The Anticancer Drug Bleomycin Shows Potent Antifungal Activity by Altering Phospholipid Biosynthesis.

Invasive fungal infections are difficult to treat with limited drug options, mainly because fungi are eukaryotes and share many cellular mechanisms with the human host. Most current antifungal drugs are either fungistatic or highly toxic. Therefore, there is a critical need to identify important fungal specific drug targets for novel antifungal development. Numerous studies have shown the fungal phosphatidylserine (PS) biosynthetic pathway to be a potential target. It is synthesized from CDP-diacylglycerol and serine, and the fungal PS synthesis route is different from that in mammalian cells, in which preexisting phospholipids are utilized to produce PS in a base-exchange reaction. In this study, we utilized a Saccharomyces cerevisiae heterologous expression system to screen for inhibitors of Cryptococcus PS synthase Cho1, a fungi-specific enzyme essential for cell viability. We identified an anticancer compound, bleomycin, as a positive candidate that showed a phospholipid-dependent antifungal effect. Its inhibition on fungal growth can be restored by ethanolamine supplementation. Further exploration of the mechanism of action showed that bleomycin treatment damaged the mitochondrial membrane in yeast cells, leading to increased generation of reactive oxygen species (ROS), whereas supplementation with ethanolamine helped to rescue bleomycin-induced damage. Our results indicate that bleomycin does not specifically inhibit the PS synthase enzyme; however, it may affect phospholipid biosynthesis through disruption of mitochondrial function, namely, the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which helps cells maintain membrane composition and functionality. IMPORTANCE Invasive fungal pathogens cause significant morbidity and mortality, with over 1.5 million deaths annually. Because fungi are eukaryotes that share much of their cellular machinery with the host, our armamentarium of antifungal drugs is highly limited, with only three classes of antifungal drugs available. Drug toxicity and emerging resistance have limited their use. Hence, targeting fungi-specific enzymes that are important for fungal survival, growth, or virulence poses a strategy for novel antifungal development. In this study, we developed a heterologous expression system to screen for chemical compounds with activity against Cryptococcus phosphatidylserine synthase, Cho1, a fungi-specific enzyme that is essential for viability in C. neoformans. We confirmed the feasibility of this screen method and identified a previously unexplored role of the anticancer compound bleomycin in disrupting mitochondrial function and inhibiting phospholipid synthesis.

Defining ER-mitochondria contact dynamics in Plasmodium falciparum by targeting component of phospholipid synthesis pathway, phosphatidylserine synthase (PfPSS).

The malaria parasite completes the asexual cycle inside the host erythrocyte, which requires extensive membrane biogenesis for its development and multiplication. Metabolic pathways for the synthesis of membrane phospholipids (PL), including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS), are crucial for parasite survival. Here, we have studied the P. falciparum enzyme responsible for PS synthesis, Phosphatidylserine synthase (PfPSS), GFP targeting approach confirmed it to be localized in the parasite ER as well as in ER-protrusions. Detailed high resolution microscopy, using these transgenic parasites expressing PfPSS-GFP, redefined the dynamics of ER during the intraerythrocytic life cycle and its association with the mitochondria. We report for the first time presence of ER-mitochondria contact (ERMC) in Plasmodium; ERMC is formed by PfPSS containing ER-protrusions, which associate with the mitochondria surface throughout the parasite growth cycle. Further, ERMC is found to be stable and refractory to ER and mitochondrial stresses, suggesting that it is formed through strong tethering complexes. PfPSS was found to interact with other major key enzyme involved in PL synthesis, choline/Etn-phosphotransferase (CEPT), which suggest that ER is the major site for PL biosynthesis. Overall, this study defines the morphological organisation of ERMC which mediates PL synthesis/transport in the Plasmodium.

Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases.

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the "Kennedy pathway" is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.

Mapping the Substrate-Binding Sites in the Phosphatidylserine Synthase in Candida albicans.

The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the CHO1 gene, is a potential drug target for pathogenic fungi, such as Candida albicans. However, both substrate-binding sites of C. albicans Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif, which is present within Cho1. We tested the CAPT motif for its role in PS synthesis by mutating conserved residues using alanine substitution mutagenesis. PS synthase assays revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1 function. In contrast, there were no clear motifs in Cho1 for binding serine. Therefore, to identify the serine binding site, PS synthase sequences from three fungi were aligned with sequences of a similar enzyme, phosphatidylinositol (PI) synthase, from the same fungi. This revealed a motif that was unique to PS synthases. Using alanine substitution mutagenesis, we found that some of the residues in this motif are required for Cho1 function. Two alanine substitution mutants, L184A and R189A, exhibited contrasting impacts on PS synthase activity, and were characterized for their Michaelis-Menten kinetics. The L184A mutant displayed enhanced PS synthase activity and showed an increased Vmax. In contrast, R189A showed decreased PS synthase activity and increased Km for serine, suggesting that residue R189 is involved in serine binding. These results help to characterize PS synthase substrate binding, and should direct rational approaches for finding Cho1 inhibitors that may lead to better antifungals.

Phosphatidylserine flux into mitochondria unveiled by organelle-targeted Escherichia coli phosphatidylserine synthase PssA.

Most phospholipids are synthesised in the endoplasmic reticulum and distributed to other cellular membranes. Although the vesicle transport contributes to the phospholipid distribution among the endomembrane system, exactly how phospholipids are transported to, from and between mitochondrial membranes remains unclear. To gain insights into phospholipid transport routes into mitochondria, we expressed the Escherichia coli phosphatidylserine (PS) synthase PssA in various membrane compartments with distinct membrane topologies in yeast cells lacking a sole PS synthase (Cho1). Interestingly, PssA could complement loss of Cho1 when targeted to the endoplasmic reticulum (ER), peroxisome, or lipid droplet membranes. Synthesised PS could be converted to phosphatidylethanolamine (PE) by Psd1, the mitochondrial PS decarboxylase, suggesting that phospholipids synthesised in the peroxisomes and low doses (LDs) can efficiently reach mitochondria. Furthermore, we found that PssA which has been integrated into the mitochondrial inner membrane (MIM) from the matrix side could partially complement the loss of Cho1. The PS synthesised in the MIM was also converted to PE, indicating that PS flops across the MIM to become PE. These findings expand our understanding of the intracellular phospholipid transport routes via mitochondria.

Phosphatidylserine Synthase from Salicornia europaea Is Involved in Plant Salt Tolerance by Regulating Plasma Membrane Stability.

Salinity-induced lipid alterations have been reported in many plant species; however, how lipid biosynthesis and metabolism are regulated and how lipids work in plant salt tolerance are much less studied. Here, a constitutively much higher phosphatidylserine (PS) content in the plasma membrane (PM) was found in the euhalophyte Salicornia europaea than in Arabidopsis. A gene encoding PS synthase (PSS) was subsequently isolated from S. europaea, named SePSS, which was induced by salinity. Multiple alignments and phylogenetic analysis suggested that SePSS belongs to a base exchange-type PSS, which localises to the endoplasmic reticulum. Knockdown of SePSS in S. europaea suspension cells resulted in reduced PS content, decreased cell survival rate, and increased PM depolarization and K+ efflux under 400 or 800 mM NaCl. By contrast, the upregulation of SePSS leads to increased PS and phosphatidylethanolamine levels and enhanced salt tolerance in Arabidopsis, along with a lower accumulation of reactive oxygen species, less membrane injury, less PM depolarization and higher K+/Na+ in the transgenic lines than in wild-type (WT). These results suggest a positive correlation between PS levels and plant salt tolerance, and that SePSS participates in plant salt tolerance by regulating PS levels, hence PM potential and permeability, which help maintain ion homeostasis. Our work provides a potential strategy for improving plant growth under multiple stresses.

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea.

The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination.IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

Characterization of a Phosphatidylserine Synthase of Vibrio parahaemolyticus.

Phosphatidylserine synthase (Pss) is involved in the metabolic pathway in phospholipid synthesis in different organisms. In this study, Pss expression in Vibrio parahaemolyticus was verified through liquid chromatography tandem-mass spectrometry. To analyze the characteristics of Pss, the recombinant Pss was overexpressed and purified from Escherichia coli. The optimum temperature and pH of Pss were 40 °C and 8, respectively. When reacting with divalent metal, Pss activity decreased. In addition, Pss could not only use cytidine diphosphate diacylglycerol (CDP-DAG, 16:0), but also CDP-DAG (18:1) as a substrate to produce cytidine 5'-monophosphate. Furthermore, the 3D structure of Pss was predicted, and the results revealed that histidine and lysine of the two HKD motifs were present in the catalytic site. Moreover, CDP-DAG (16:0) was docked with the Pss model. To investigate whether the two HKD motifs in Pss are important to its activity, site-directed mutagenesis of histidine was performed. The results revealed that the activities of both H131A and H352A were diminished. Little is known regarding the catalytic site of type I Pss. This is the first report on the biochemical characterization of Pss in V. parahaemolyticus.

Engineering yeast phospholipid metabolism for de novo oleoylethanolamide production.

Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l-1 and a yield on glucose of 405.8 µg g-1. Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.

Phosphatidylserine synthase regulates cellular homeostasis through distinct metabolic mechanisms.

Phosphatidylserine (PS), synthesized in the endoplasmic reticulum (ER) by phosphatidylserine synthase (PSS), is transported to the plasma membrane (PM) and mitochondria through distinct routes. The in vivo functions of PS at different subcellular locations and the coordination between different PS transport routes are not fully understood. Here, we report that Drosophila PSS regulates cell growth, lipid storage and mitochondrial function. In pss RNAi, reduced PS depletes plasma membrane Akt, contributing to cell growth defects; the metabolic shift from phospholipid synthesis to neutral lipid synthesis results in ectopic lipid accumulation; and the reduction of mitochondrial PS impairs mitochondrial protein import and mitochondrial integrity. Importantly, reducing PS transport from the ER to PM by loss of PI4KIIIα partially rescues the mitochondrial defects of pss RNAi. Together, our results uncover a balance between different PS transport routes and reveal that PSS regulates cellular homeostasis through distinct metabolic mechanisms.

Phosphatidylserine synthesis is essential for viability of the human fungal pathogen Cryptococcus neoformans.

Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs.

Disruption of Phosphatidylserine Synthesis or Trafficking Reduces Infectivity of Ebola Virus.

The outer leaflet of the viral membrane of Ebola virus (EBOV) virions is enriched with phosphatidylserine (PtdSer), which is thought to play a central role in viral tropism, entry, and virus-associated immune evasion. We investigated the effects of inhibiting synthesis and/or export of PtdSer to the cell surface of infected cells on viral infectivity. Knockdown of both PtdSer synthase enzymes, PTDSS1 and PTDSS2, effectively decreased viral production. Decreased PtdSer expression resulted in an accumulation of virions at the plasma membrane and adjacent of intracellular organelles, suggesting that virion budding is impaired. The addition of inhibitors that block normal cellular trafficking of PtdSer to the plasma membrane resulted in a similar accumulation of virions and reduced viral replication. These findings demonstrate that plasma membrane-associated PtdSer is required for efficient EBOV budding, increasing EBOV infectivity, and could constitute a potential therapeutic target for the development of future countermeasures against EBOV.

Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis.

The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis.

Role of phosphatidylserine synthase in shaping the phospholipidome of Candida albicans.

Phosphatidylserine (PS) synthase (Cho1p) and the PS decarboxylase enzymes (Psd1p and Psd2p), which synthesize PS and phosphatidylethanolamine (PE), respectively, are crucial for Candida albicans virulence. Mutations that disrupt these enzymes compromise virulence. These enzymes are part of the cytidine diphosphate-diacylglycerol pathway (i.e. de novo pathway) for phospholipid synthesis. Understanding how losses of PS and/or PE synthesis pathways affect the phospholipidome of Candida is important for fully understanding how these enzymes impact virulence. The cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutations cause similar changes in levels of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol and PS. However, only slight changes were seen in PE and phosphatidylcholine (PC). This finding suggests that the alternative mechanism for making PE and PC, the Kennedy pathway, can compensate for loss of the de novo synthesis pathway. Candida albicans Cho1p, the lipid biosynthetic enzyme with the most potential as a drug target, has been biochemically characterized, and analysis of its substrate specificity and kinetics reveal that these are similar to those previously published for Saccharomyces cerevisiae Cho1p.

Phosphorylation of lipid metabolic enzymes by yeast protein kinase C requires phosphatidylserine and diacylglycerol.

Protein kinase C in Saccharomyces cerevisiae, i.e., Pkc1, is an enzyme that plays an important role in signal transduction and the regulation of lipid metabolic enzymes. Pkc1 is structurally similar to its counterparts in higher eukaryotes, but its requirement of phosphatidylserine (PS) and diacylglycerol (DAG) for catalytic activity has been unclear. In this work, we examined the role of these lipids in Pkc1 activity with protein and peptide substrates. In agreement with previous findings, yeast Pkc1 did not require PS and DAG for its activity on the peptide substrates derived from lipid metabolic proteins such as Pah1 [phosphatidate (PA) phosphatase], Nem1 (PA phosphatase phosphatase), and Spo7 (protein phosphatase regulatory subunit). However, the lipids were required for Pkc1 activity on the protein substrates Pah1, Nem1, and Spo7. Compared with DAG, PS had a greater effect on Pkc1 activity, and its dose-dependent interaction with the protein kinase was shown by the liposome binding assay. The Pkc1-mediated degradation of Pah1 was attenuated in the cho1Δ mutant, which is deficient in PS synthase, supporting the notion that the phospholipid regulates Pkc1 activity in vivo.

Phosphatidylserine synthesis at membrane contact sites promotes its transport out of the ER.

Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments.

SB-224289 Antagonizes the Antifungal Mechanism of the Marine Depsipeptide Papuamide A.

In order to expand the repertoire of antifungal compounds a novel, high-throughput phenotypic drug screen targeting fungal phosphatidylserine (PS) synthase (Cho1p) was developed based on antagonism of the toxin papuamide A (Pap-A). Pap-A is a cyclic depsipeptide that binds to PS in the membrane of wild-type Candida albicans, and permeabilizes its plasma membrane, ultimately causing cell death. Organisms with a homozygous deletion of the CHO1 gene (cho1ΔΔ) do not produce PS and are able to survive in the presence of Pap-A. Using this phenotype (i.e. resistance to Pap-A) as an indicator of Cho1p inhibition, we screened over 5,600 small molecules for Pap-A resistance and identified SB-224289 as a positive hit. SB-224289, previously reported as a selective human 5-HT1B receptor antagonist, also confers resistance to the similar toxin theopapuamide (TPap-A), but not to other cytotoxic depsipeptides tested. Structurally similar molecules and truncated variants of SB-224289 do not confer resistance to Pap-A, suggesting that the toxin-blocking ability of SB-224289 is very specific. Further biochemical characterization revealed that SB-224289 does not inhibit Cho1p, indicating that Pap-A resistance is conferred by another undetermined mechanism. Although the mode of resistance is unclear, interaction between SB-224289 and Pap-A or TPap-A suggests this screening assay could be adapted for discovering other compounds which could antagonize the effects of other environmentally- or medically-relevant depsipeptide toxins.

Phosphatidylserine Synthase Controls Cell Elongation Especially in the Uppermost Internode in Rice by Regulation of Exocytosis.

The uppermost internode is one of the fastest elongating organs in rice, and is expected to require an adequate supply of cell-wall materials and enzymes to the cell surface to enhance mechanical strength. Although it has been reported that the phenotype of shortened uppermost internode 1 (sui1) is caused by mutations in PHOSPHATIDYLSERINE SYNTHASE (OsPSS), the underlying mechanism remains unclear. Here we show that the OsPSS-1, as a gene expressed predominantly in elongating cells, regulates post-Golgi vesicle secretion to intercellular spaces. Mutation of OsPSS-1 leads to compromised delivery of CESA4 and secGFP towards the cell surface, resulting in weakened intercellular adhesion and disorganized cell arrangement in parenchyma. The phenotype of sui1-4 is caused largely by the reduction in cellulose contents in the whole plant and detrimental delivery of pectins in the uppermost internode. We found that OsPSS-1 and its potential product PS (phosphatidylserine) localized to organelles associated with exocytosis. These results together suggest that OsPSS-1 plays a potential role in mediating cell expansion by regulating secretion of cell wall components.

Molecular mechanism of dietary phospholipid requirement of Atlantic salmon, Salmo salar, fry.

The phospholipid (PL) requirement in fish is revealed by enhanced performance when larvae are provided PL-enriched diets. To elucidate the molecular mechanism underlying PL requirement in Atlantic salmon, Salmo salar, were fed a minimal PL diet and tissue samples from major lipid metabolic sites were dissected from fry and parr. In silico analysis and cloning techniques demonstrated that salmon possess a full set of enzymes for the endogenous production of PL. The gene expression data indicated that major PL biosynthetic genes of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylinositol (PtdIns) display lower expression in intestine during the early developmental stage (fry). This is consistent with the hypothesis that the intestine of salmon is immature at the early developmental stage with limited capacity for endogenous PL biosynthesis. The results also indicate that intact PtdCho, PtdEtn and PtdIns are required in the diet at this stage. PtdCho and sphingomyelin constitute the predominant PL in chylomicrons, involved in the transport of dietary lipids from the intestine to the rest of the body. As sphingomyelin can be produced from PtdCho in intestine of fry, our findings suggest that supplementation of dietary PtdCho alone during early developmental stages of Atlantic salmon would be sufficient to promote chylomicron formation. This would support efficient transport of dietary lipids, including PL precursors, from the intestine to the liver where biosynthesis of PtdEtn, PtdSer, and PtdIns is not compromised as in intestine facilitating efficient utilisation of dietary energy and the endogenous production of membrane PL for the rapidly growing and developing animal.

Lipid Biosynthetic Genes Affect Candida albicans Extracellular Vesicle Morphology, Cargo, and Immunostimulatory Properties.

Microbial secretion is integral for regulating cell homeostasis as well as releasing virulence factors during infection. The genes encoding phosphatidylserine synthase (CHO1) and phosphatidylserine decarboxylase (PSD1 and PSD2) are Candida albicans genes involved in phospholipid biosynthesis, and mutations in these genes affect mitochondrial function, cell wall thickness, and virulence in mice. We tested the roles of these genes in several agar-based secretion assays and observed that the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ strains manifested less protease and phospholipase activity. Since extracellular vesicles (EVs) are surrounded by a lipid membrane, we investigated the effects of these mutations on EV structure, composition, and biological activity. The cho1Δ/Δ mutant releases EVs comparable in size to wild-type EVs, but EVs from the psd1Δ/Δ psd2Δ/Δ strain are much larger than those from the wild type, including a population of >100-nm EVs not observed in the EVs from the wild type. Proteomic analysis revealed that EVs from both mutants had a significantly different protein cargo than that of EVs from the wild type. EVs were tested for their ability to activate NF-κB in bone marrow-derived macrophage cells. While wild-type and psd1Δ/Δ psd2Δ/Δ mutant-derived EVs activated NF-κB, the cho1Δ/Δ mutant-derived EV did not. These studies indicate that the presence and absence of these C. albicans genes have qualitative and quantitative effects on EV size, composition, and immunostimulatory phenotypes that highlight a complex interplay between lipid metabolism and vesicle production.