TgPL2, a patatin‐like phospholipase domain‐containing protein,is involved in the maintenance of apicoplast lipids homeostasis in Toxoplasma

Patatin‐like phospholipases are involved in numerous cellular functions, including lipid metabolism and membranes remodeling. The patatin‐like catalytic domain, whose phospholipase activity relies on a serine‐aspartate dyad and an anion binding box, is widely spread among prokaryotes and eukaryotes. We describe TgPL2, a novel patatin‐like phospholipase domain‐containing protein from the parasitic protist Toxoplasma gondii. TgPL2 is a large protein, in which the key motifs for enzymatic activity are conserved in the patatin‐like domain. Using immunofluorescence assays and immunoelectron microscopy analysis, we have shown that TgPL2 localizes to the apicoplast, a non‐photosynthetic plastid found in most apicomplexan parasites. This plastid hosts several important biosynthetic pathways, which makes it an attractive organelle for identifying new potential drug targets. We thus addressed TgPL2 function by generating a conditional knockdown mutant and demonstrated it has an essential contribution for maintaining the integrity of the plastid. In absence of TgPL2, the organelle is rapidly lost and remaining apicoplasts appear enlarged, with an abnormal accumulation of membranous structures, suggesting a defect in lipids homeostasis. More precisely, analyses of lipid content upon TgPL2 depletion suggest this protein is important for maintaining levels of apicoplast‐generated fatty acids, and also regulating phosphatidylcholine and lysophosphatidylcholine levels in the parasite.     

A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages

The apicomplexan parasite Toxoplasma gondii is able to suppress nitric oxide production in activated macrophages. A screen of over 6000 T. gondii insertional mutants identified two clones, which were consistently unable to suppress nitric oxide production from activated macrophages. One strain, called 89B7, grew at the same rate as wild‐type parasites in naïve macrophages, but unlike wild type, the mutant was degraded in activated macrophages. This degradation was marked by a reduction in the number of parasites within vacuoles over time, the loss of GRA4 and SAG1 protein staining by immunofluorescence assay, and the vesiculation and breakdown of the internal parasite ultrastructure by electron microscopy. The mutagenesis plasmid in the 89B7 clone disrupts the promoter of a 3.4 kb mRNA that encodes a predicted 68 kDa protein with a cleavable signal peptide and a patatin‐like phospholipase domain. Genetic complementation with the genomic locus of this patatin‐like protein restores the parasites ability to suppress nitric oxide and replicate in activated macrophages. A haemagglutinin‐tagged version of this patatin‐like protein shows punctate localization into atypical T. gondii structures within the parasite. This is the first study that defines a specific gene product that is needed for parasite survival in activated but not naïve macrophages.     

Characterization of a Nilaparvata lugens (Stål) brummer gene and analysis of its role in lipid metabolism

The brummer (bmm) genes encode the lipid storage droplet‐associated triacylglycerols (TAG) lipases, which belong to the Brummer/Nutrin subfamily. These enzymes hydrolyze the ester bonds in TAG in lipid metabolism and act in insect energy homeostasis. Exposure to some agricultural chemicals leads to increased fecundity, which necessarily involves lipid metabolism, in some planthopper species. However, the biological roles of bmm in planthopper lipid storage and mobilization have not been investigated. Here, the open reading frame (ORF) of bmm (Nlbmm) was cloned and sequenced from the brown planthopper (BPH; Nilaparvata lugens). The ORF is 1014 bp encoding 338 amino acid residues. Nlbmm contained patatin domains and shared considerable evolutionary conservation with other insect bmms. Nlbmm is highly expressed in the fat body, consistent with its roles in lipid metabolism. Injection with Nlbmm double‐stranded RNA (dsNlbmm) led to reduced Nlbmm mRNA accumulation, but did not influence expression of several genes related to lipid synthesis including acyl‐CoA‐binding protein (ACBP), acetyl‐CoA carboxylase (ACC), and a lipophorin receptor (LpR). Nlbmm knockdown led to increased TAG contents in whole bodies, accumulation of total fat body lipid, and decreased hemolymph lipid content. Nlbmm knockdown did not influence the synthesis and distribution of glycerol. We infer that Nlbmm acts in TAG breakdown and fat metabolism in N. lugens.     

Phospholipase activity in Cryptococcus neoformans

Phospholipases have only been detected in a few fungi and yeasts, in particular in Candida albicans. Secreted phospholipases are considered by some researchers to be a potential factor of virulence and pathogenicity in C. albicans. Twenty-three Cryptococcus neoformans strains were tested in order to observe phospholipase production. Twenty-two of the 23 strains tested were able to produce phospholipases, and the ratio diameter of the colony to total diameter of the colony plus zone of precipitation (Pz) ranged between 0.271 and 0.949. C. neoformans, just like C. albicans, can be divided on the basis of the Pz into different strains according to their virulence and pathogenicity. There also appeared to be a correlation between the phospholipase production and the size of the capsule in the strains isolated from AIDS patients. For this reason, further studies on C. neoformans phospholipase activity would be useful in evaluating the virulence of different strains.     

Structure-function relationship for the highly toxic crotoxin from Crotalus durissus terrificus

The three-dimensional structure of the highly toxic crotoxin from Crotalus durissus terrificus was modelled based on sequence analysis and the refined structure of calcium-free phospholipase of Crotalus atrox venom. Small-angle x-ray scattering experiments were performed on aqueous solutions of crotoxin. The radial distribution function derived from these scattering experiments and the one calculated from the model structure are in good agreement. Crotoxin consists of a basic and an acidic subunit. The model strongly suggests that the overall folding motif of phospholipases has been preserved in both subunits. The basic domain has an intact active site. The residues that are expected to contact the lipid tails of the phospholipid are different from other phospholipases, but they are all hydrophobic. The acidic domain consists of three independent chains interconnected by disulfide bonds. Compared to other phospholipases the active site for the greater part has been preserved in this domain, but it is not very well shielded from solvent. Most residues normally in contact with the lipid tails of the phospholipid are missing, which might explain the acidic subunit's lack of phospholipase activity. A homology between the third chain of the acidic domain and neurophysins suggests that the acidic domain may act as a chaperone for the basic domain. Correspondence to: Y P. Mascarenhas     

Fitness in invasive social wasps: the role of variation in viral load,immune response and paternity in predicting nest size and reproductive output

Within any one habitat, the relative fitness of organisms in a population can vary substantially. Social insects like the common wasp are among the most successful invasive animals, but show enormous variation in nest size and other fitness‐related traits. Some of this variation may be caused by pathogens such as viruses that can have serious consequences in social insects, which range from reduced productivity to colony death. Both individual immune responses and colony‐level traits such as genetic diversity are likely to influence effects of pathogen infections on colony fitness. Here we investigate how infections with Kashmir Bee Virus (KBV), immune response and intracolony genetic diversity (due to queen polyandry) affect nest size in the invasive common wasp Vespula vulgaris. We show that KBV is highly prevalent in wasps and expression of antiviral immune genes is significantly increased with higher viral loads across individuals. Patriline membership within a nest did not influence KBV susceptibility or immune response. A permutational MANCOVA revealed that polyandry, viral load and expression of the immune gene Dicer were significant predictors of variation in nest size. High intracolony genetic diversity due to polyandry has previously been hypothesized to improve colony‐level resistance to parasites and pathogens. Consistent with this hypothesis, we observed genetically diverse colonies to be significantly larger and to produce more queens, although this effect was not driven by the pathogen we investigated. Invasive wasps clearly suffer from pathogens and expend resources, as indicated here by elevated immune gene expression, toward reducing pathogen‐impact on colony fitness.     

A Plasmodium Phospholipase Is Involved in Disruption of the Liver Stage Parasitophorous Vacuole Membrane

The coordinated exit of intracellular pathogens from host cells is a process critical to the success and spread of an infection. While phospholipases have been shown to play important roles in bacteria host cell egress and virulence, their role in the release of intracellular eukaryotic parasites is largely unknown. We examined a malaria parasite protein with phospholipase activity and found it to be involved in hepatocyte egress. In hepatocytes, Plasmodium parasites are surrounded by a parasitophorous vacuole membrane (PVM), which must be disrupted before parasites are released into the blood. However, on a molecular basis, little is known about how the PVM is ruptured. We show that Plasmodium berghei phospholipase, PbPL, localizes to the PVM in infected hepatocytes. We provide evidence that parasites lacking PbPL undergo completely normal liver stage development until merozoites are produced but have a defect in egress from host hepatocytes. To investigate this further, we established a live-cell imaging-based assay, which enabled us to study the temporal dynamics of PVM rupture on a quantitative basis. Using this assay we could show that PbPL-deficient parasites exhibit impaired PVM rupture, resulting in delayed parasite egress. A wild-type phenotype could be re-established by gene complementation, demonstrating the specificity of the PbPL deletion phenotype. In conclusion, we have identified for the first time a Plasmodium phospholipase that is important for PVM rupture and in turn for parasite exit from the infected hepatocyte and therefore established a key role of a parasite phospholipase in egress.     

Mutants of phospholipase A (pPLA‐I) have a red light and auxin phenotype

pPLA‐I is the evolutionarily oldest patatin‐related phospholipase A (pPLA) in plants, which have previously been implicated to function in auxin and defence signalling. Molecular and physiological analysis of two allelic null mutants for pPLA‐I [ppla‐I‐1 in Wassilewskija (Ws) and ppla‐I‐3 in Columbia (Col) ] revealed pPLA‐I functions in auxin and light signalling. The enzyme is localized in the cytosol and to membranes. After auxin application expression of early auxin‐induced genes is significantly slower compared with wild type and both alleles show a slower gravitropic response of hypocotyls, indicating compromised auxin signalling. Additionally, phytochrome‐modulated responses like abrogation of gravitropism, enhancement of phototropism and growth in far red‐enriched light are decreased in both alleles. While early flowering, root coils and delayed phototropism are only observed in the Ws mutant devoid of phyD, the light‐related phenotypes observed in both alleles point to an involvement of pPLA‐I in phytochrome signalling.     

Lipid droplet hijacking by intracellular pathogens

Lipid droplets were long considered to be simple storage structures, but they have recently been shown to be dynamic organelles involved in diverse biological processes, including emerging roles in innate immunity. Various intracellular pathogens, including viruses, bacteria, and parasites, specifically target host lipid droplets during their life cycle. Viruses such as hepatitis C, dengue, and rotaviruses use lipid droplets as platforms for assembly. Bacteria, such as mycobacteria and Chlamydia, and parasites, such as trypanosomes, use host lipid droplets for nutritional purposes. The possible use of lipid droplets by intracellular pathogens, as part of an anti‐immunity strategy, is an intriguing question meriting further investigation in the near future.     

Rice phospholipase A superfamily: organization, phylogenetic and expression analysis during abiotic stresses and development

Background

Phospholipase A (PLA) is an important group of enzymes responsible for phospholipid hydrolysis in lipid signaling. PLAs have been implicated in abiotic stress signaling and developmental events in various plants species. Genome-wide analysis of PLA superfamily has been carried out in dicot plant Arabidopsis. A comprehensive genome-wide analysis of PLAs has not been presented yet in crop plant rice.

Methodology/Principal Findings

A comprehensive bioinformatics analysis identified a total of 31 PLA encoding genes in the rice genome, which are divided into three classes; phospholipase A₁ (PLA₁), patatin like phospholipases (pPLA) and low molecular weight secretory phospholipase A₂ (sPLA₂) based on their sequences and phylogeny. A subset of 10 rice PLAs exhibited chromosomal duplication, emphasizing the role of duplication in the expansion of this gene family in rice. Microarray expression profiling revealed a number of PLA members expressing differentially and significantly under abiotic stresses and reproductive development. Comparative expression analysis with Arabidopsis PLAs revealed a high degree of functional conservation between the orthologs in two plant species, which also indicated the vital role of PLAs in stress signaling and plant development across different plant species. Moreover, sub-cellular localization of a few candidates suggests their differential localization and functional role in the lipid signaling.

Conclusion/Significance

The comprehensive analysis and expression profiling would provide a critical platform for the functional characterization of the candidate PLA genes in crop plants.     

Isolation and preliminary characterisation of two toxic phospholipases A2 from the venom of the Malayan cobra (Naja naja sputatrix)

Two toxic phospholipases A have been isolated from the venom of the Malayan cobra (Naja naja sputatrix). The phospholipases A were purified by successive ion-change chromatography on SP-Sephadex C-25, Sephadex G-75 gel filtration chromatography and successive Bio-Rex 70 ion-exchange chromatography. The purified toxic phospholipases A were homogeneous electrophoretically. They were designated as sputatrix phospholipase A-I and sputatrix phospholipase A-II. Positional specificity studies showed that they belong to the A₂-type phospholipase A. The medium lethal dose 50% (LD₅₀) values of the two phospholipases A are 0.27 and 0.28 μg/g, respectively, by intravenous injection and 1.05 and 1.00 μg./g, respectively, by intraperitoneal injection. The molecular weights of the two enzymes are 14 000 as determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. Amino acid composition of sputatrix phospholipase A-I differs from sputatrix phospholipase A-II only by having one extra amino acid: a glutamic acid. Amino acid compositions of the two enzymes are also similar to those of other cobra venom phospholipases A.     

Non‐specific phospholipases C,NPC2 and NPC6, are required for root growth in Arabidopsis

Mutants in lipid metabolism often show a lethal phenotype during reproduction that prevents investigating a specific role of the lipid during different developmental processes. We focused on two non‐specific phospholipases C, NPC2 and NPC6, whose double knock‐out causes a gametophyte‐lethal phenotype. To investigate the role of NPC2 and NPC6 during vegetative growth, we produced transgenic knock‐down mutant lines that circumvent the lethal effect during gametogenesis. Despite no defect observed in leaves, root growth was significantly retarded, with abnormal cellular architecture in root columella cells. Furthermore, the short root phenotype was rescued by exogenous supplementation of phosphocholine, a product of non‐specific phospholipase C (NPC) ‐catalyzed phosphatidylcholine hydrolysis. The expression of phospho‐base N‐methyltransferase 1 (PMT1), which produces phosphocholine and is required for root growth, was induced in the knock‐down mutant lines and was attenuated after phosphocholine supplementation. These results suggest that NPC2 and NPC6 may be involved in root growth by producing phosphocholine via metabolic interaction with a PMT‐catalyzed pathway, which highlights a tissue‐specific role of NPC enzymes in vegetative growth beyond the gametophyte‐lethal phenotype.     

Histamine releasing factor and elongation factor 1 alpha secreted via malaria parasites extracellular vesicles promote immune evasion by inhibiting specific T cell responses

Protozoan pathogens secrete nanosized particles called extracellular vesicles (EVs) to facilitate their survival and chronic infection. Here, we show the inhibition by Plasmodium berghei NK65 blood stage‐derived EVs of the proliferative response of CD4⁺ T cells in response to antigen presentation. Importantly, these results were confirmed in vivo by the capacity of EVs to diminish the ovalbumin‐specific delayed type hypersensitivity response. We identified two proteins associated with EVs, the histamine releasing factor (HRF) and the elongation factor 1α (EF‐1α) that were found to have immunosuppressive activities. Interestingly, in contrast to WT parasites, EVs from genetically HRF‐ and EF‐1α‐deficient parasites failed to inhibit T cell responses in vitro and in vivo. At the level of T cells, we demonstrated that EVs from WT parasites dephosphorylate key molecules (PLCγ1, Akt, and ERK) of the T cell receptor signalling cascade. Remarkably, immunisation with EF‐1α alone or in combination with HRF conferred a long‐lasting antiparasite protection and immune memory. In conclusion, we identified a new mechanism by which P. berghei‐derived EVs exert their immunosuppressive functions by altering T cell responses. The identification of two highly conserved immune suppressive factors offers new conceptual strategies to overcome EV‐mediated immune suppression in malaria‐infected individuals.     

Maternal pathogen exposure causes diet‐ and pathogen‐specific transgenerational costs

Transgenerational effects, whereby the environment experienced by a parent leads to an altered offspring phenotype, have now been described in a variety of taxa. In invertebrates, much of the research on these effects has concentrated on the role of parental exposure to pathogens or immune elicitors in determining offspring immune investment or disease resistance. To date, however, studies of transgenerational effects in invertebrates have generally been restricted to single infections or immune elicitors in ideal laboratory environments. Animals in field situations will commonly experience sub‐optimal environments and co‐infection by multiple species of parasites and pathogens, leading to increased relative costs of immune investment and changing fitness benefits from offspring responses to the parental environment. Here we investigate a more ecologically realistic scenario involving both multiple infections and resource limitation, using the Indian meal moth Plodia interpunctella as a model host, challenged with the entomopathogenic bacterium Bacillus thuringiensis and fungus Beauveria bassiana. Mothers were exposed to low doses of one or both pathogens, or a control. Offspring from each family were reared on either good‐ or poor‐quality food and then exposed to one or both pathogens. Maternal exposure to pathogens led to reduced pathogen resistance in offspring, depending on the combination of maternal and offspring pathogen‐specific infections and resource limitation in the offspring generation. Much research to date has focussed on trans‐generational immune priming, in which parental exposure to pathogens or immune elicitors leads to upregulated immune reactivity in their offspring. The lack of any such effects in our system suggests that the production of less resistant offspring following parental exposure to pathogens might be an important alternative, driven by costs of resistance rather than adaptive benefits.     

Patatin-related phospholipase A: nomenclature, subfamilies and functions in plants

The release of fatty acids from membrane glycerolipids has been implicated in a variety of cellular processes, but the enzymes involved and their regulation are poorly understood in plants. One large group of acyl-hydrolyzing enzymes is structurally related to patatins. Patatins are potato tuber proteins with acyl-hydrolyzing activity, and the patatin catalytic domain is widely spread in bacterial, yeast, plant and animal enzymes. Recent results have indicated that patatin-related enzymes are involved in different cellular functions, including plant responses to auxin, elicitors or pathogens, and abiotic stresses and lipid mobilization during seed germination. In this review, we highlight recent developments regarding these enzymes and propose the nomenclature pPLA for the patatin-related phospholipase A enzyme.     

Molecular dynamics simulations reveal structural insights into inhibitor binding modes and functionality in human Group IIA phospholipase A2

Human Group IIA phospholipase A₂ (hGIIA) promotes inflammation in immune‐mediated pathologies by regulating the arachidonic acid pathway through both catalysis‐dependent and ‐independent mechanisms. The hGIIA crystal structure, both alone and inhibitor‐bound, together with structures of closely related snake‐venom‐derived secreted phospholipase enzymes has been well described. However, differentiation of biological and nonbiological contacts and the relevance of structures determined from snake venom enzymes to human enzymes are not clear. We employed molecular dynamics (MD) and docking approaches to understand the binding of inhibitors that selectively or nonselectively block the catalysis‐independent mechanism of hGIIA. Our results indicate that hGIIA behaves as a monomer in the solution environment rather than a dimer arrangement that is in the asymmetric unit of some crystal structures. The binding mode of a nonselective inhibitor, KH064, was validated by a combination of the experimental electron density and MD simulations. The binding mode of the selective pentapeptide inhibitor FLSYK to hGIIA was stipulated to be different to that of the snake venom phospholipases A₂ of Daboia russelli pulchella (svPLA₂). Our data suggest that the application of MD approaches to crystal structure data is beneficial in evaluating the robustness of conclusions drawn based on crystal structure data alone. Proteins 2017; 85:827–842. © 2016 Wiley Periodicals, Inc.     

Biology and Biochemistry of Plant Phospholipases

Phospholipases are a complex group of enzymes that hydrolyze phospholipids. The plant phospholipase family is composed of multiple members with varying positional specificity, and each type is represented by multiple isoforms distinguishable by their structural, catalytic, and physiological characteristics. A large number of phospholipase genes and gene families have been identified and the biochemical properties of several members have been characterized, revealing considerable molecular and catalytic diversity. Forward and reverse genetics has further revealed that phospholipases are widely involved in physiological processes including lipid metabolism, cell signaling, and responses to biotic and abiotic stresses. Such studies have highlighted the potential biotechnological value of phospholipases as targets for improving stress tolerance. The catalytic diversity of various phospholipase isoforms is also of increasing interest for industrial biocatalysis. This review focuses on recently acquired information on biochemical, molecular and functional aspects of plant phospholipases.