Figure 5.
Mutation of K134 of Rab5, a residue identified as ubiquitinated upon infection with L. pneumophila, affects both Rab5 recruitment to the LCV and bacterial proliferation within cells. (a) HeLa-FcγRII cells expressing 3x-FLAG-Rab5A (WT, K22R, K33R, or K134R) and HA-UB were infected with the WT L. pneumophila Lp01 strain for 1 h at an MOI of 50. After infection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against HA and FLAG. (b) HeLa-FcγRII cells expressing mRFP-Rab5A (WT) or (K134R) were infected with the WT L. pneumophila Lp01 strain for 1 h at an MOI of 10. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. Bar, 5 µm. Data are representative of three independent experiments (100 vacuoles were scored in each experiment). Results are shown as means ± SD. P value is determined using an unpaired two-tailed Student’s test. ****P < 0.0001. (c) HeLa-FcγRII cells were transfected with myc-RabGAP-5, GFP or GFP-Lpg2525, and 3x-FLAG-Rab5A (Q79L, left panel) or -Rab5A (Q79L/K134R, right panel). At 24 h after transfection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against myc, GFP, and FLAG. An asterisk indicates nonspecific bands. Data are representative of three independent experiments. Results are shown as means ± SD. P value is determined using one-way ANOVA with Tukey’s multiple comparisons. **P < 0.01. (d) HeLa-FcγRII cells expressing mRFP-Rab5A (WT) or (K134R) were infected with the WT L. pneumophila Lp01 strain for 8 h at an MOI of 5. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. Bar, 5 µm. The graph shows the average number of L. pneumophila in a single vacuole. Data are representative of three independent experiments (30 vacuoles were scored in each experiment). Results are shown as means ± SEM. P value is determined using an unpaired two-tailed Student’s test. *P < 0.05. IB, immunoblotting. Source data are available for this figure: SourceData F5. Refer to the image caption for details.

Mutation of K134 of Rab5, a residue identified as ubiquitinated upon infection with L. pneumophila, affects both Rab5 recruitment to the LCV and bacterial proliferation within cells. (a) HeLa-FcγRII cells expressing 3x-FLAG-Rab5A (WT, K22R, K33R, or K134R) and HA-UB were infected with the WT L. pneumophila Lp01 strain for 1 h at an MOI of 50. After infection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against HA and FLAG. (b) HeLa-FcγRII cells expressing mRFP-Rab5A (WT) or (K134R) were infected with the WT L. pneumophila Lp01 strain for 1 h at an MOI of 10. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. Bar, 5 µm. Data are representative of three independent experiments (100 vacuoles were scored in each experiment). Results are shown as means ± SD. P value is determined using an unpaired two-tailed Student’s test. ****P < 0.0001. (c) HeLa-FcγRII cells were transfected with myc-RabGAP-5, GFP or GFP-Lpg2525, and 3x-FLAG-Rab5A (Q79L, left panel) or -Rab5A (Q79L/K134R, right panel). At 24 h after transfection, cell lysates were prepared and immunoprecipitated with the FLAG-M2 beads. The precipitated proteins were analyzed by IB with antibodies against myc, GFP, and FLAG. An asterisk indicates nonspecific bands. Data are representative of three independent experiments. Results are shown as means ± SD. P value is determined using one-way ANOVA with Tukey’s multiple comparisons. **P < 0.01. (d) HeLa-FcγRII cells expressing mRFP-Rab5A (WT) or (K134R) were infected with the WT L. pneumophila Lp01 strain for 8 h at an MOI of 5. After infection, cells were fixed and stained with anti-Legionella antiserum for detection of extracellular bacteria, then permeabilized and further stained with DAPI for detection of intracellular bacteria. Bar, 5 µm. The graph shows the average number of L. pneumophila in a single vacuole. Data are representative of three independent experiments (30 vacuoles were scored in each experiment). Results are shown as means ± SEM. P value is determined using an unpaired two-tailed Student’s test. *P < 0.05. IB, immunoblotting. Source data are available for this figure: SourceData F5.

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