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. 2018 Nov 5;217(11):3863-3872.
doi: 10.1083/jcb.201801208. Epub 2018 Oct 1.

Legionella remodels the plasma membrane-derived vacuole by utilizing exocyst components as tethers

Kohei Arasaki  1   2 , Hana Kimura  2 , Mitsuo Tagaya  2 , Craig R Roy  3
Affiliations

Legionella remodels the plasma membrane-derived vacuole by utilizing exocyst components as tethers

Kohei Arasaki et al. J Cell Biol. .

Abstract

During the initial stage of infection, Legionella pneumophila secretes effectors that promote the fusion of endoplasmic reticulum (ER)-derived vesicles with the Legionella-containing vacuole (LCV). This fusion leads to a remodeling of the plasma membrane (PM)-derived LCV into a specialized ER-like compartment that supports bacterial replication. Although the effector DrrA has been shown to activate the small GTPase Rab1, it remains unclear how DrrA promotes the tethering of host vesicles with the LCV. Here, we show that Sec5, Sec15, and perhaps Sec6, which are subunits of the exocyst that functions in the tethering of exocytic vesicles with the PM, are required for DrrA-mediated, ER-derived vesicle recruitment to the PM-derived LCV. These exocyst components were found to interact specifically with a complex containing DrrA, and the loss of Sec5 or Sec15 significantly suppressed the recruitment of ER-derived vesicles to the LCV and inhibited intracellular replication of Legionella Importantly, Sec15 is recruited to the LCV, and Rab1 activation is necessary for this recruitment.

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Figures

Figure 1.
Figure 1.
Loss of Sec5 or Sec15 inhibits the DrrA-mediated recruitment of ER-derived vesicles to the PM. (A–C) Acceptor (A), donor (B), or both acceptor and donor (C) cells were transfected with or without siRNA targeting the indicated proteins. 48 h after transfection, donor cells were transfected with a plasmid encoding Luciferase-KDEL and incubated for 24 h. An ER-derived vesicle recruitment assay using semi-intact cells and recombinant His6-DrrA was conducted as described previously (Arasaki et al., 2012). Values are the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 compared with no-siRNA. (D) HEK293-FcγRII cells (acceptor) and 3x-FLAG-Sec22b–expressing HEK293-FcγRII cells (donor) were transfected with siRNA targeting the indicated proteins. 48 h after transfection, acceptor cells were additionally transfected with plasmids encoding GFP-DrrA61–647 and GFP-Stx3 and incubated for 24 h, and then the cells were permeabilized. The permeabilized cells were incubated with vesicles containing 3x-FLAG-Sec22b prepared from the donor cells, and protein complexes were precipitated using anti-FLAG M2 agarose. The precipitated proteins were analyzed by Western blotting using antibodies against GFP and FLAG. Values below the GFP strip represent the average of the GFP/FLAG intensity ratio (n = 3) normalized to that in no-siRNA. IP, immunoprecipitation; KD, knockdown.
Figure 2.
Figure 2.
Legionella effector DrrA interacts with the exocyst in a component-specific manner. (A) HEK293-FcγRII cells were cotransfected with plasmids encoding 3x-FLAG or 3x-FLAG-exocyst components and GFP-Stx2, 3, or 4. 24 h after transfection, cell lysates were prepared and immunoprecipitated (IP). (B) HEK293-FcγRII cells were cotransfected with plasmids encoding 3x-FLAG, 3x-FLAG-Exo70 or -Sec15, and GFP-DrrA61–647 or -DrrA451–647. 24 h after transfection, cell lysates were prepared and immunoprecipitated. (C) HEK293-FcγRII cells were cotransfected with plasmids encoding GFP-DrrA61–647 and 3x-FLAG constructs. 24 h after transfection, cell lysates were prepared and immunoprecipitated. (D) HEK293-FcγRII cells were transfected with a plasmid encoding 3x-FLAG-Sec15. 24 h after transfection, cells were infected with wild-type Legionella or ΔdrrA mutant strain for 1 h at MOI 50. After infection, cell lysates were prepared and immunoprecipitated.
Figure 3.
Figure 3.
Expression of mutant Sec15 inhibits recruitment of Sec22b to the LCV. (A) HEK293-FcγRII cells were cotransfected with plasmids encoding GFP, GFP-DrrA61–647 or -DrrA451–647, and FLAG or FLAG-Rab1A. 24 h after transfection, cell lysates were prepared and immunoprecipitated (IP). (B) HEK293-FcγRII cells were transfected with plasmids encoding 3x-FLAG-Sec15 (wild-type) or -Sec15N691A and GFP-Rab1. 24 h after transfection, cells were infected with wild-type Legionella for 1 h at MOI 50. After infection, cell lysates were prepared and immunoprecipitated. (C) HEK293-FcγRII cells were cotransfected with 3x-FLAG-Sec15 (wild-type) or -Sec15N691A and RFP-Rab1 or -Sec22b. 24 h after transfection, cells were infected with wild-type Legionella for 1 h at MOI 5, fixed, and stained with antibodies against FLAG and Legionella. Localization of RFP-Rab1 and RFP-Sec22b was assessed for the vacuoles containing wild-type Legionella. Values are the mean ± SD (n = 3, 50 vacuoles in each experiment). n.s., not significant. *P < 0.05 compared with wild-type. Bar, 10 µm.
Figure 4.
Figure 4.
Silencing of Sec5 or Sec15 suppresses intracellular replication of Legionella. (A) HEK293-FcγRII cells were transfected with or without siRNA targeting the indicated proteins. 48 h after transfection, cells were additionally transfected with RFP-Rab1 or RFP-Sec22b for 24 h. After transfection, cells were infected with wild-type Legionella for 1 h at MOI 5, and then localization of RFP-Rab1 and RFP-Sec22b was assessed for vacuoles containing wild-type Legionella. Values are the mean ± SD (n = 3, 100 vacuoles in each experiment). **P < 0.01 and ***P < 0.001 compared with no-siRNA. (B) 3x-FLAG-Sec22b–expressing HEK293-FcγRII cells were transfected with or without siRNA targeting indicated proteins. 72 h after transfection, cells were infected with wild-type Legionella for 1 h at MOI 50. After infection, cell lysates were prepared and immunoprecipitated (IP). Values below the GFP strip represent the average of the Stx3/FLAG intensity ratio (n = 3) normalized to that in no-siRNA. (C) HEK293-FcγRII cells were transfected with or without siRNA targeting the indicated proteins. 72 h after transfection, cells were infected with wild-type Legionella for 10 h at MOI 5. Intracellular replication of Legionella was assessed by counting bacteria residing in a single vacuole in the infected cells. The data are represented as the percentage of vacuoles containing 1 bacterium (white bars), 2–5 bacteria (light gray bars), 6–10 bacteria (dark gray bars), or >11 bacteria (black bars). Values are the mean ± SD (n = 3, 100 vacuoles in each experiment). **P < 0.01 compared with no-siRNA.
Figure 5.
Figure 5.
DrrA-mediated Rab1 activity is required for the recruitment of Sec15 to the LCV. (A) HeLa-FcγRII cells were cotransfected with GFP-DrrA61–647 or -DrrA451–647 and 3x-FLAG-Sec15 (wild-type) or -Sec15N691A. 24 h after transfection, cells were fixed and stained with an antibody against FLAG. Arrows indicate DrrA-positive structures. Bar, 5 µm. (B) HeLa-FcγRII cells were transfected with 3x-FLAG-Sec15. 24 h after transfection, cells were infected with wild-type Legionella or ΔdrrA mutant strain for 1 h at MOI 10 and fixed, and extracellular Legionella were stained. After staining, cells were permeabilized and stained with an antibody against FLAG. Intracellular Legionella were detected by Hoechst 33342. Bar, 1 µm. Values are the mean ± SD (n = 3, 100 vacuoles in each experiment). *P < 0.05 compared with ΔdrrA. (C) HeLa-FcγRII cells were cotransfected with GFP-Sec15 and FLAG-Rab1 wild-type or -Rab1N121I. 24 h after transfection, cells were infected with wild-type Legionella for 1 h at MOI 10, fixed, and stained with an antibody against FLAG. Legionella were detected by Hoechst 33342. Bar, 2 µm. Values are the mean ± SD (n = 3, 100 vacuoles positive for Rab1 in each experiment). **P < 0.01 compared with Rab1N121I.
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