Figure 3.

Analysis of residues required for translocation. Wild-type S. typhimurium or strains carrying mutations in SPI2 TTSS (ssaT) or SPI1 TTSS (ΔprgH-K) expressing various CyaA fusions were used to infect RAW264.7 cells at a multiplicity of infection (MOI) of 10. Macrophages were infected for 1 h with bacteria in late logarithmic growth for SPI1 TTSS expression (C) or for 1 h with late stationary phase bacteria plus 5 or 6 h of gentamycin treatment for SPI2 TTSS expression (A and B). Infected macrophages were lysed in 0.1 M HCl; cellular cAMP levels were determined by enzyme immunoassay and were normalized for protein content determined by the Bradford assay and are presented as pmol of cAMP per μg of protein. SspH1₁₄₀ and SspH1_{1–31,36–140}CyaA (Δ32–35) were used in B and C. Note that cAMP production may be variable between assays, and infection of bacteria expressing SspH1₁₄₀CyaA results in cAMP increases that are similar to those seen with SspH1₂₀₈CyaA in a direct comparison (data not shown).