Hemolytic activity and invasiveness of <i>S</i>. Typhimurium SL1344 strains.

<p><b>A and B:</b> Sheep red blood cells were infected with <i>S</i>. Typhimurium SL1344 and derived mutant strains and hemolysis activity was followed measuring hemoglobin release at 542 nm. The hemolytic activity of the Δ<i>sipB</i> mutant strain is shown as a negative control. Hemolysis activity was assayed in triplicate and the error bar represents standard deviation. A representative experiment is shown. Unpaired t-tests were used to determine whether the values were significantly different. p-values: ns, not significant; ***, p ≤ 0.0005. <b>A</b>. Hemolytic activity of the wild-type and Δ<i>iacP S</i>. Typhimurium SL1344 strains that harbored the control empty plasmid pP_TET or the corresponding plasmids containing <i>iacP</i> or <i>iacP</i>_<i>S38T</i>. SipB was detected by western blot in bacterial crude extracts from the corresponding strains to show that similar amounts of SipB were produced (shown underneath the graph). <b>B</b>. Hemolytic activity of <i>S</i>. Typhimurium SL1344 strains, in which the indicated gene was deleted or modified by point substitution at the original locus. SipB was detected by western blot in bacterial crude extracts from the corresponding strains before starting the hemolysis assay (shown underneath the graph, top panel), and in sRBC membranes, isolated by sucrose density gradient, at the end of the hemolysis assay (shown underneath the graph, bottom panel). To load as much material as possible while ensuring that there was no leakage between the wells, the samples were separated by empty lanes. Those have been removed for the figure, which is symbolized by the white dotted lines. <b>C</b>: Invasion assays were performed on HeLa cells with <i>S</i>. Typhimurium SL1344 strains used for hemolysis assays. Invasion rates were normalized to the internalization level of WT, which was set to 100%. Each experiment was performed in triplicate and values are the mean of 6 independent experiments ± standard error of the mean. Unpaired t-tests were used to determine whether the values were significantly different. p-values: ns, not significant; **, p ≤ 0.05; ***, p ≤ 0.0001. <b>D</b>. Competitive index between the SL1344 wild-type strain (strain JV112) and Δ<i>sipB</i> (strain JV114) or between the SL1344 wild-type strain (strain JV112) and Δ<i>iacP</i>_intra (strain JV129) in mice inoculated perorally. The black bar indicates the mean CI. Values for the mean CI ± SEM (t-test p-value) were 0.0716 ± 0.0225 (p-value < 0.0001) and 0.641 ± 0.1034 (p-value = 0.0255) for Δ<i>sipB</i>/WT and Δ<i>iacP</i>/WT, respectively.