Figure 7

Recretion of selected InlA mutations in EGD-e. A. Comparison of the invasion attributes of EGD-e and EGD-e InlA^m* (Ser192Asn/Trp369Ser). Exponential phase L. monocytogenes cells (OD = 0.8) were invaded (MOI of 25:1) in triplicate for 1 h before overlaying with gentamicin. Invasion was expressed as the average cfu count per well (with standard deviation) or invasion relative to EGD-e (below graph). The graph is representative of the data from three independent experiments. B. The relative virulence of EGD-e compared against EGD-e InlA^m* (tagged with pIMC3kan and pIMC3ery respectively) was accessed by competitive index after i.v. infection (1 × 10⁴ cfu of each strain) of 15 Balb/c mice. On each subsequent day 5 mice were euthanized and spleens and livers aseptically removed and enumerated. Data are presented as the mean and standard deviation of 5 mice, competitive indices and statistical analysis was conducted using the one sample t test as described previously [18]. NS = Not significant. C. Oral inoculations of Balb/c mice with EGD-e::pIMC3kan and EGD-e InlA^m* ::pIMCery mixed at a 1:1 ratio in a total inoculum of 1 × 10¹⁰ cfu/200 μl containing 100 mg of CaCO₃. *** = p < 0.005. D. Competitive index virulence in a Balb/c oral infection model with EGD-e InlA^m* ::pIMC3ery competed against EGD-e::pIMC3kan, EGD-e A::pIMC3kan (InlA-N259Y), EGD-e B::pIMC3kan (InlA-Q190L), EGD-e C::pIMC3kan (InlA-T164A/K301I/G303E) or EGD-e D::pIMC3kan (InlA-S173I/L185F/L188I) as described in C. The invasion levels were significantly (p < 0.005) different than EGD-e InlA^m* for all competed strains.