Fig. 6: Iron-dependent camSOD activity enables S. aureus to resist host stresses.
From: An evolutionary path to altered cofactor specificity in a metalloenzyme
a The ΔsodM mutant strain (lacking the gene encoding camSOD-orange squares) of S. aureus shows greatly diminished growth in the presence of paraquat (PQ) relative to wild type (WT-blue circles) under manganese-depleted conditions imposed by the presence of the human protein complex calprotectin (CP)8,16,17. S. aureus cells expressing the Leu159Gly variant of camSOD from the native sodM locus (green triangles) also show reduced growth relative to the wild type. Note that the asterisk represents p < 0.05 via two-way ANOVA with Tukey’s post-test performed in Graphpad Prism when compared to wild-type bacteria grown in the same concentration of calprotectin. Multiple independent clones of the Leu159Gly variant (n = 8) were assayed at the same time as those of the wild type (n = 3) and the ΔsodM mutant strain (n = 3). Each data point represents an independent biological replicate. Black bars represent the mean, with error bars showing standard error of the mean (SEM). The diminished ability of the camSOD variant to use iron (Table 1) renders S. aureus less capable of overcoming the host immune response. b, c In-gel SOD activity assays using extracts (5.4 μg) prepared from b wild type S. aureus and c the strain expressing the Leu159Gly camSOD variant, cultured under identical conditions to those used in a, demonstrate that both forms of the SodM enzyme are detectable at similar levels (upper panels). Treatment of the extracts (left) or recombinant enzymes (right) with H2O2 (lower panels), a specific inhibitor of the iron-loaded form8, demonstrated that the camSOD was loaded with iron in wild-type cells. The MnSOD enzyme is labeled as SodA, camSOD as SodM. This is a representative gel from a triplicate analysis of independent biological replicates (n = 3). Note that molecular weight markers were not used in these native gels, with SOD bands identified in the cell extracts by comparison of their mobility with the purified recombinant proteins. Source data for all panels are provided as a source data file.
