Protein-Mediated Changes in Membrane Fluidity and Ordering: Insights into the Molecular Mechanism and Implications for Cellular Function.

Probing protein-membrane interactions is vital for understanding biological functionality for various applications such as drug development, targeted drug delivery, and creation of functional biomaterials for medical and industrial purposes. In this study, we have investigated interaction of Human Serum Albumin (HSA) with two different lipids, dipalmitoylphosphatidylglycerol (dDPPG) and dipalmitoylphosphatidylcholine (dDPPC), using Vibrational Sum Frequency Generation spectroscopy at different membrane fluidity values. In the liquid-expanded (LE) state of the lipid, HSA (at pH 3.5) deeply intercalated lipid chains through a combination of electrostatic and hydrophobic interactions, which resulted in more ordering of the lipid chains. However, in the liquid-condensed (LC) state, protein intercalation is decreased due to tighter lipid packing. Moreover, our findings revealed distinct differences in HSA's interaction with dDPPG and dDPPC lipids. The interaction with dDPPC remained relatively weak compared to dDPPG. These results shed light on the significance of protein mediated changes in lipid characteristics, which hold considerable implications for understanding membrane protein behavior, lipid-mediated cellular processes, and lipid-based biomaterial design.

Biosynthesis of phosphatidylglycerol in photosynthetic organisms.

Phosphatidylglycerol (PG) is a unique phospholipid class with its indispensable role in photosynthesis and growth in land plants, algae, and cyanobacteria. PG is the only major phospholipid in the thylakoid membrane of cyanobacteria and plant chloroplasts and a main lipid component in photosynthetic protein-cofactor complexes such as photosystem I and photosystem II. In plants and algae, PG is also essential as a substrate for the biosynthesis of cardiolipin, which is a unique lipid present only in mitochondrial membranes and crucial for the functions of mitochondria. PG biosynthesis pathways in plants include three membranous organelles, plastids, mitochondria, and the endoplasmic reticulum in a complex manner. While the molecular biology underlying the role of PG in photosynthetic functions is well established, many enzymes responsible for the PG biosynthesis are only recently cloned and functionally characterized in the model plant species including Arabidopsis thaliana and Chlamydomonas reinhardtii and cyanobacteria such as Synechocystis sp. PCC 6803. The characterization of those enzymes helps understand not only the metabolic flow for PG production but also the crosstalk of biosynthesis pathways between PG and other lipids. This review aims to summarize recent advances in the understanding of the PG biosynthesis pathway and functions of involved enzymes.

The patatin-like phospholipase PfPNPLA2 is involved in the mitochondrial degradation of phosphatidylglycerol during Plasmodium falciparum blood stage development.

Plasmodium falciparum is an Apicomplexa responsible for human malaria, a major disease causing more than ½ million deaths every year, against which there is no fully efficient vaccine. The current rapid emergence of drug resistances emphasizes the need to identify novel drug targets. Increasing evidences show that lipid synthesis and trafficking are essential for parasite survival and pathogenesis, and that these pathways represent potential points of attack. Large amounts of phospholipids are needed for the generation of membrane compartments for newly divided parasites in the host cell. Parasite membrane homeostasis is achieved by an essential combination of parasite de novo lipid synthesis/recycling and massive host lipid scavenging. Latest data suggest that the mobilization and channeling of lipid resources is key for asexual parasite survival within the host red blood cell, but the molecular actors allowing lipid acquisition are poorly characterized. Enzymes remodeling lipids such as phospholipases are likely involved in these mechanisms. P. falciparum possesses an unusually large set of phospholipases, whose functions are largely unknown. Here we focused on the putative patatin-like phospholipase PfPNPLA2, for which we generated an glmS-inducible knockdown line and investigated its role during blood stages malaria. Disruption of the mitochondrial PfPNPLA2 in the asexual blood stages affected mitochondrial morphology and further induced a significant defect in parasite replication and survival, in particular under low host lipid availability. Lipidomic analyses revealed that PfPNPLA2 specifically degrades the parasite membrane lipid phosphatidylglycerol to generate lysobisphosphatidic acid. PfPNPLA2 knockdown further resulted in an increased host lipid scavenging accumulating in the form of storage lipids and free fatty acids. These results suggest that PfPNPLA2 is involved in the recycling of parasite phosphatidylglycerol to sustain optimal intraerythrocytic development when the host resources are scarce. This work strengthens our understanding of the complex lipid homeostasis pathways to acquire lipids and allow asexual parasite survival.

New roles of LPGAT1: From mitochondrial import of phosphatidylglycerol to MEGDEL disease.

Dysregulation of mitochondrial lipidome is associated with several human pathologies. Sun et al.1 show that LPGAT1 cooperates with TIMM14 to regulate phosphatidylglycerol transport from the endoplasmic reticulum to the mitochondria, and uncover the involvement of LPGAT1 deficiency in MEGDEL syndrome.

Structural insights into cardiolipin replacement by phosphatidylglycerol in a cardiolipin-lacking yeast respiratory supercomplex.

Cardiolipin is a hallmark phospholipid of mitochondrial membranes. Despite established significance of cardiolipin in supporting respiratory supercomplex organization, a mechanistic understanding of this lipid-protein interaction is still lacking. To address the essential role of cardiolipin in supercomplex organization, we report cryo-EM structures of a wild type supercomplex (IV1III2IV1) and a supercomplex (III2IV1) isolated from a cardiolipin-lacking Saccharomyces cerevisiae mutant at 3.2-Å and 3.3-Å resolution, respectively, and demonstrate that phosphatidylglycerol in III2IV1 occupies similar positions as cardiolipin in IV1III2IV1. Lipid-protein interactions within these complexes differ, which conceivably underlies the reduced level of IV1III2IV1 and high levels of III2IV1 and free III2 and IV in mutant mitochondria. Here we show that anionic phospholipids interact with positive amino acids and appear to nucleate a phospholipid domain at the interface between the individual complexes, which dampen charge repulsion and further stabilize interaction, respectively, between individual complexes.

Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids.

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

Cytidine diphosphate diacylglycerol synthase is essential for mitochondrial structure and energy production in Arabidopsis thaliana.

Cytidine diphosphate diacylglycerol (CDP-DAG), an important intermediate for glycerolipid biosynthesis, is synthesized under the catalytic activity of CDP-DAG synthase (CDS) to produce anionic phosphoglycerolipids such as phosphatidylglycerol (PG) and cardiolipin (CL). Previous studies showed that Arabidopsis CDSs are encoded by a small gene family, termed CDS1-CDS5, the members of which are integral membrane proteins in endoplasmic reticulum (ER) and in plastids. However, the details on how CDP-DAG is provided for mitochondrial membrane-specific phosphoglycerolipids are missing. Here we present the identification of a mitochondrion-specific CDS, designated CDS6. Enzymatic activity of CDS6 was demonstrated by the complementation of CL synthesis in the yeast CDS-deficient tam41Δ mutant. The Arabidopsis cds6 mutant lacking CDS6 activity showed decreased mitochondrial PG and CL biosynthesis capacity, a severe growth deficiency finally leading to plant death. These defects were rescued partly by complementation with CDS6 or supplementation with PG and CL. The ultrastructure of mitochondria in cds6 was abnormal, missing the structures of cristae. The degradation of triacylglycerol (TAG) in lipid droplets and starch in chloroplasts in the cds6 mutant was impaired. The expression of most differentially expressed genes involved in the mitochondrial electron transport chain was upregulated, suggesting an energy-demanding stage in cds6. Furthermore, the contents of polar glycerolipids in cds6 were dramatically altered. In addition, cds6 seedlings lost the capacity for cell proliferation and showed a higher oxidase activity. Thus, CDS6 is indispensable for the biosynthesis of PG and CL in mitochondria, which is critical for establishing mitochondrial structure, TAG degradation, energy production and seedling development.

Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis.

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human ß-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while ΔmprF2 and ΔmprF1 ΔmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2 and ΔmprF1 ΔmprF2. In the mprF mutants, particularly ΔmprF1 ΔmprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the ΔmprF1 ΔmprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both ΔmprF2 and ΔmprF1 ΔmprF2, compared to the wild type and ΔmprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. IMPORTANCE The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

Complex electrostatic effects on the selectivity of membrane-permeabilizing cyclic lipopeptides.

Cyclic lipopeptides (CLiPs) have many biological functions, including the selective permeabilization of target membranes, and technical and medical applications. We studied the anionic CLiP viscosin from Pseudomonas along with a neutral analog, pseudodesmin A, and the cationic viscosin-E2K to better understand electrostatic effects on target selectivity. Calcein leakage from liposomes of anionic phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) is measured in comparison with net-neutral phosphatidylcholine by time-resolved fluorescence. By contrast to the typical selectivity of cationic peptides against anionic membranes, we find viscosin more active against PG/PE at 30 µM lipid than viscosin-E2K. At very low lipid concentration, the selectivity is reversed. An equi-activity analysis reveals the reciprocal partition coefficients, 1/K, and the CLiP-to-lipid mole ratio within the membrane as leakage after 1 h reaches 50%, Re50. As expected, 1/K to PG/PE is much lower (higher affinity) for viscosin-E2K (3 µM) than viscosin (15 µM). However, the local damage to the PG/PE membrane caused by a viscosin molecule is much stronger than that of viscosin-E2K. This can be explained by the strong membrane expansion due to PG/viscosin repulsion inducing asymmetry stress between the two leaflets and, ultimately, transient limited leakage at Re50 = 0.08. PG/viscosin-E2K attraction opposes expansion and leakage starts only as the PG charges in the outer leaflet are essentially compensated by the cationic peptide (Re50 = 0.32). In the high-lipid regime (at lipid concentrations cL ≫ 1/K), virtually all CLiP is membrane bound anyway and Re50 governs selectivity, favoring viscosin. In the low-lipid regime at cL ≪ 1/K, virtually all CLiP is in solution, 1/K becomes important and the "cation attacks anionic membrane" selectivity gets restored. Overall, activity and selectivity data can only properly be interpreted if the lipid regime is known and predictions for other lipid concentrations or cell counts require knowledge of 1/K and Re50.

Two Different Phospholipases C, Isc1 and Pgc1, Cooperate To Regulate Mitochondrial Function.

The absence of Isc1, the yeast homologue of mammalian neutral sphingomyelinase type 2, leads to severe mitochondrial dysfunction. We show that the deletion of another type C phospholipase, the phosphatidylglycerol (PG)-specific phospholipase Pgc1, rescues this defect. Phosphatidylethanolamine (PE) levels and cytochrome c oxidase activity, which were reduced in isc1Δ cells, were restored to wild-type levels in the pgc1Δ isc1Δ mutant. The Pgc1 substrate PG inhibited the in vitro activities of Isc1 and the phosphatidylserine decarboxylase Psd1, an enzyme crucial for PE biosynthesis. We also identify a mechanism by which the balance between the current demand for PG and its consumption is controlled. We document that the product of PG hydrolysis, diacylglycerol, competes with the substrate of PG-phosphate synthase, Pgs1, and thereby inhibits the biosynthesis of excess PG. This feedback loop does not work in the absence of Pgc1, which catalyzes PG degradation. Finally, Pgc1 activity is partially inhibited by products of Isc1-mediated hydrolysis. The described functional interconnection of the two phospholipases contributes significantly to lipid homeostasis throughout the cellular architecture. IMPORTANCE In eukaryotic cells, mitochondria are constantly adapting to changes in the biological activity of the cell, i.e., changes in nutrient availability and environmental stresses. We propose a model in which this adaptation is mediated by lipids. Specifically, we show that mitochondrial phospholipids regulate the biosynthesis of cellular sphingolipids and vice versa. To do this, lipids move by free diffusion, which does not require energy and works under any condition. This model represents a simple way for the cell to coordinate mitochondrial structure and performance with the actual needs of overall cellular metabolism. Its simplicity makes it a universally applicable principle of cellular regulation.

A metabolomics study of the intervention effect of Tartary buckwheat on hyperlipidemia mice.

Tartary buckwheat can improve hyperlipidemia and affect the changes of metabolic pathways to the body. In this study, we use LC/MS to obtain metabolic fingerprints of plasma samples collected from control (LFD), high-fat diet (HFD), Tartary buckwheat protein (BWP), and Tartary buckwheat starch (BWS). Using the metabolic network database, through OPLS-DA, the potential biomarkers and pathways of BWP and BWS intervention in hyperlipidemia mice are initially determined. The results showed that there are 30 metabolites in total, among which linoleic acid, glycerol, phosphatidyl, ethanolamine, and galactose ceramide are the most important differentially expressed metabolites in BWP and BWS plasma samples. These metabolites are involved in eight metabolic pathways, such as linoleic acid metabolism, arachidonic acid metabolism. Tartary buckwheat can alleviate the symptoms of hyperlipidemia in mice by affecting the above-mentioned metabolic pathways. This research has a profound impact on the development of nutritious foods of buckwheat. PRACTICAL APPLICATIONS: Tartary buckwheat, also known as wild buckwheat, is a typical embodiment homology of medicine and food. We have clarified that the protein and starch extracted from tartary buckwheat have the function of reducing blood lipids. It is expected to be applied to functional food materials in the health food market. Also, the effects of tartary buckwheat protein and starch in improving metabolic pathways can be generally applied as a physiological active compound of functional food supplements.

Impacts of phosphatidylglycerol on plastid gene expression and light induction of nuclear photosynthetic genes.

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis, and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development, with decreased chlorophyll accumulation, impaired thylakoid formation, and down-regulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species (ROS)-responsive genes were up-regulated in pgp1-2. However, ROS production was not enhanced in the mutant even under strong light, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered down-regulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor gene regulating chloroplast development, in pgp1-2 up-regulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in plastid gene expression and plant development.

Crucial importance of length of fatty-acyl chains bound to the sn-2 position of phosphatidylglycerol for growth and photosynthesis of Synechocystis sp. PCC 6803.

Phosphatidylglycerol (PG) in thylakoid membrane is essential for growth and photosynthesis of photosynthetic organisms. Although the sn-2 position of PG in thylakoid membrane is exclusively esterified with C16 fatty acids, the functional importance of the C16 fatty-acyl chains at the sn-2 position has not been clarified. In this study, we chemically synthesized non-metabolizable PG molecules: we introduced linoleic acid (18:2, fatty acid containing 18 carbons with 2 double bonds) and one of the saturated fatty acids with different chain length (12:0, 14:0, 16:0, 18:0 and 20:0) by ether linkage to the sn-1 and sn-2 positions, respectively. With the synthesized ether-linked PG molecules, we checked whether they could complement the growth and photosynthesis of pgsA mutant cells of Synechocystis sp. PCC 6803 to understand the importance of length of fatty chains at the sn-2 position of PG. The pgsA mutant is incapable of synthesizing PG, so it requires exogenous PG added to medium for growth. The growth rate and photosynthetic activity of mutant cells depended on the length of fatty chains: the PG molecular species binding 16:0 most effectively complemented the growth and photosynthesis of mutant cells, and other PG molecular species with fatty chains shorter or longer than 16:0 were less effective; especially, those binding 12:0 inhibited the growth and photosynthetic activity of the mutant cells. These data demonstrate that length of fatty chains bound to the sn-2 position of PG is critical for PG performance in growth and photosynthesis.

Blocking phosphatidylglycerol degradation in yeast defective in cardiolipin remodeling results in a new model of the Barth syndrome cellular phenotype.

Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.

Specific Incorporation of Polyunsaturated Fatty Acids into the sn-2 Position of Phosphatidylglycerol Accelerates Photodamage to Photosystem II under Strong Light.

Free fatty acids (FFAs) are generated by the reaction of lipases with membrane lipids. Generated polyunsaturated fatty acids (PUFAs) containing more than two double bonds have toxic effects in photosynthetic organisms. In the present study, we examined the effect of exogenous FFAs in the growth medium on the activity of photosystem II (PSII) under strong light in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PUFAs but not monounsaturated fatty acids accelerated the rate of photodamage to PSII by inactivating electron transfer at the oxygen-evolving complex. Moreover, supplemented PUFAs were specifically incorporated into the sn-2 position of phosphatidylglycerol (PG), which usually contains C16 fatty acids at the sn-2 position in Synechocystis cells. The disruption of the gene for an acyl-ACP synthetase reduced the effect of PUFAs on the photoinhibition of PSII. Thus, the specific incorporation of PUFAs into PG molecules requires acyl-ACP synthetase and leads to an unstable PSII, thereby accelerating photodamage to PSII. Our results are a breakthrough into elucidating the molecular mechanism of the toxicity of PUFAs to photosynthetic organisms.

Polyunsaturated fatty acids cause physiological and behavioral changes in Vibrio alginolyticus and Vibrio fischeri.

Vibrio alginolyticus and Vibrio (Aliivibrio) fischeri are Gram-negative bacteria found globally in marine environments. During the past decade, studies have shown that certain Gram-negative bacteria, including Vibrio species (cholerae, parahaemolyticus, and vulnificus) are capable of using exogenous polyunsaturated fatty acids (PUFAs) to modify the phospholipids of their membrane. Moreover, exposure to exogenous PUFAs has been shown to affect certain phenotypes that are important factors of virulence. The purpose of this study was to investigate whether V. alginolyticus and V. fischeri are capable of responding to exogenous PUFAs by remodeling their membrane phospholipids and/or altering behaviors associated with virulence. Thin-layer chromatography (TLC) analyses and ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC/ESI-MS) confirmed incorporation of all PUFAs into membrane phosphatidylglycerol and phosphatidylethanolamine. Several growth phenotypes were identified when individual fatty acids were supplied in minimal media and as sole carbon sources. Interestingly, several PUFAs acids inhibited growth of V. fischeri. Significant alterations to membrane permeability were observed depending on fatty acid supplemented. Strikingly, arachidonic acid (20:4) reduced membrane permeability by approximately 35% in both V. alginolyticus and V. fischeri. Biofilm assays indicated that fatty acid influence was dependent on media composition and temperature. All fatty acids caused decreased swimming motility in V. alginolyticus, while only linoleic acid (18:2) significantly increased swimming motility in V. fischeri. In summary, exogenous fatty acids cause a variety of changes in V. alginolyticus and V. fischeri, thus adding these bacteria to a growing list of Gram-negatives that exhibit versatility in fatty acid utilization and highlighting the potential for environmental PUFAs to influence phenotypes associated with planktonic, beneficial, and pathogenic associations.

The cell envelope of Staphylococcus aureus selectively controls the sorting of virulence factors.

Staphylococcus aureus bi-component pore-forming leukocidins are secreted toxins that directly target and lyse immune cells. Intriguingly, one of the leukocidins, Leukocidin AB (LukAB), is found associated with the bacterial cell envelope in addition to secreted into the extracellular milieu. Here, we report that retention of LukAB on the bacterial cells provides S. aureus with a pre-synthesized active toxin that kills immune cells. On the bacteria, LukAB is distributed as discrete foci in two distinct compartments: membrane-proximal and surface-exposed. Through genetic screens, we show that a membrane lipid, lysyl-phosphatidylglycerol (LPG), and lipoteichoic acid (LTA) contribute to LukAB deposition and release. Furthermore, by studying non-covalently surface-bound proteins we discovered that the sorting of additional exoproteins, such as IsaB, Hel, ScaH, and Geh, are also controlled by LPG and LTA. Collectively, our study reveals a multistep secretion system that controls exoprotein storage and protein translocation across the S. aureus cell wall.

Florigen sequestration in cellular membranes modulates temperature-responsive flowering.

Plants respond to temperature changes by modulating florigen activity to optimize the timing of flowering. We show that the Arabidopsis thaliana mobile florigen FLOWERING LOCUS T (FT) interacts with the negatively charged phospholipid phosphatidylglycerol (PG) at cellular membranes and binds the lipid bilayer. Perturbing PG biosynthesis in phloem companion cells leads to temperature-insensitive early flowering. Low temperatures facilitate FT sequestration in the cellular membrane of the companion cell, thus reducing soluble FT levels and delaying flowering. A mutant in PHOSPHATIDYLGLYCEROLPHOSPHATE SYNTHASE 1 accumulates more soluble FT at lower temperatures and exhibits reduced temperature sensitivity. Thus, cellular membranes sequester FT through their ability to bind the phospholipid PG, and this sequestration modulates the plant's response to temperature changes.

Selective Antifungal Activity and Fungal Biofilm Inhibition of Tryptophan Center Symmetrical Short Peptide.

Candida albicans, an opportunistic fungus, causes dental caries and contributes to mucosal bacterial dysbiosis leading to a second infection. Furthermore, C.albicans forms biofilms that are resistant to medicinal treatment. To make matters worse, antifungal resistance has spread (albeit slowly) in this species. Thus, it has been imperative to develop novel, antifungal drug compounds. Herein, a peptide was engineered with the sequence of RRFSFWFSFRR-NH2; this was named P19. This novel peptide has been observed to exert disruptive effects on fungal cell membrane physiology. Our results showed that P19 displayed high binding affinity to lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the plasma membrane phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidylglycerol (PG), further indicating that the molecular mechanism of P19 was not associated with the receptor recognition, but rather related to competitive interaction with the plasma membrane. In addition, compared with fluconazole and amphotericin B, P19 has been shown to have a lower potential for resistance selection than established antifungal agents.

Strain-specific metabolic responses to long-term caloric restriction in female ILSXISS recombinant inbred mice.

The role that genetic background may play in the responsiveness of organisms to interventions such as caloric restriction (CR) is underappreciated but potentially important. We investigated the impact of genetic background on a suite of metabolic parameters in female recombinant inbred ILSXISS mouse strains previously reported to show divergent lifespan responses to 40% CR (TejJ89-lifespan extension; TejJ48-lifespan unaffected; TejJ114-lifespan shortening). Body mass was reduced across all strains following 10 months of 40% CR, although this loss (relative to ad libitum controls) was greater in TejJ114 relative to the other strains. Gonadal white adipose tissue (gWAT) mass was similarly reduced across all strains following 40% CR, but brown adipose tissue (BAT) mass increased only in strains TejJ89 and TejJ48. Surprisingly, glucose tolerance was improved most notably by CR in TejJ114, while both strains TejJ89 and TejJ114 were hyperinsulinemic following CR relative to their AL controls. We subsequently undertook an unbiased metabolomic approach in gWAT and BAT tissue derived from strains TejJ89 and TejJ114 mice under AL and 40% CR. In gWAT from TejJ89 a significant reduction in several long chain unsaturated fatty acids was observed following 40% CR, but gWAT from TejJ114 appeared relatively unresponsive to CR with far fewer metabolites changing. Phosphatidylethanoloamine lipids within the BAT were typically elevated in TejJ89 following CR, while some phosphatidylglycerol lipids were decreased. However, BAT from strain TejJ114 again appeared unresponsive to CR. These data highlight strain-specific metabolic differences exist in ILSXISS mice following 40% CR. We suggest that precisely how different fat depots respond dynamically to CR may be an important factor in the variable longevity under 40% CR reported in these mice.