Figure 2

S . Gallinarum's invasion defect is related to a T3SS-1 defect in spite of a functional T3SS-1 apparatus. A. Strain SG 287/91 secretes proteins in the culture medium through its T3SS-1. Secretion assays were performed in LB NaCl 0.3 M. Proteins secreted in the supernatant by SE LA5, SG 287/91 and their T3SS-1 isogenic mutants were precipitated, separated using SDS-PAGE and stained with Neuhoff blue. The experiment was performed three times. B. Strain SG 287/91 translocates SopD effector into the host cell cytosol through its T3SS-1. Translocation assays were performed using strains SG 287/91 and 287/91invA carrying a plasmid encoding a fusion SopD-TEM-1 β-lactamase (pCX340sopD). When SopD was translocated into the cells (avian CLEC213 or human HeLa cells), β-lactamase hydrolyzed a green fluorescent substrate previously loaded in the cells (CCF4/AM), releasing a blue fluorescent substrate. The experiment was performed three times. C. Strain SG 287/91 is associated with less actin foci than strain SE LA5. Fluorescent SE LA5 or SG 287/91 bacteria were deposited on HeLa EGFP-actin-transfected cells for 0.5 h with an MOI of 100 and 1000 respectively to obtain a similar number of adhered bacteria. White arrows indicate actin foci associated with bacteria. The number of actin foci associated with bacteria per 100 transfected cells was determined visually using an Apotome system microscope. The experiment was performed in triplicate. Photographs illustrate the impact of S. Enteritidis (left) and S. Gallinarum (right) on the actin cytoskeleton.