Figure 2. Hierarchical injection of SipA and SptP.

(A) Phases of the SipA- and SptP injection process; (B) Typical time lapse movie of the infection process. The bacteria harbored a pGFP plasmid to facilitate detection (GFP fluorescence) in the presence of membrane ruffling (phase contrast). (C) Representative images of M1269 and M1223 (pHilA, pGFP) in the early/intermediate or late phase of SipA and SptP injection into COS7 cells. Cells were fixed, permeabilized with lysozyme, and immunostained for LPS (blue), SipA (red) and SptP (green). (D) Time course of SipA and SptP depletion from hilA-overexpressing bacteria during the infection of COS7 cells. Infection was monitored as in (B) and intrabacterial SipA- and SptP pools were stained as in (C). For each bacterium, the graph shows the time between docking and fixation, the presence/absence of SipA (red) and SptP (green) in the bacterial cytosol. Gray lines connect SipA and SptP data from the same bacterium. Circles represent data obtained from M1223 (pHilApGFP; sipA_HAsptP_M45) and triangles data from M1269 (pHilApGFP; sipA_M45sptP_HA). The data was fitted using a rolling average algorithm (red and green lines, see Materials and Methods) to determine when injection was completed with 50% probability (t_50%). tu = time units.