A Single Legionella Effector Catalyzes a Multistep Ubiquitination Pathway to Rearrange Tubular Endoplasmic Reticulum for Replication

Abstract

Intracellular pathogens manipulate host organelles to support replication within cells. For Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. We show here that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as alterations in Rtn4 associated with the replication compartment. These rearrangements are triggered via Sde-promoted ubiquitin transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin transfer requires two sequential enzymatic activities from a single Sde polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4 or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. Therefore, a single bacterial protein drives a multistep biochemical pathway to control ubiquitination and tubular ER function independently of the host ubiquitin machinery.

Keywords: ADP-ribosylation; Legionella pneumophila; Sde proteins; endoplasmic reticulum; nucleotidase; phosphodiesterase; replication vacuole formation; reticulon; transferase; type IV secretion; ubiquitin.

Fig. 1. Sde family members promote Rtn4 rearrangements in response to L. pneumophila challenge

(A,B) Bone marrow derived macrophages (BMDMs) from A/J mice were challenged, followed by fixation, permeabilization with 0.1% Triton X100 and probing with α-Rtn4 (green), α-L. pneumophila (red), and Hoechst (blue). Scalebar: 5 µm. (C) BMDMs challenged for 1 h with LP02 (WT) or an icm/dot⁻(dotA3) mutant were fixed, permeabilized as noted, and probed. Scalebar: 5 µm. Arrows indicate location of bacterium within infected cells. (D,E) Altered electrophoretic migration of Rtn4 after L. pneumophila challenge. HeLa cells were challenged for 2 h, solubilized in SDS at room temperature, fractionated by SDS-PAGE, and probed. Lanes: Un, uninfected. (F) Sde family members result in Rtn4 electrophoretic variants. 40–46 h after transfection into Cos1 cells of noted plasmids, cells were extracted, gel fractionated and blots probed with α-Rtn4. (G) The chromosomal arrangement of the sde genes. (H) Rtn4 rearrangements in BMDM dependent on presence of Sde family members at 1 hpi (See Fig. S1A). Scalebar: 5µm. (I) Deletion of sde family (KK099) prevents Rtn4 rearrangements about the LCV. BMDM were challenged for 1 h prior to probing as in panel A. (J,K) Sde family members promote immediate Rtn4 rearrangements after host cell contact. Cos7 cells harboring Rtn4b-GFP were challenged with L. pneumophila and images from live cells were captured over a 10 min period (See Movies S1 & S2). Scalebar: 5µm. Images displayed at 1.15X the captured sizes. See Fig. S1; Tables S1 and S2.

Fig. 2. Sde-dependent ER rearrangements generate Rtn4-staining pseudovesicles or linear stacks

(A–D): Cos7 cells harboring Rtn4b-GFP-APEX2 (See Fig. S1B) challenged for 1 h with either LP02 (A,B) WT or (C,D) the Δsde strain (KK099), subjected to DAB staining followed by TEM. Panels A,B are TEM images of different sections from the same cell. Panel B is a high magnification image of Rtn4-rich region abutting bacterium that can be seen in panel A. Arrows point to (A) membranes in direct apposition to the LCV or (B) projections of Rtn4-associated membranes. Panels E,F: BMDM challenged for 1 h with either (E) LP02 (WT) or (F) Δsde strain (KK034). (E(inset)) Boxed area at higher magnification. Arrowhead points to projections from round structure. (F) Stacks of ER surrounding Δsde strain (KK034). (See Fig. S1, S2, and S3; Table S3).

Fig. 3. Sde family members promote Rtn4 ubiquitination

(A) GFP-SdeC or GFP (vector) were transiently expressed in HeLa cells for 24 h, followed by Rtn4 IP. Eluates were fractionated by SDS-PAGE and stained. (B) HA-Ub was transiently co-expressed with either GFP or GFP-SdeC in HeLa cells for 24 h, then subjected to IP with α-Rtn4 IP, fractionated by SDS-PAGE, and probed for Ub-modified Rtn4 with α-HA (E=eluate, FT=Flowthrough, T=Total). (C) HEK293T cells were transiently transfected with HA-Ub for 24 h, the cell culture medium was replaced with 10µM MG132 (Millipore) medium 30–60 min. prior to challenge with L. pneumophila and the infection was allowed to proceed (MPI, minutes post infection), prior to IP with α-Rtn4. (D) Domain structure of Sde family proteins, with endpoints noted for SdeC (Qiu et al., 2016). See Table S4.

Fig. 4. Sde family mono ADP-ribosyltransferase activity is required for Rtn4 restructuring and ubiquitination

(A) A/J BMDMs were challenged for 1 h, fixed, permeabilized with 1% Triton X100 and probed with α-Rtn4 (green), α-L. pneumophila (red), and Hoescht (blue). 50 L. pneumophila vacuoles were assessed for Rtn4 colocalization per coverslip. (B) Representative micrographs of Rtn4 association with the LCV at 1hpi (hour post infection), scale bar=5µm. (C) HEK293T cells transiently transfected with HA-Ub were challenged with L. pneumophila, and extracts were subjected to IP with α-Rtn4. Eluates were analyzed for Ub by probing with α-HA. (D) HEK293T extracts were incubated at 37°C with 10nM recombinant SdeC, and 20µM recombinant human HA-Ub monomer. Reactions were separated by SDS-PAGE and probed for α-εAdo (ADPr), and α-HA (Ub). (E) SdeC was incubated with K63-linked Ub tetramers at 37°C. Reactions were fractionated by SDS-PAGE, and assayed for altered migration and ADPr of Ub, by silver staining and immunoblotting. Lanes; WT: WT SdeC; C118S: DUB mutant; E859A: ART mutant; ---: No SdeC; WT(No HA-UB): WT SdeC No HA-UB added (See Figs. S4 and S5).

Fig. 5. Sde family NP domain is required for Rtn4 rearrangements, intracellular growth, and functions cooperatively with ART domain to conjugate Ub

(A,B) A/J BMDM were challenged for 1 h followed by fixation, permeabilization with 1% Triton X100, and probed as in Fig. 4A–B. L. pneumophila vacuoles were assessed for Rtn4 colocalization (See Fig. S4A). (B) Representative micrographs of Rtn4 association with the LCV from part A, scale bar 5 µm. (C) Dictyostelium discoideum was challenged with WT (Lp02) or mutant L. pneumophila expressing luciferase (PahpC::lux). L. pneumophila intracellular growth (luminescence) was monitored hourly. Mean ± SEM for every 5 h increment; results representative of ≥3 replicate experiments (See Fig. S4B–D). (D) HEK293T extract was incubated at 37°C for the indicated time with recombinant SdeC, εNAD, and recombinant human HA-Ub. Rtn4 Ub and εADPr were assessed by immunoblot with indicated antibodies. (E) Recombinant Ub or poly-His-Ub monomers were incubated with εNAD and recombinant SdeC at 37°C for the indicated time. Ub ADPr was assessed as in panel D. (F) A four component system is sufficient to ubiquitinate Rtn4. Purified GST-Rtn4 was incubated with noted components and SdeC derivatives for 1 h. Proteins were fractionated by SDS-PAGE and visualized by silver stain (See Fig. S6). Lanes: WT, WT SdeC; E859A, ART mutant; H416A, NP mutant (See Figs. S4 and S6).

Fig. 6. ART and diphosphohydrolase-dependent ribose-monophosphate modification of Ub

(A) SdeC in presence of εNAD results in a modification of 212 amu. Ub (black), Ub incubated with the NP mutant SdeC (H416A, gray) or SdeC (WT, red) were subjected to LC-MS analysis and the deconvoluted masses of the peaks for each sample displayed. (B) Proposed pathway to generated modification of 212 amu. (C,D) Trypsin/AspN treatment of modified Ub species followed by extracted ion chromatography (XIC) analysis reveals predicted modifications. Shown are XIC chromatograms of species having displayed m/z values for both major modifications displayed in panel B. (E) Treatment of species 3 with alkaline phosphatase results in a product predicted for ribosylated Ub. Ub was treated with noted enzymes, followed by LC-MS, and the deconvoluted masses of the peaks for each sample are displayed. (F) Likely products that lead to the generation of 132 amu modification. (G) Electrospray ionization MS/MS spectrum of trypsin/AspN Ub fragment having +212 amu modification resulting from SdeC treatment. The b-type ion fragments are displayed above the trypsin/AspN peptide that has an increase of 212.01 amu over the predicted size of the unmodified Ub peptide. Each predicted b-type ion was identified and displayed along with identified y-type ion fragments. Ions marked #212 denote fragment sizes that correspond to the predicted b-type ions having an added 212 amu.