Mtb ESX-1 secretion is linked to virulence and hemolysis. (A) Bacterial growth in the J774 macrophage cell line as assessed by intracellular bacterial fluorescence. n = 12 fields per condition. *P < 0.05, two-way ANOVA with Dunnett’s test. (B) Bacterial burdens in 4-day-postinfection (dpi) zebrafish larvae as assessed by bacterial fluorescence. n = 30 larvae per condition. *P < 0.05, one-way ANOVA with Dunnett’s test. (C) Survival of adult zebrafish infected with 100 colony-forming units (CFU) of Mm. n = 12 fish per condition. *P < 0.05, log-rank (Mantel–Cox) test. (D) Immunoblot of Mm lysates and culture filtrates, representative of four experimental replicates. (E) Protein quantification from D by image densitometry, relative to WT culture filtrates. *P < 0.05, one-way ANOVA with Dunnett’s test relative to ::WT. (F) Contact-dependent sheep RBC lysis by 3.0 × 108 CFU Mm. n = 4 experimental replicates. (E and F) *P < 0.05, one-way ANOVA with Dunnett’s test relative to ∆RD1. Error bars, SD.