Figure 8
Elongation factor Tu of LVS participates in binding to human monocyte-like THP-1 cells and in their infection. A: THP-1 cells were infected for 30 min by opsonized LVS-GFP that had been pre-incubated either with RPMI, NIS or anti-EF-Tu Ab (diluted 1/2,000). THP-1 cells were also pre-incubated with 50 μg His-EF-Tu and then infected for 30 min by opsonized LVS-GFP pre-incubated with NIS. Fluorescence microscopy was analyzed at 63× magnification. Fifteen fields containing average of 100 cells were examined to quantify number of bound bacteria. Results shown are means ± SD from three independent experiments. *, p < 0.01 for group of bacteria treated with anti-EF-Tu Ab compared to group incubated with RPMI. B: Intracellular replication of LVS. THP-1 cells were infected for 30 min with opsonized LVS that were first incubated either with RPMI (■), mouse NIS (◆) or anti-EF-Tu Ab (●), both diluted 1/2,000. Cells were then washed with RPMI containing gentamicin and further incubated in RPMI-FCS and gentamicin for indicated times. Quantification of intracellular bacteria was performed as described in Methods. Results shown are means from five independent experiments, each in triplicate ± SD indicated by error bars. C: LVS were incubated either with Human serum, H.I. serum or with No Human serum. LVS in these different conditions were further incubated either with RPMI, mouse NIS or anti-EF-Tu Ab, both diluted 1/2,000. THP-1 cells were then infected by the different LVS preparations. THP-1 cells, pre-incubated with anti-CR3 MAb were infected with opsonized LVS that had been pretreated or not by anti-EF-Tu Ab. THP-1 cells were also preincubated with 50 μg His-EF-Tu before infection by opsonized LVS. Cells were washed in RPMI with gentamicin and incubated in RPMI-FCS and gentamicin for 22 h. Quantification of intracellular bacteria was performed as described in Methods. N.E. (not examined): anti-CR3 MAb, anti-CR3 MAb and anti-EF-Tu Ab and His-EF-Tu samples were not tested in H.I serum and No human serumconditions. Results shown are means from five independent experiments, each in triplicate ± SD indicated by error bars.