The novel bacterial effector protein Cb EPF1 mediates ER-LD membrane contacts to regulate host lipid droplet metabolism

Abstract

Effective intracellular communication between cellular organelles is pivotal for maintaining cellular homeostasis. Tether proteins, which are responsible for establishing membrane contact sites between cell organelles, enable direct communication between organelles and ultimately influence organelle function and host cell homeostasis. While recent research has identified tether proteins in several bacterial pathogens, their functions have predominantly been associated with mediating inter-organelle communication specifically between the bacteria containing vacuole (BCV) and the host endoplasmic reticulum (ER). However, this study reveals a novel bacterial effector protein, CbEPF1, which acts as a molecular tether beyond the confines of the BCV and facilitates interactions between host cell organelles. Coxiella burnetii, an obligate intracellular bacterial pathogen, encodes the FFAT motif-containing protein CbEPF1 which localizes to host lipid droplets (LDs). CbEPF1 establishes inter-organelle contact sites between host LDs and the ER through its interactions with VAP family proteins. Intriguingly, CbEPF1 modulates growth of host LDs in a FFAT motif-dependent manner. These findings highlight the potential for bacterial effector proteins to impact host cellular homeostasis by manipulating inter-organelle communication beyond conventional BCVs.

Keywords: Coxiella burnetii; Effector proteins; FFAT motif; Inter-organelle contacts; Lipid droplets; Molecular tethers.

Figure 1:. CbEPF1-GFP localizes to host ER and LDs.

(A) Two putative FFAT motifs were identified in the C-terminal region of CbEPF1 protein. The sequence and position of the FFAT motifs are shown, with amino acid numbers indicated. Conventional FFAT motif sequence is shown below predicted CbEPF1 FFAT motifs. AH represents a predicted site of amphipathic helix. (B) Live cell microscopy shows that ectopically expressed CbEPF1-GFP localized to the host ER and LDs in mCherry-Coxiella infected HeLa cells. The ER was labeled with BFP-KDEL and LDs were labeled with LipidTOX-red. The Coxiella Containing Vacuole (CCV) membrane is outlined in red in the phase image. Scale bar 10 μm (overview) and 2 μm (magnified). (C) CbEPF1-GFP associated with LD biogenesis in the ER. Arrowheads indicate early-stage LD biogenesis sites (t = 4–6 mins) and arrows mark larger LDs with CbEPF1-GFP localized on the entire LD surface (t >13 mins). Images were acquired every 1 min (t = 0–20 min) by spinning disc confocal microscopy. Scale bar 0.2 μm. (D) Intensity profile of BFP-KDEL, CbEPF1-GFP, and LipidTOX-red from Fig. 1C during LD biogenesis in the ER from t=0–20 min. From 4 min time point, the increase in LipidTOX-red intensity indicates emergence and growth of LD.

Figure 2:. CbEPF1 associates with host LDs and induces inter-organelle contact sites between the host ER and LDs.

(A) HeLa cells expressing CbEPF1-GFP and BFP-KDEL were treated with OA (30 μM) and imaged every 6 hrs by live cell spinning disc microscopy. Representative deconvoluted images show CbEPF1-GFP relocates from the ER to the LD surface as LDs grow (arrowhead). Scale bar 10 μm (overview) and 1 μm (magnified). (B) HeLa cells expressing GFP or CbEPF1-GFP and BFP-KDEL were treated with OA (100 μM, overnight) to induce LD biogenesis. LDs were visualized with LipidTOX red. ER shows limited association with LDs in GFP expressing cells (top panel). CbEPF1-GFP expression induces extended contacts between ER and LDs (bottom panel). Arrows show ER wrapping around CbEPF1 localized LDs. Arrow heads show absence of ER interaction at the regions of LDs where CbEPF1-GFP localization is absent. Scale bar 10 μm (overview) and 1 μm (magnified). (C) Representative image of 3D rendering of a Z-stack image illustrating the association between LDs (magenta) and ER (blue). In cells expressing GFP alone (i), the ER exhibits minor contacts with LDs. In contrast, cells expressing CbEPF1-GFP (ii) display extended ER-LD contacts, with ER-LD interactions specifically localized to regions where CbEPF1-GFP (green) is present on LDs.

Figure 3:. CbEPF1 FFAT motifs interact with the VAP MSP domain

(A) Schematic representation of the FFAT motif containing protein interaction with the ER protein VAP to form inter-organelle contact sites between ER and LDs. (B) CbEPF1 amino acid sequence contains two FFAT motifs. Number indicates amino acid position; essential position (2^nd residue) in FFAT motif is underlined. Mutations in FFAT motif(s) are highlighted in orange. (C) Bacterial adenylate cyclase-based two hybrid assay (BACTH) shows CbEPF1 interacts with VAPB in an MSP-FFAT motif dependent manner. (D) HeLa cells expressing mCherry-VAPB along with GFP or CbEPF1-GFP were treated with OA (100 μM, overnight) and LDs were stained with Lipi Blue. The white arrow shows mCherry-VAPB as ring like structure around CbEPF1-GFP localized LDs. Scale bars: 10 μm (overview) and 2 μm (magnified). (E) Immunoprecipitation of GFP and CbEPF1-GFP (WT, F1mt, F2mt, and F3mt) from lysates of HEK293 induced with OA (100 μM, overnight). WB represents western blot analysis using respective primary antibody.

Figure 4:. Functional FFAT motifs are required for CbEPF1-induced ER-LDs contact sites.

(A) CbEPF1-F1mt-GFP and CbEPF1-F2mt-GFP induce ER-LD contact sites while CbEPF1-F3mt-GFP failed to induce ER-LD contact sites. Scale bar 10 μm (overview) and 1 μm (magnified). (B) CbEPF1-F3mt-GFP expression in cells causes clustering of LDs. Scale bar 10 μm (overview) and 1 μm (magnified). (C) Representative image of 3D rendering of a Z-stack image illustrating the association between LDs (magenta) and ER (blue) in CbEPF1-FFAT mutant-GFP expressing cells. CbEPF1-F1mt-GFP or CbEPF1-F2mt-GFP expressing cells exhibited ER-LD contacts, while the CbEPF1-F3mt-GFP expressing cells showed exclusion of ER around LD clusters.

Figure 5:. CbEPF1 regulates host LD metabolism

(A) HeLa cells expressing CbEPF1-GFP or CbEPF1-FFAT mutant-GFP contain significantly high number of LDs compared to control cells. The number of LDs/cell in HeLa cells transiently expressing respective protein (GFP, n = 22 cells; CbEPF1-GFP, n = 29 cells; CbEPF1-F1mt-GFP, n = 25 cells; CbEPF1-F2mt-GFP, n = 23 cells, CbEPF1-F3mt-GFP, n = 25 cells) were quantified and shown as Mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (B) HeLa cells with CbEPF1-GFP or CbEPF1-F1mt-GFP or CbEPF1-F2mt-GFP expression show larger LDs when induced with OA (100 μM) compared to GFP or CbEPF1-F3mt-GFP expressing cells. The size of LDs in cells expressing respective protein (GFP, n = 90 LDs from 6 cells; CbEPF1-GFP, n = 90 LDs from 8 cells; CbEPF1-F1mt-GFP, n = 80 LDs from 6 cells; CbEPF1-F2mt-GFP, n = 79 LDs from 7 cells, CbEPF1-F3mt-GFP, n = 90 LDs from 7 cells) were quantified and shown as Mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (C) Representative images for LD size in HeLa cells expressing GFP or CbEPF1-GFP or CbEPF1-FFAT mutants, treated with OA (100 μM, overnight). Scale bars: 10 μm (overview) and 1 μm (magnified).

Figure 6:. Model of the CbEPF1-mediated inter-organelle contact sites between host ER and LDs to regulate host LD metabolism.

Schematic representation of how CbEPF1 establish ER-LD contacts in the Coxiella infected cells. CbEPF1, localized on host LDs, interacts with VAPs in the ER to establish ER-LD contact sites (left side). The extent of CbEPF1-mediated ER-LD contacts may be contingent on protein expression levels (right side). In overexpression conditions, CbEPF1 promotes substantial ER wrapping around entire LDs. Conversely, in native cells with lower protein expression, CbEPF1-mediated ER-LD contacts may be limited to specific short regions. The Coxiella Containing Vacuole (CCV) and type 4b secretion system (T4BSS) are depicted in the Coxiella-infected cell model.