> grown bacterial cultures in THB. (B) Hemolysis of red blood cells by bacterial culture supernatants. (C) Degradation of calf thymus DNA by bacterial culture supernatants, run on a 1% agarose gel. (D) Bacterial survival after 30 min incubation with human neutrophils.</p

Primers used for sequencing the <i>ropB</i> locus (including <i>speB</i>), the <i>covR/S</i> locus, <i>luxS</i>, <i>rofA</i> and <i>ropA</i>.

<p>Primers used for sequencing the <i>ropB</i> locus (including <i>speB</i>), the <i>covR/S</i> locus, <i>luxS</i>, <i>rofA</i> and <i>ropA</i>.</p

Microarray analysis emphasizes the down-regulation of the SpeB operon.

<p>The figure shows the top 10 genes up- and down-regulated in the <i>ropB</i> mutant bacteria compared to WT when the bacteria are inoculated <i>in vivo</i> (P<0.05). The values plotted represent log ratios (log mutant/WT), and genes co-clustered in operons are highlighted (black bars, SpeB operon; white bars, SLS operon).</p

Growth of Bacteria in Human Blood.

a<p>Growth for 2 h at 37°C using blood of donor A.</p>b<p>Growth for 2 h at 37°C using blood of donor B.</p

Relative levels of C3 deposition on WT and Δ<i>dltA</i> mutant bacteria.

<p>WT bacteria (open histogram) and Δ<i>dltA</i> mutants (filled histogram) were stained with a fluorescein-labeled antibody against human C3 and analyzed by flow cytometry.</p

Comparing the relationship of complement deposition to M protein levels by flow cytometry.

<p>WT cells, 5448 (panel A) and 8004 (panel C), and their isogenic mutants, 5448Δ<i>dltA</i> (panel B) and 8004Δ<i>dltA</i> (panel D), were labeled with anti-C3 (FITC-A) and anti-M1 (Cy5-A). Numbers are given representing the % of cells in each gated area.</p

Localization of C3 and M1 by immunofluorescence.

<p>WT 8004 cells (Panels A and C) and 8004Δ<i>dltA</i> cells (Panels B and D) were stained either with human anti-C3 (Panels A and B) or anti-M1 (Panels C and D) antibodies. DAPI staining (blue) was used to visualize nuclei. Note that the fluorescence associated with M proteins (red) decreases in the <i>dltA</i> mutant and the fluorescence associated with C3 dramatically increases (green).</p

Analysis of the amounts of M protein and SIC released into the medium.

<p>Protein was precipitated from the supernatant of WT and <i>dltA</i> mutant bacteria grown to mid-log phase using TCA. A) Samples were separated by SDS-PAGE and blotted to nitrocellulose. Bands containing M1 protein were visualized using an anti-M1 antibody. B) Samples from 8004 and 8004Δ<i>dltA</i> were separated by 2-D gel electrophoresis. Only the portion of the gel surrounding SIC is shown. SIC was identified by mass spectrometry.</p