Rac1 activity and host cell type regulate translocon formation.

(A) Lack of Rac1 inhibitor YopE and Rac1Q61L overexpression enhance translocon formation. Native HeLa cells were infected with WA-314 or WA-314ΔYopE and myc-Rac1Q61L overexpressing HeLa cells were infected with WA-314 at a MOI of 50 for 60 min. Cells were stained for YopB, myc and DAPI as indicated. Dashed lines indicate host cell circumference. Insets show strongly increased contrast settings to visualize the host cell bodies. Scale bars: 5 μm. The percentage of YopB positive bacteria of total cell associated bacteria was determined. Bars represent mean ± S.D. n = 6505 WA-314 and n = 5640 WA-314ΔYopE bacteria were investigated in 4 independent experiments (upper graph); n = 3423 bacteria (WA-314) associated to control transfected cells and n = 7882 bacteria associated to myc-Rac1Q61L transfected cells were investigated in 3 independent experiments (lower graph). (B) Digitonin extraction reveals increased incorporation of YopB and YopD into host cell membranes upon Rac1Q61L overexpression or lack of Rac1 inhibitor YopE. Experimental conditions as in (A) but with a MOI of 100. HeLa cells were lyzed with digitonin and resulting supernatants (containing membrane integrated and soluble Yops from host cells) and cell pellets (containing intact bacteria and digitonin insoluble cell components) were analyzed with Western blot for the indicated proteins. YopH serves as marker for effector translocation, myc indicates myc-Rac1QL61 expression, calnexin serves as host cell loading control and HSP60 serves as bacterial loading and lysis control. Data are representative of 3 independent experiments. (C) Quantification of YopB spots per bacterium using STED microscopy. HeLa cells and human primary macrophages (Mφ) were infected with WA-314 or WA-314ΔYopE. Cells were immunostained with anti-YopB antibody followed by AbberiorStarRed or Abberior635P secondary antibody and recorded by STED microscopy. The number of YopB spots per bacterium was analyzed using Imaris software (Methods). Bars represent mean ± S.D. of n = 20 WA-314 (HeLa), n = 27 WA-314ΔYopE (HeLa), n = 52 WA-314 (Mφ) and n = 7 WA-314ΔYopE (Mφ) bacteria investigated in 2–3 independent experiments. *p (D) YopB positive WA-314 and WA-314ΔYopE in macrophages at different infection times. Primary human macrophages were infected with WA-314 or WA-314ΔYopE at a MOI of 10 for the indicated time periods. The percentage of YopB positive bacteria of total cell associated bacteria was calculated. Data represent mean ± S.D. of n = 3047 WA-314 and n = 1822 WA-314ΔYopE in 2–3 independent experiments.