Figure 4.

Early plasminogen activator protease (Pla)–mediated T3S in the airways occurs independent of Pla proteolytic activity. A, Quantification of cells targeted for T3S by YopE-Bla reporter variants of CO92, CO92 Δpla, CO92 Δpla + pla wild type (WT), CO92 Δpla + plaS99A, or CO92 Δpla + plaD206A. Mice were infected intranasally with 5 × 10⁴ colony-forming units (CFUs) of the different strains, and bronchoalveolar lavage fluid (BALF) was stained with CCF2-AM and subjected to flow cytometry. Plots represent the number of cells targeted for Yop delivery per 100 live cells. B, BALF from A was immunostained with a panel of antibodies to identify the cell types targeted for Yop delivery using flow cytometry. Plots represent the number of cells that were alveolar macrophages (F4/80⁺CD11c^highCD11b^low), interstitial macrophages (F4/80⁺CD11c^lowCD11b^high), monocytes (F4/80⁻CD11b^highCD11c^lowLy-6G⁻), dendritic cells (F4/80⁻CD11c^highCD11b^{high or low}), and neutrophils (F4/80⁻CD11b^highLy-6G⁺) for every 100 Yop-targeted cells (blue). C, Quantification of the release of tumor necrosis factor (TNF) α, interleukin 6 (IL-6), and monocyte chemoattractant protein (MCP) 1 from MH-S cells infected with CO92, CO92 Δpla, CO92 Δpla + pla WT, CO92 Δpla + plaS99A, or CO92 Δpla + plaD206A at a multiplicity of infection of 1, after 2 and 4 hours after infection. All experiments were performed 3 times; error bars represent standard deviations (n = 5). *P < .01; †P < .001 (Welch t test or 2-way analysis of variance).