Figure 6. Contribution of uncharacterized FPI genes to T6SS function.

(a) T6SS sheath assembly (arrowheads) in F. novicida U112 iglA-sfGFP ΔpdpE, ΔanmK, ΔpdpC, ΔpdpD, ΔpdpD/anmK, ΔpdpC/pdpD and ΔpdpC/pdpD/anmK. GFP channel and 3.3 × 3.3 μm fields of view are shown. Scale bars, 1 μm. (b) Quantification of cytosolic bacteria in unprimed wild-type BMDMs 4 h after infection with F. novicida U112 iglA-sfGFP wild type, ΔpdpB, ΔpdpE, ΔanmK, ΔpdpC, ΔpdpD, ΔpdpD/anmK or ΔpdpC/pdpD/anmK (normalized to wild type). (c) Release of LDH and IL-1β from primed wild-type BMDMs 10 h after infection with F. novicida U112 iglA-sfGFP wild type, ΔpdpB, ΔanmK, ΔpdpC, ΔpdpD, ΔpdpD/anmK, ΔpdpC/pdpD or ΔpdpC/pdpD/anmK (NI—noninfected control). (d,e) Bacterial burden (as colony-forming units (CFUs) per gram tissue) in the spleen and liver (d) and serum IL-18 levels (e) of wild-type C57BL/6JRj mice infected subcutaneously for 2 days with 1 × 10⁴ F. novicida U112 iglA-sfGFP wild type, ΔpdpB, ΔpdpE, ΔpdpC, ΔpdpD/anmK and ΔpdpC/pdpD/anmK. Small horizontal lines indicate the mean. Each symbol represents an individual mouse (n=5 per experiment). Graphs show pooled data from two independent biological replicates with n=5 per experiment (n=10 total per group); small horizontal lines indicate the mean. (b–e) Data are pooled from three independent experiments (b) (mean and s.d. are shown) or two independent experiments (d,e) or are representatives of three independent experiments (c) (mean and s.d. of triplicate wells are shown). **P<0.01, ***P<0.001 and ****P<0.0001 (two-tailed unpaired t-test with Welch’s correction (b,c) or Mann–Whitney test (d,e)).