Fig. 4. PsseJ-LysE and flhDC are necessary for delivery to tumors.

a To control cell invasion and protein release, IDf+ Salmonella were created by transforming ΔflhD Salmonella with PsseJ-LysE, PBAD-flhDC, and Plac-GFP. b IDf+ Salmonella were administered to 4T1 cancer cells and PBAD-flhDC was induced with 20 mM arabinose. Controls either lacked PsseJ-LysE, were uninduced, or both. Invasion was detected with anti-Salmonella antibodies and delivery was detected by the presence of released GFP. c GFP (green, arrows) was only delivered from Salmonella with activated PBAD-flhDC and transformed with PsseJ-LysE. d Induced IDf+ Salmonella delivered significantly more GFP than all controls (P < 0.0001; n = 6). e IDf+ Salmonella (2 × 10⁸ CFU/mouse) were intravenously injected into BALB/c mice with subcutaneous 4T1 tumors via the tail vein (n = 3). In flhDC+ mice, 100 µg of arabinose was injected IP at 48 and 72 h. At 96 h, delivered GFP (arrows) was measured in excised tumors with immunohistochemistry. f In the transition zone of the tumors in e, induction of flhDC increased the fraction of cells with delivered GFP (P = 0.0004; n = 15). Data are shown as means ± SEM. The statistical comparisons in d were to a single condition performed with ANOVA followed by Dunnett’s method. The statistical comparison in f is a two-tailed, unpaired Student’s t-test. Asterisks indicate significance (***P < 0.001; ****P < 0.0001). The results in b are from a single experiment and the images in c are representative of 6 independent biological samples. Scale bars in c and e are 10 µm.