Fig. 6. IB and EB Ot enter host cells using distinct mechanisms.

Images (A) and quantification (B) of the effect of endocytosis inhibitors on entry of IB and EB Ot into HUVEC cells. IB and EB Ot were seeded onto fresh HUVEC cells that had been treated with inhibitors of clathrin-mediated endocytosis (100 µM dynasore) or macropinocytosis (50 µM amiloride or 100 µM zoniporide). Cells were fixed 3 h post-infection. Dynasore inhibits entry of IB but not EB, whilst amiloride and zoniporide inhibited entry of both IB and EB. Red = actin (phalloidin), blue = Hoechst (DNA), green = Ot (anti-TSA56 antibody). Scale bar = 10 µm. Similar results were found in L929 cells (Supplementary Fig. 10). Images (C) and quantification (D) of the effect of 0.2 µM siRNA against clathrin on entry of EB and IB Ot into HUVEC cells. Experiments were carried out as in A, B. In B, D. n = 30 fields randomly imaged over 3 independent experiments. In each field the percentage of infected host cells was counted. Individual values and means are shown here. Statistical significance in B was determined by comparing all groups using one-way ANOVA, followed by a Dunnett’s multiple comparison tests with the control within the IB or EB group, and in D by performing a Mann–Whitney test comparing the scramble control and the clathrin siRNA treated condition for IB and EB separately. E Blocking the bacterial surface adhesins TSA56 and ScaC with anti-TSA56 and anti-ScaC antibodies strongly inhibits entry of IB but not EB Ot in HUVEC cells. Bacterial entry in the presence of vehicle control or antibodies was determined by quantifying the number of intracellular bacteria per host cell 30 min post-infection using immunofluorescence microscopy of bacteria labeled with HPG. Statistical significance was determined by comparing all groups using one-way ANOVA, followed by a Dunnett’s multiple comparison tests with the control within the IB or EB group. ****p ≤ 0.0001; ns not significant. *p value ≤ 0.05; **p value ≤ 0.01; ***p value ≤ 0.001; ****p value ≤ 0.0001.