Host Phospholipid Research Articles

Antiphospholipid autoantibodies in Lyme disease arise after scavenging of host phospholipids by Borrelia burgdorferi.

A close association with its vertebrate and tick hosts allows Borrelia burgdorferi, the bacterium responsible for Lyme disease, to eliminate many metabolic pathways and instead scavenge key nutrients from the host. A lipid-defined culture medium was developed to demonstrate that exogenous lipids are an essential nutrient of B. burgdorferi, which can accumulate intact phospholipids from its environment to support growth. Antibody responses to host phospholipids were studied in mice and humans using an antiphospholipid ELISA. Several of these environmentally acquired phospholipids including phosphatidylserine and phosphatidic acid, as well as borrelial phosphatidylcholine, are the targets of antibodies that arose early in infection in the mouse model. Patients with acute infections demonstrated antibody responses to the same lipids. The elevation of antiphospholipid antibodies predicted early infection with better sensitivity than did the standardized 2-tier tests currently used in diagnosis. Sera obtained from patients with Lyme disease before and after antibiotic therapy showed declining antiphospholipid titers after treatment. Further study will be required to determine whether these antibodies have utility in early diagnosis of Lyme disease, tracking of the response to therapy, and diagnosis of reinfection, areas in which current standardized tests are inadequate.

Multifaceted membrane binding head of the SARS-CoV-2 spike protein.

The SARS-CoV-2 spike protein presents a surface with enormous membrane binding potential to host tissues and organelles of infected cells. Its exposed trimeric head binds not only the angiotensin-converting enzyme 2 (ACE2), but also host phospholipids which are missing from all existing structures. Hence, the membrane interaction surfaces that mediate viral fusion, entry, assembly and egress remain unclear. Here the spike:membrane docking sites are identified based on membrane optimal docking area (MODA) analysis of 3D structures of spike proteins in closed and open conformations at endocytic and neutral pH levels as well as ligand complexes. This reveals multiple membrane binding sites in the closed spike head that together prefer convex membranes and are modulated by pH, fatty acids and post-translational modifications including glycosylation. The exposure of the various membrane interaction sites adjusts upon domain repositioning within the trimer, allowing formation of intermediate bilayer complexes that lead to the prefusion state while also enabling ACE2 receptor recognition. In contrast, all antibodies that target the spike head would block the membrane docking process that precedes ACE2 recognition. Together this illuminates the engagements of the spike protein with plasma, endocytic, ER or exocytic vesicle membranes that help to drive the cycle of viral infection, and offers novel sites for intervention.

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

PE_PGRS3 ensures provision of the vital phospholipids cardiolipin and phosphatidylinositols by promoting the interaction between M. tuberculosis and host cells

ABSTRACT PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) constitute a large family of complex modular proteins whose role is still unclear. Among those, we have previously shown, using the heterologous expression in Mycobacterium smegmatis, that PE_PGRS3 containing a unique arginine-rich C-terminal domain, promotes adhesion to host cells. In this study, we investigate the role of PE_PGRS3 and its C-terminal domain directly in Mtb using functional deletion mutants. The results obtained here show that PE_PGRS3 is localized on the mycobacterial cell wall and its arginine-rich C-terminal region protrudes from the mycobacterial membrane and mediates Mtb entry into epithelial cells. Most importantly, this positively charged helical domain specifically binds phosphorylated phosphatidylinositols and cardiolipin, whereas it is unable to bind other phospholipids. Interestingly, administration of cardiolipin and phosphatidylinositol but no other phospholipids was able to turn-off expression of pe_pgrs3 activated by phosphate starvation conditions. These findings suggest that PE_PGRS3 has the key role to serve as a bridge between mycobacteria and host cells by interacting with specific host phospholipids and extracting them from host cells, for their direct integration or as a source of phosphate, during phases of TB pathogenesis when Mtb is short of phosphate supply.

Stimulated phospholipid synthesis is key for hepatitis B virus replications

Chronic hepatitis B Virus (HBV) infection has high morbidity, high pathogenicity and unclear pathogenesis. To elucidate the relationship between HBV replication and host phospholipid metabolites, we measured 10 classes of phospholipids in serum of HBV infected patients and cells using ultra performance liquid chromatograph-triple quadruple mass spectrometry. We found that the levels of phosphatidylcholine (PC), phosphatidylethanolamine, and lyso-phosphatidic acid were increased in HBsAg (+) serum of infected patients compared with HBsAg (−), while phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and sphingomyelin were decreased, which were confirmed in an HBV infected HepG2.2.15 cell line. We further evaluated the enzyme levels of PC pathways and found that PCYT1A and LPP1 for PC synthesis were up-regulated after HBV infection. Moreover, HBV replication was inhibited when PCYT1A and LPP1 were inhibited. These results indicated that the PC synthesis in HBV infected host are regulated by PCYT1A and LPP1, which suggests that PCYT1A, LPP1 could be new potential targets for HBV treatment.

An engineered mutant of a host phospholipid synthesis gene inhibits viral replication without compromising host fitness

Viral infections universally rely on numerous hijacked host factors to be successful. It is therefore possible to control viral infections by manipulating host factors that are critical for viral replication. Given that host genes may play essential roles in certain cellular processes, any successful manipulations for virus control should cause no or mild effects on host fitness. We previously showed that a group of positive-strand RNA viruses enrich phosphatidylcholine (PC) at the sites of viral replication. Specifically, brome mosaic virus (BMV) replication protein 1a interacts with and recruits a PC synthesis enzyme, phosphatidylethanolamine methyltransferase, Cho2p, to the viral replication sites that are assembled on the perinuclear endoplasmic reticulum (ER) membrane. Deletion of the CHO2 gene inhibited BMV replication by 5-fold; however, it slowed down host cell growth as well. Here, we show that an engineered Cho2p mutant supports general PC synthesis and normal cell growth but blocks BMV replication. This mutant interacts and colocalizes with BMV 1a but prevents BMV 1a from localizing to the perinuclear ER membrane. The mislocalized BMV 1a fails to induce the formation of viral replication complexes. Our study demonstrates an effective antiviral strategy in which a host lipid synthesis gene is engineered to control viral replication without comprising host growth.

Helminth Defense Molecules as Design Templates for Membrane Active Antibiotics.

A design template for membrane active antibiotics against microbial and tumor cells is described. The template is an amino acid sequence that combines the properties of helminth defense molecules, which are not cytolytic, with the properties of host-defense peptides, which disrupt microbial membranes. Like helminth defense molecules, the template folds into an amphipathic helix in both mammalian host and microbial phospholipid membranes. Unlike these molecules, the template exhibits antimicrobial and anticancer properties that are comparable to those of antimicrobial and anticancer antibiotics. The selective antibiotic activity of the template builds upon a functional synergy between three distinctive faces of the helix, which is in contrast to two faces of membrane-disrupting amphipathic structures. This synergy enables the template to adapt pore formation mechanisms according to the nature of the target membrane, inducing the lysis of microbial and tumor cells.

Therapeutic Targets in Chlamydial Fatty Acid and Phospholipid Synthesis.

Chlamydia trachomatis is an obligate intracellular pathogen with a reduced genome reflecting its host cell dependent life style. However, C. trachomatis has retained all of the genes required for fatty acid and phospholipid synthesis that are present in free-living bacteria. C. trachomatis assembles its cellular membrane using its own biosynthetic machinery utilizing glucose, isoleucine, and serine. This pathway produces disaturated phospholipid molecular species containing a branched-chain 15-carbon fatty acid in the 2-position, which are distinct from the structures of host phospholipids. The enoyl reductase step (FabI) is a target for antimicrobial drug discovery, and the developmental candidate, AFN-1252, blocks the activity of CtFabI. The x-ray crystal structure of the CtFabI•NADH•AFN-1252 ternary complex reveals the interactions between the drug, protein, and cofactor. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infection cycle reduces infectious titers, and treatment at the time of infection prevents the first cell division. Fatty acid synthesis is essential for C. trachomatis proliferation within its eukaryotic host, and CtFabI is a validated therapeutic target against C. trachomatis.

Matrix-glycoprotein interactions required for budding of a plant nucleorhabdovirus and induction of inner nuclear membrane invagination.

Nucleorhabdoviruses such as Sonchus yellow net virus (SYNV) replicate in the nuclei and undergo morphogenesis at the inner nuclear membrane (IM) in plant cells. Mature particles are presumed to form by budding of the Matrix (M) protein-nucleocapsid complexes through host IMs to acquire host phospholipids and the surface glycoproteins (G). To address mechanisms underlying nucleorhabdovirus budding, we generated recombinant SYNV G mutants containing a truncated amino-terminal (NT) or carboxyl-terminal (CT) domain. Electron microscopy and sucrose gradient centrifugation analyses showed that the CT domain is essential for virion morphogenesis whereas the NT domain is also required for efficient budding. SYNV infection induces IM invaginations that are thought to provide membrane sites for virus budding. We found that in the context of viral infections, interactions of the M protein with the CT domain of the membrane-anchored G protein mediate M protein translocation and IM invagination. Interestingly, tethering the M protein to endomembranes, either by co-expression with a transmembrane G protein CT domain or by artificial fusion with the G protein membrane targeting sequence, induces IM invagination in uninfected cells. Further evidence to support functions of G-M interactions in virus budding came from dominant negative effects on SYNV-induced IM invagination and viral infections that were elicited by expression of a soluble version of the G protein CT domain. Based on these data, we propose that cooperative G-M interactions promote efficient SYNV budding.

Both B-1a and B-1b cells exposed to Mycobacterium tuberculosis lipids differentiate into IgM antibody-secreting cells.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. The cellular immune response to mycobacteria has been characterized extensively, but the antibody response remains underexplored. The present study aimed to examine whether host or bacterial phospholipids induce secretion of IgM, and specifically anti-phospholipid IgM, antibodies by B cells and to identify the responsible B-cell subset. Here we show that peritoneal B cells responded to lipid antigens by secreting IgM antibodies. Specifically, stimulation with M. tuberculosis H37Rv total lipids resulted in significant induction of total and anti-phosphatidylcholine IgM. Similarly, IgM antibody production increased significantly with stimulation by whole Mycobacterium bovis bacillus Calmette-Guérin. The B-1 subset was the dominant source of IgM antibodies after exposure to cardiolipin. Both CD5+ B-1a and CD5- B-1b cell subsets secreted total IgM antibodies after exposure to M. tuberculosis H37Rv total lipids in vitro. Overall, our results suggest that the poly-reactive B-1 cell repertoire contributes to non-specific anti-phospholipid IgM antibody secretion in response to M. tuberculosis lipids.

Host-based lipid inflammation drives pathogenesis in Francisella infection

Mass spectrometry imaging (MSI) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host-pathogen interface during a septic bacterial infection. Using Francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. Here, we demonstrate detection and distribution of endotoxin in a lethal murine F. novicida infection model, in addition to determining the temporally and spatially resolved innate lipid inflammatory response in both 2D and 3D renderings using MSI. Further, we show that the cyclooxygenase-2-dependent lipid inflammatory pathway is responsible for lethality in F. novicida infection due to overproduction of proinflammatory effectors including prostaglandin E2. The results of this study emphasize that spatial determination of the host lipid components of the immune response is crucial to identifying novel strategies to effectively address highly pathogenic and lethal infections stemming from bacterial, fungal, and viral origins.

Chlamydia trachomatis utilizes the mammalian CLA1 lipid transporter to acquire host phosphatidylcholine essential for growth.

Phosphatidylcholine is a constituent of Chlamydia trachomatis membranes that must be acquired from its mammalian host to support bacterial proliferation. The CLA1 (SR-B1) receptor is a bi-directional phosphatidylcholine/cholesterol transporter that is recruited to the inclusion of Chlamydia-infected cells along with ABCA1. C. trachomatis growth was inhibited in a dose-dependent manner by BLT-1, a selective inhibitor of CLA1 function. Expression of a BLT-1-insensitive CLA1(C384S) mutant ameliorated the effect of the drug on chlamydial growth. CLA1 knockdown using shRNAs corroborated an important role for CLA1 in the growth of C. trachomatis. Trafficking of a fluorescent phosphatidylcholine analogue to Chlamydia was blocked by the inhibition of CLA1 or ABCA1 function, indicating a critical role for these transporters in phosphatidylcholine acquisition by this organism. Our analyses using a dual-labelled fluorescent phosphatidylcholine analogue and mass spectrometry showed that the phosphatidylcholine associated with isolated Chlamydia was unmodified host phosphatidylcholine. These results indicate that C. trachomatis co-opts host phospholipid transporters normally used to assemble lipoproteins to acquire host phosphatidylcholine essential for growth.

Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome and is thought to rely on its mammalian host cell for nutrients. Although several lines of evidence suggest C. trachomatis utilizes host phospholipids, the bacterium encodes all the genes necessary for fatty acid and phospholipid synthesis found in free living Gram-negative bacteria. Bacterially derived phospholipids significantly increased in infected HeLa cell cultures. These new phospholipids had a distinct molecular species composition consisting of saturated and branched-chain fatty acids. Biochemical analysis established the role of C. trachomatis-encoded acyltransferases in producing the new disaturated molecular species. There was no evidence for the remodeling of host phospholipids and no change in the size or molecular species composition of the phosphatidylcholine pool in infected HeLa cells. Host sphingomyelin was associated with C. trachomatis isolated by detergent extraction, but it may represent contamination with detergent-insoluble host lipids rather than being an integral bacterial membrane component. C. trachomatis assembles its membrane systems from the unique phospholipid molecular species produced by its own fatty acid and phospholipid biosynthetic machinery utilizing glucose, isoleucine, and serine.

The role of a phospholipase (PLD) in virulence of Purpureocillium lilacinum (Paecilomyces lilacinum)

Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, understanding the role of phospholipases on the virulence of the filamentous fungus Purpureocillium lilacinum has been still rather limited. In this study, pld gene was characterized. It encodes the protein phospholipase D (PLD) in P. lilacinum. This gene, 3303 bp open reading frame fragment (ORF), encodes a protein of 1100 amino acids with high similarity to the same gene from Penicillium oxalicum and Aspergillus fumigatus. Secondary structure prediction showed two PLD phosphodiesterase domains (437–464 bp and 885–912 bp). The pld gene was significantly regulated during infection of Meloidogyne incognita eggs by P. lilacinum. The expression of pld gene using RT-PCR was the highest at 36 and 48 h, which introduce evidence that the presence of M. incognita may induce the expression of the pld gene in P. lilacinum. In addition, maltose and l-alanine were found to increase the expression of pld gene. An acidic environment (pH 3.0–4.0) and moderate temperatures (27–29 °C) are favorable for pld expression in P. lilacinum.

Dispersing single-walled carbon nanotubes using common phospholipids with a small amount of polyethylene glycol-phospholipid additives

Nearly 60% of the single-walled carbon nanotubes (SWCNTs) in a sample were rendered dispersible in water using common double-chain phospholipids, such as phosphatidylcholine, with a small amount of polyethylene glycol (PEG)-derivatized phospholipid. The dispersion isotherms were measured versus the PEG-phospholipid concentration in various host phospholipids. In every case, the isotherms followed a step-shaped curve characterized by the critical dispersion concentration (CDC) and the saturated dispersed amount (SDA). The CDC remains nearly constant (5μmol/L) and independent of the type and concentration of the host phospholipid. The SDA increases as the alkyl chains of the host phosphatidylcholine decrease in length. The most efficient result is obtained with a glycerol-phospholipid at a 10nmol/L CDC. The dispersion mechanism does not involve SWCNT encapsulation by the vesicles; instead, dispersion occurs by co-adsorbed phospholipids aggregating on the surface.

Characterization of the GbdR regulon in Pseudomonas aeruginosa.

Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa's response to the host, particularly changes regulated by the host-derived quaternary amines choline and glycine betaine (GB). We previously identified GbdR as an AraC family transcription factor that directly regulates choline acquisition from host phospholipids (via binding to plcH and pchP promoters), is required for catabolism of the choline metabolite GB, and is an activator that induces transcription in response to GB or dimethylglycine. Our goal was to characterize the GbdR regulon in P. aeruginosa by using genetics and chemical biology in combination with transcriptomics and in vitro DNA-binding assays. Here we show that GbdR activation regulates transcription of 26 genes from 12 promoters, 11 of which have measureable binding to GbdR in vitro. The GbdR regulon includes the genes encoding GB, dimethylglycine, sarcosine, glycine, and serine catabolic enzymes and the BetX and CbcXWV quaternary amine transport proteins. We characterized the GbdR consensus binding site and used it to identify that the recently characterized acetylcholine esterase gene, choE (PA4921), is also regulated by GbdR. The regulon member not directly controlled by GbdR is the secreted lipase gene lipA, which was also the only regulon member repressed under GbdR-activating conditions. Determination of the GbdR regulon provides deeper understanding of how GbdR links bacterial metabolism and virulence. Additionally, identification of two uncharacterized regulon members suggests roles for these proteins in response to choline metabolites.

Molecular Dynamics Simulations of Depth Distribution of Spin-Labeled Phospholipids within Lipid Bilayer

Spin-labeled lipids are commonly used as fluorescence quenchers in studies of membrane penetration of dye-labeled proteins and peptides using depth-dependent quenching. Accurate calculations of depth of the fluorophore rely on the use of several spin labels placed in the membrane at various positions. The depth of the quenchers (spin probes) has to be determined independently; however, experimental determination of transverse distributions of spin probe depths is difficult. In this Article, we use molecular dynamics (MD) simulations to study the membrane behavior and depth distributions of spin-labeled phospholipids in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. To probe different depths within the bilayer, a series containing five Doxyl-labeled lipids (n-Doxyl PC) has been studied, in which a spin moiety was covalently attached to nth carbon atoms (where n = 5, 7, 10, 12, and 14) of the sn-2 stearoyl chain of the host phospholipid. Our results demonstrate that the chain-attached spin labels are broadly distributed across the model membrane and their environment is characterized by a high degree of mobility and structural heterogeneity. Despite the high thermal disorder, the depth distributions of the Doxyl labels were found to correlate well with their attachment positions, indicating that the distribution of the spin label within the model membrane is dictated by the depth of the nth lipid carbon atom and not by intrinsic properties of the label. In contrast, a much broader and heterogeneous distribution was observed for a headgroup-attached Tempo spin label of Tempo-PC lipids. MD simulations reveal that, due to the hydrophobic nature, a Tempo moiety favors partitioning from the headgroup region deeper into the membrane. Depending on the concentration of Tempo-PC lipids, the probable depth of the Tempo moiety could span a range from 14.4 to 18.2 Å from the membrane center. Comparison of the MD-estimated immersion depths of Tempo and n-Doxyl labels with their suggested experimental depth positions allows us to review critically the possible sources of error in depth-dependent fluorescence quenching studies.

Phospholipid catabolism by gut microbiota and the risk of cardiovascular disease

Phospholipid catabolism by gut microbiota and the risk of cardiovascular disease

Identification of Two Legionella pneumophila Effectors that Manipulate Host Phospholipids Biosynthesis

The intracellular pathogen Legionella pneumophila translocates a large number of effector proteins into host cells via the Icm/Dot type-IVB secretion system. Some of these effectors were shown to cause lethal effect on yeast growth. Here we characterized one such effector (LecE) and identified yeast suppressors that reduced its lethal effect. The LecE lethal effect was found to be suppressed by the over expression of the yeast protein Dgk1 a diacylglycerol (DAG) kinase enzyme and by a deletion of the gene encoding for Pah1 a phosphatidic acid (PA) phosphatase that counteracts the activity of Dgk1. Genetic analysis using yeast deletion mutants, strains expressing relevant yeast genes and point mutations constructed in the Dgk1 and Pah1 conserved domains indicated that LecE functions similarly to the Nem1-Spo7 phosphatase complex that activates Pah1 in yeast. In addition, by using relevant yeast genetic backgrounds we examined several L. pneumophila effectors expected to be involved in phospholipids biosynthesis and identified an effector (LpdA) that contains a phospholipase-D (PLD) domain which caused lethal effect only in a dgk1 deletion mutant of yeast. Additionally, LpdA was found to enhance the lethal effect of LecE in yeast cells, a phenomenon which was found to be dependent on its PLD activity. Furthermore, to determine whether LecE and LpdA affect the levels or distribution of DAG and PA in-vivo in mammalian cells, we utilized fluorescent DAG and PA biosensors and validated the notion that LecE and LpdA affect the in-vivo levels and distribution of DAG and PA, respectively. Finally, we examined the intracellular localization of both LecE and LpdA in human macrophages during L. pneumophila infection and found that both effectors are localized to the bacterial phagosome. Our results suggest that L. pneumophila utilize at least two effectors to manipulate important steps in phospholipids biosynthesis.

Temperate membrane-containing halophilic archaeal virus SNJ1 has a circular dsDNA genome identical to that of plasmid pHH205

A temperate haloarchaeal virus, SNJ1, was induced from the lysogenic host, Natrinema sp. J7-1, with mitomycin C, and the virus produced plaques on lawns of Natrinema sp. J7-2. Optimization of the induction conditions allowed us to increase the titer from ∼104PFU/ml to ∼1011PFU/ml. Single-step growth curves exhibited a burst size of ∼100PFU/cell. The genome of SNJ1 was observed to be a circular, double-stranded DNA (dsDNA) molecule (16,341bp). Surprisingly, the sequence of SNJ1 was identical to that of a previously described plasmid, pHH205, indicating that this plasmid is the provirus of SNJ1. Several structural protein-encoding genes were identified in the viral genome. In addition, the comparison of putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses, as well as the presence of lipid constituents from the host phospholipid pool, strongly suggest that SNJ1 belongs to the PRD1-type lineage of dsDNA viruses, which have an internal membrane.