Host Lipid Membrane Research Articles

Cholesterol-Dependent Membrane Deformation by Metastable Viral Capsids Facilitates Entry.

Nonenveloped viruses employ unique entry mechanisms to breach and infect host cells. Understanding these mechanisms is crucial for developing antiviral strategies. Prevailing perspective suggests that nonenveloped viruses release membrane pore-forming peptides to breach host membranes. However, the precise involvement of the viral capsid in this entry remains elusive. Our study presents direct observations elucidating the dynamically distinctive steps through which metastable reovirus capsids disrupt host lipid membranes as they uncoat into partially hydrophobic intermediate particles. Using both live cells and model membrane systems, our key finding is that reovirus capsids actively deform and permeabilize lipid membranes in a cholesterol-dependent process. Unlike membrane pore-forming peptides, these metastable viral capsids induce more extensive membrane perturbations, including budding, bridging between adjacent membranes, and complete rupture. Notably, cholesterol enhances subviral particle adsorption, resulting in the formation of pores equivalent to the capsid size. This cholesterol dependence is attributed to the lipid condensing effect, particularly prominent at an intermediate cholesterol level. Furthermore, our results reveal a positive correlation between membrane disruption extent and efficiency of viral variants in establishing infection. This study unveils the crucial role of capsid-lipid interaction in nonenveloped virus entry, providing new insights into how cholesterol homeostasis influences virus infection dynamics.

Lipid Specificity of the Fusion of Bacterial Extracellular Vesicles with the Host Membrane.

Bacterial membrane vesicles (MVs) facilitate the long-distance delivery of virulence factors crucial for pathogenicity. The entry and trafficking mechanisms of virulence factors inside host cells are recently emerging; however, whether bacterial MVs can fuse and modulate the physicochemical properties of the host lipid membrane and membrane lipid parameter for fusion remains unknown. In this study, we reconstituted the interaction of bacterial MVs with host cell lipid membranes and quantitatively showed that bacterial MV interaction increases the fluidity, dipole potential, and compressibility of a biologically relevant multicomponent host membrane upon fusion. The presence of cylindrical lipids, such as phosphatidylcholine, and a moderate acyl chain length of C16 help the MV interaction. While significant binding of bacterial MVs to the raft-like lipid membranes with phase-separated regions of the membrane was observed, however, MVs prefer binding to the liquid-disordered regions of the membrane. Furthermore, the elevated levels of cholesterol tend to hinder the interaction of bacterial MVs, as evident from the favorable excess Gibbs free energy of mixing bacterial MVs with host lipid membranes. The findings provide new insights that might have implications for the regulation of host machinery by bacterial pathogens through manipulation of the host membrane properties.

Evaluation of fendiline treatment in VP40 system with nucleation-elongation process: a computational model of Ebola virus matrix protein assembly.

Ebola virus (EBOV) infection can cause deadly hemorrhagic fever, which has a mortality rate of ~50%-90% without treatment. The recent outbreaks in Uganda and the Democratic Republic of the Congo illustrate its threat to human health. Though two antibody-based treatments were approved, mortality rates in the last outbreak were still higher than 30%. This can partly be due to the requirement of advanced medical facilities for current treatments. As a result, it is very important to develop and evaluate new therapies for EBOV infection, especially those that can be easily applied in the developing world. The significance of our research is that we evaluate the potential of lipid-targeted treatments in reducing EBOV assembly and egress. We achieved this goal using the VP40 system combined with a computational approach, which both saves time and lowers cost compared to traditional experimental studies and provides innovative new tools to study viral protein dynamics.

Membrane Condensation and Curvature Induced by SARS-CoV-2 Envelope Protein.

The envelope (E) protein of SARS-CoV-2 participates in virion encapsulation and budding at the membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). The positively curved membrane topology required to fit an 80 nm viral particle is energetically unfavorable; therefore, viral proteins must facilitate ERGIC membrane curvature alteration. To study the possible role of the E protein in this mechanism, we examined the structural modification of the host lipid membrane by the SARS-CoV-2 E protein using synchrotron-based X-ray methods. Our reflectometry results on solid-supported planar bilayers show that E protein markedly condenses the surrounding lipid bilayer. For vesicles, this condensation effect differs between the two leaflets such that the membrane becomes asymmetric and increases its curvature. The formation of such a curved and condensed membrane is consistent with the requirements to stably encapsulate a viral core and supports a role for E protein in budding during SARS-CoV-2 virion assembly.

Influenza A Virus Infection Alters Lipid Packing and Surface Electrostatic Potential of the Host Plasma Membrane.

The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Förster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid-lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken together, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.

Hijacking of Cellular Functions by Severe Acute Respiratory Syndrome Coronavirus-2. Permeabilization and Polarization of the Host Lipid Membrane by Viroporins.

Like all viral infections, SARS-CoV-2 acts at multiple levels, hijacking fundamental cellular functions and assuring its replication and immune system evasion. In particular, the viral 3′ Open Reading Frame (ORF3a) codes for a hydrophobic protein, which embeds in the cellular membrane, where it acts as an ion viroporin and is related to strong inflammatory response. Here we report equilibrium and enhanced sampling molecular dynamic simulation of the SARS-CoV-2 ORF3a in a model lipid bilayer, showing how the protein permeabilizes the lipid membrane, via the formation of a water channel, which in turn assures ion transport. We report the free energy profile for both K+ and Cl– transfer from the cytosol to the extracellular domain. The important role of ORF3a in the viral cycle and its high conservation among coronaviruses may also make it a target of choice for future antiviral development, further justifying the elucidation of its mechanism at the atomistic level.

Membrane-Bound Configuration and Lipid Perturbing Effects of Hemagglutinin Subunit 2 N-Terminus Investigated by Computer Simulations.

Hemagglutinin (HA) mediated fusion of influenza virus envelope with host lipid membrane is a critical step warrantying virus entry to the cell. Despite tremendous advances in structural biology methods, the knowledge concerning the details of HA2 subunit insertion into the target membrane and its subsequent bilayer perturbing effect is still rather limited. Herein, based on a set of molecular dynamics simulations, we investigate the structure and interaction with lipid membrane of the N-terminal HA2 region comprising a trimer of fusion peptides (HAfps) tethered by flexible linkers to a fragment of coiled-coil stem structure. We find that, prior to insertion into the membrane, HAfps within the trimers do not sample space individually but rather associate into a compact hydrophobic aggregate. Once within the membrane, they fold into tight helical hairpins, which remain at the lipid-water interface. However, they can also assume stable, membrane-spanning configurations of significantly increased membrane-perturbing potential. In this latter case, HAfps trimers centre around the well-hydrated transmembrane channel-forming distinct, symmetric assemblies, whose wedge-like shape may play a role in promoting membrane curvature. We also demonstrate that, following HAfps insertion, the coiled-coil stem spontaneously tilts to almost membrane-parallel orientation, reflecting experimentally observed configuration adopted in the course of membrane fusion by complete HA2 units at the rim of membrane contact zones.

Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe.

ABSTRACTThe intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-β-cyclodextrin reduced the interaction. The stomatin-prohibitin-flotillin-HflC/K domain of FLOT2, FLOT21–183, was sufficient for the unique FLOT2 localization and interaction with NPC1. NPC1, FLOT2, and FLOT21–183 trafficked to the lumen of Anaplasma inclusions. A loss-of-function mutant, NPC1P691S (mutation in the sterol-sensing domain), did not colocalize or interact with FLOT2 or with Anaplasma inclusions and inhibited infection. Ezetimibe is a drug that blocks cholesterol absorption in the small intestine by inhibiting plasma membrane Niemann-Pick C1-like 1 interaction with FLOTs. Ezetimibe blocked the interaction between NPC1 and FLOT2 and inhibited Anaplasma infection. Ezetimibe did not directly inhibit Anaplasma proliferation but inhibited host membrane lipid and cholesterol traffic to the bacteria in the inclusion. These data suggest that Anaplasma hijacks NPC1 vesicles containing cholesterol bound to FLOT2 to deliver cholesterol into Anaplasma inclusions to assimilate cholesterol for its proliferation. These results provide insights into mechanisms of intracellular cholesterol transport and a potential approach to inhibit Anaplasma infection by blocking cholesterol delivery into the lumen of bacterial inclusions.

Membranotropic and biological activities of the membrane fusion peptides from SARS-CoV spike glycoprotein: The importance of the complete internal fusion peptide domain.

Fusion peptides (FP) are prominent hydrophobic segments of viral fusion proteins that play critical roles in viral entry. FPs interact with and insert into the host lipid membranes, triggering conformational changes in the viral protein that leads to the viral-cell fusion. Multiple membrane-active domains from the severe acute respiratory syndrome (SARS) coronavirus (CoV) spike protein have been reported to act as the functional fusion peptide such as the peptide sequence located between the S1/S2 and S2’ cleavage sites (FP1), the S2’-adjacent fusion peptide domain (FP2), and the internal FP sequence (cIFP). Using a combined biophysical approach, we demonstrated that the α-helical coiled-coil-forming internal cIFP displayed the highest membrane fusion and permeabilizing activities along with membrane ordering effect in phosphatidylcholine (PC)/phosphatidylglycerol (PG) unilamellar vesicles compared to the other two N-proximal fusion peptide counterparts. While the FP1 sequence displayed intermediate membranotropic activities, the well-conserved FP2 peptide was substantially less effective in promoting fusion, leakage, and membrane ordering in PC/PG model membranes. Furthermore, Ca2+ did not enhance the FP2-induced lipid mixing activity in PC/phosphatidylserine/cholesterol lipid membranes, despite its strong erythrocyte membrane perturbation. Nonetheless, we found that the three putative SARS-CoV membrane-active fusion peptide sequences here studied altered the physical properties of model and erythrocyte membranes to different extents. The importance of the distinct membranotropic and biological activities of all SARS-CoV fusion peptide domains and the pronounced effect of the internal fusion peptide sequence to the whole spike-mediated membrane fusion process are discussed.

Cholesterol-dependent cytolysins promote immune evasion by clustering host membrane lipids

Abstract Pathogens causing necrotizing soft tissue infections, such as Streptococcus pyogenes and Clostridium perfringens, use cholesterol dependent cytolysins (CDCs) to evade immune responses by blocking pro-inflammatory TNFα production in macrophages. This evasion may be an unintended consequence of membrane repair responses to the CDC. CDCs trigger lipid-dependent sequestration and shedding of CDC pores on microvesicles. However, the molecular mechanisms by which CDCs exploit membrane repair to disrupt immune signaling is unknown. One target may be cholesterol-rich microdomains (CRMs) because they are focal points for toxin binding and immune signaling. We hypothesized that when CDCs oligomerize, they cluster membrane lipids in CRMs, along with immune receptors, into microscale assemblies that are spontaneously shed as microvesicles. We tested this hypothesis by artificially clustering sphingolipids using the sphingolipid-binding protein Ostreolysin A (OlyA), fused with tandem FKBP dimerization domains. We challenged murine bone marrow derived macrophages with and without FKBP homodimerizer and OlyA prior to LPS challenge and measured intracellular TNFα by flow cytometry. We found that oligomerization reduced OlyA binding, consistent with shedding. OlyA oligomerization further suppressed TNFα production, suggesting that microscale lipid assemblies interfere with cellular signaling. Overall, these results suggest that one cost to repair by shedding toxins on microvesicles is the loss of cellular signaling needed to promote inflammation. This may represent one strategy CDC-producing pathogens use to promote immune evasion and cause disease.

Characterizing Membrane Association and Periplasmic Transfer of Bacterial Lipoproteins through Molecular Dynamics Simulations

SummaryEscherichia coli lipoprotein precursors at the inner membrane undergo three maturation stages before transport by the Lol system to the outer membrane. Here, we develop a pipeline to simulate the membrane association of bacterial lipoproteins in their four maturation states. This has enabled us to model and simulate 81 of the predicted 114 E. coli lipoproteins and reveal their interactions with the host lipid membrane. As part of this set we characterize the membrane contacts of LolB, the lipoprotein involved in periplasmic translocation. We also consider the means and bioenergetics for lipoprotein localization. Our calculations uncover a preference for LolB over LolA and therefore indicate how a lipoprotein may be favorably transferred from the inner to outer membrane. Finally, we reveal that LolC has a role in membrane destabilization, thereby promoting lipoprotein transfer to LolA.

Cholesterol Alters Physical Properties of the Target Membrane to Facilitate Influenza Membrane Fusion at the Single-Particle Level

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including: initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. Because the same viral protein, hemagglutinin, is responsible for IAV binding and fusing to the host membrane, it has been difficult to investigate the role of cholesterol in the host lipid membrane on viral fusion without convolving the two processes. Here, we use synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles without binding hemagglutinin. To avoid potential issues with adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles, and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, while the rate of lipid mixing is independent of cholesterol composition. We also link the effect of cholesterol on IAV fusion to physical properties of the membrane.

Dengue virus reduces AGPAT1expression to alter phospholipids and enhance infection in Aedes aegypti.

More than half of the world population is at risk of dengue virus (DENV) infection because of the global distribution of its mosquito vectors. DENV is an envelope virus that relies on host lipid membranes for its life-cycle. Here, we characterized how DENV hijacks the mosquito lipidome to identify targets for novel transmission-blocking interventions. To describe metabolic changes throughout the mosquito DENV cycle, we deployed a Liquid chromatography-high resolution mass spectrometry (LC-HRMS) workflow including spectral similarity annotation in cells, midguts and whole mosquitoes at different times post infection. We revealed a major aminophospholipid reconfiguration with an overall early increase, followed by a reduction later in the cycle. We phylogenetically characterized acylglycerolphosphate acyltransferase (AGPAT) enzyme isoforms to identify those that catalyze a rate-limiting step in phospholipid biogenesis, the acylation of lysophosphatidate to phosphatidate. We showed that DENV infection decreased AGPAT1, but did not alter AGPAT2 expression in cells, midguts and mosquitoes. Depletion of either AGPAT1 or AGPAT2 increased aminophospholipids and partially recapitulated DENV-induced reconfiguration before infection in vitro. However, only AGPAT1 depletion promoted infection by maintaining high aminophospholipid concentrations. In mosquitoes, AGPAT1 depletion also partially recapitulated DENV-induced aminophospholipid increase before infection and enhanced infection by maintaining high aminophospholipid concentrations. These results indicate that DENV inhibition of AGPAT1 expression promotes infection by increasing aminophospholipids, as observed in the mosquito's early DENV cycle. Furthermore, in AGPAT1-depleted mosquitoes, we showed that enhanced infection was associated with increased consumption/redirection of aminophospholipids. Our study suggests that DENV regulates aminophospholipids, especially phosphatidylcholine and phosphatidylethanolamine, by inhibiting AGPAT1 expression to increase aminophospholipid availability for virus multiplication.

Coupling between Inclusions and Membranes at the Nanoscale.

The activity of cell membrane inclusions (such as ion channels) is influenced by the host lipid membrane, to which they are elastically coupled. This coupling concerns the hydrophobic thickness of the bilayer (imposed by the length of the channel, as per the hydrophobic matching principle) but also its slope at the boundary of the inclusion. However, this parameter has never been measured so far. We combine small-angle x-ray scattering data and a complete elastic model to measure the slope for the model gramicidin channel and show that it is surprisingly steep in two membrane systems with very different elastic properties. This conclusion is confirmed and generalized by the comparison with recent results in the simulation literature and with conductivity measurements.

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion.

Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. The RSV F protein comprises a trimer of disulfide-bonded F1 and F2 subunits that is present on the virion surface in a metastable prefusion state. This prefusion form is readily triggered to undergo refolding to bring two heptad repeats (heptad repeat A [HRA] and HRB) into close proximity to form a six-helix bundle that stabilizes the postfusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75 to 97), an amphipathic α-helix that lies at the interface of the prefusion F trimer and is a major structural feature of the F2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation, and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Here, we propose a model in which HRC plays a stabilizing role within the globular head for the prefusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the postfusion state.IMPORTANCE RSV is recognized as the most important viral pathogen among pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of prefusion stabilized F protein-based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here, we present a comprehensive mutagenesis-based study of a region of the RSV F2 subunit (amino acids 75 to 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the prefusion form.

The Middle Fragment of Helicobacter pylori CagA Induces Actin Rearrangement and Triggers Its Own Uptake into Gastric Epithelial Cells.

Cytotoxin-associated gene product A (CagA) is a major virulence factor secreted by Helicobacter pylori. CagA activity in the gastric epithelium is associated with higher risk of gastric cancer development. Bacterial type IV secretion system (T4SS)-mediated translocation of CagA into the cytosol of human epithelial cells occurs via a poorly understood mechanism that requires CagA interaction with the host membrane lipid phosphatidylserine (PS) and host cell receptor integrin α5β1. Here we have characterized the isolated recombinant middle fragment of CagA (CagA-M) that contains the positively-charged PS-binding region (aa 613–636) and a putative β1 integrin binding site, but lacks the EPIYA region, secretion signal peptide and the CagA multimerization motif. We show that CagA-M, when immobilized on latex beads, is capable of binding to, and triggering its own uptake into, gastric epithelial cells in the absence of infection with cagA-positive H. pylori. Using site-directed mutagenesis, fluorescent and electron microscopy, and highly-specific inhibitors, we demonstrate that the cell-binding and endocytosis-like internalization of CagA-M are dependent on (1) binding to PS; (2) β1 integrin activity; and (3) actin dynamics. Interaction of CagA-M with the host cells is accompanied by the development of long filopodia-like protrusions (macrospikes). This novel morphology is different from the hummingbird phenotype induced by the translocation of full-length CagA. The determinants within CagA-M and within the host that are important for endocytosis-like internalization into host cells are very similar to those observed for T4SS-mediated internalization of full-length CagA, suggesting that the latter may involve an endocytic pathway.

Model for a novel membrane envelope in a filamentous hyperthermophilic virus.

Biological membranes create compartments, and are usually formed by lipid bilayers. However, in hyperthermophilic archaea that live optimally at temperatures above 80°C the membranes are monolayers which resemble fused bilayers. Many double-stranded DNA viruses which parasitize such hosts, including the filamentous virus AFV1 of Acidianus hospitalis, are enveloped with a lipid-containing membrane. Using cryo-EM, we show that the membrane in AFV1 is a ~2 nm-thick monolayer, approximately half the expected membrane thickness, formed by host membrane-derived lipids which adopt a U-shaped 'horseshoe' conformation. We hypothesize that this unusual viral envelope structure results from the extreme curvature of the viral capsid, as 'horseshoe' lipid conformations favor such curvature and host membrane lipids that permit horseshoe conformations are selectively recruited into the viral envelope. The unusual envelope found in AFV1 also has many implications for biotechnology, since this membrane can survive the most aggressive conditions involving extremes of temperature and pH.

Azobenzene-Cholesterol as a Photoactivator in Biomimetic Membranes: 2. Membrane Structure

Properties of biomembranes are modified in presence of additives such as cholesterol (chol) or proteins. Azobenzene-cholesterol (azo-chol) is a photoactive variety of chol since its azo-headgroup exhibits a reversible change of the conformation upon illumination (365nm: trans to cis; 455nm: cis to trans). Grazing incidence X-ray diffraction (GIXD) was applied to resolve the lateral structure within biomimetic monolayers consisting of a 1,2-dipalmitoyl-sn-phosphatidyl-choline (DPPC) matrix with embedded azo-chol in either of the two conformational states. A pure DPPC monolayer and also a DPPC monolayer with inactive chol were used as control systems. The results report on the symmetry of the 2D-lattice and the inter-chain distance dxy. The tilt angle of the lipid chains was calculated from the lattice parameters and the geometrical boundary conditions. The lattice parameters are modified when azo-chol in the host membrane is in all-trans state as compared to the control systems. With all-cis azo-chol, the structure is broken. The findings show the capacity of azo-chol for switching the lateral structure of the host lipid membrane though photoactivation.

Azobenzene-Cholesterol as a Photoactivator in Biomimetic Membranes: 1. Lipid Dynamics

Properties of biomembranes are modified in presence of additives such as cholesterol (chol) or proteins. Azobenzene-cholesterol (azo-chol) is a photoactive variety of chol since its azo-headgroup exhibits a reversible change of the conformation upon illumination (365nm: trans to cis; 455nm: cis to trans). Quasielastic neutron scattering (QENS) was applied to resolve lipid dynamics within biomimetic membranes consisting of a 1-palmitoyl-2-oleoyl-sn-phosphatidyl-choline (POPC) matrix with embedded azo-chol in either of the two conformational states. A pure POPC membrane and also a POPC membrane with inactive chol were used as control systems. The results report on the fast intramolecular motion of the protons in the POPC chains, a slow hop diffusion and an even slower mode both of the whole POPC molecule. The intramolecular motion and the hop diffusion are modified by the presence of azo-chol in the host membrane as compared with the control systems, but do not depend on the isomerization state. The slowest mode is though sensitive to the isomerization. The findings show the capacity of azo-chol for tuning the dynamics and most probably also the structures of the host lipid membrane though photoactivation.

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L.

Coxiella burnetii is a gram-negative intracellular bacterium that forms a large, lysosome-like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol-binding protein-related protein 1 long (ORP1L) is a mammalian lipid-binding protein that plays a dual role in cholesterol-dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N-terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co-localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L-depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.