Dual Control of Host Actin Polymerization by a Legionella Effector Pair

Figure 2

Deletion of vipA suppresses the endosomal escape and intracellular replication defects of the ΔlegK2 mutant. (a) Acquisition of vacuolar proton ATPase on LCVs. D. discoideum was infected for 1 h at an MOI of 100 with mCherry-labelled WT, ΔdotA, ΔlegK2, ΔvipA, and ΔlegK2/ΔvipA mutant L. pneumophila strains. The presence of vacuolar V-ATPase on LCVs was detected by an immunofluorescence assay with anti-VatA antibodies. (b) Detection of V-ATPase on LCVs containing L. pneumophila Paris WT strain, ΔdotA, ΔlegK2, ΔvipA, and ΔlegK2/ΔvipA L. pneumophila mutants. VatA-positive vacuoles () in amoebae infected with L. pneumophila WT, the derivative ΔdotA, ΔlegK2, ΔvipA, and ΔlegK2/ΔvipA, mutants were counted. (c) Intracellular replication of L. pneumophila in A. castellanii measured by fluorescence. A. castellanii amoebae were infected at with L. pneumophila Paris WT strain or ΔdotA, ΔlegK2, ΔvipA, and ΔlegK2/ΔvipA mutant L. pneumophila strains transformed with mCherry-expressing plasmids, and intracellular replication was monitored by quantifying mCherry-fluorescence intensity. (d, e) Intracellular replication of L. pneumophila in A. castellanii (d) and in U937-derived macrophages (e), measured by CFU. A. castellanii amoebae and U937-derived macrophages were infected at and , respectively, with L. pneumophila Paris WT strain, ΔdotA, ΔlegK2, ΔvipA, and ΔlegK2/ΔvipA mutant L. pneumophila strains, and intracellular replication was monitored by numbering CFU (colony forming units) after amoeba/macrophages lysis at different times postinfection on BCYE agar medium. (b–e) The results shown are representative of three independent experiments, and the error bars represent the standard deviations from triplicates. Statistical analyses were performed using a one-way ANOVA test for V-ATPase detection on LCVs and a two-way ANOVA test for intracellular replication: ns, no significant difference; ; ; ; .

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