Supplementary Figure 8: PCA plot of singleton strains and analysis of Cluster A ropB mutant strains.

(a) The two major clusters identified by DBSCAN are shown. (b) No subclade-specific clustering was evident within the two clusters. (c) Twenty strains with ropB mutations are outliers (colored yellow) and group away from the other strains with ropB mutations (colored orange). ropB-non-outlier strains cluster with WT-like strains (colored light blue) and strains with mutations in other major regulator genes (colored blue). (d) Cluster A ropB mutant strains separated into two groups validated by k-means clustering and were designated arbitrarily as Group I and Group II. (e) Group II ropB mutant strains had significantly decreased speB transcript levels compared to Group I strains (Mann-Whitney, two-tailed, P < 0.0001). (f) Mutations were mapped onto the crystal structure of the C-terminal region of the RopB protein. Variant amino acid positions associated with Group I or Group II organisms are labeled in red and pink, respectively. Amino acid residues present in inferred functional domains are demarcated with ovals. Mutations located in RopB functional domains were present at significantly increased frequency (test of proportions-one-tailed, P < 0.05) in Group II strains (pink labels within ovals) compared to Group I strains (red labels within ovals). PBD: peptide binding domain, NTD: N-terminal domain. The crystal structure of the NTD has not been solved. (g) Kaplan-Meier curve showing that the Group I (n = 3) and Group II (n = 4) strains differ significantly (log-rank test) in virulence in a mouse necrotizing myositis infection model (40 mice per strain). (h) Gross pathology images of infected mouse hindlimbs (n = 5 mice per strain) reflect the difference in virulence between the Group I (top) and Group II (bottom) strains, and representative images are displayed. Boxed areas demarcated in white illustrate major lesion areas.