Search

Objective To identify and analyze the phospholipase A (PLA) gene family in Talaromyces marneffei (T. marneffei), providing the basis for further research on the potential pathogenic effect and mechanism of T. marneffei PLA. Methods The members of the PLA gene family in T. marneffei were genome-wide identified by using bioinformatics, and their gene structure, chromosome distribution, protein physicochemical property and structure, conserved motif, as well as phyletic evolution, were analyzed. The expression levels of PLA genes in T. marneffei under macrophage treatment were analyzed using the qRT-PCR method. Results A total of eight PLA genes were identified from the genome of T. marneffei, unevenly located on 5 chromosomes without any tandem duplications or fragment duplications. The amino acid residues of PLA proteins ranged from 577 to 1546, molecular weights from 65 120 to 170 690, and the isoelectric point from 4.47 to 9.28. All PLA proteins were hydrophilic, with all but PLA3 classified as unstable hydrophilic proteins. Phylogenetic analysis revealed that PLA was divided into two subfamilies: PLA2 and patatin-like phospholipase, consisting of 3 and 5 members respectively. Within the same subfamily, gene structures, protein-conserved motifs, and protein structures were relatively similar, whereas significant differences existed between different subfamilies. qRT PCR results showed that the expression levels of PLA3, PLA6, and PLA8 were upregulated in T. marneffei infected with mouse macrophages. Conclusions PLA3, PLA6, and PLA8 may be related to the survival and reproduction of T. marneffei in macrophages. [ABSTRACT FROM AUTHOR]

ABSTRACT Contact-dependent hemolysins are virulence factors in a number of human pathogens, including Helicobacter pylori, Rickettsia typhi, Bartonella bacilliformis, Mycobacterium tuberculosis, entero-invasive Escherichia coli, and Shigella. Here we demonstrate that Neisseria gonorrhoeae produces an outer membrane protein, phospholipase A, that exhibits contact-dependent lytic activity on host cell membranes. This enzyme can lyse human erythrocytes over a 3-day period, whereas a phospholipase A mutant cannot. We demonstrated phospholipase A activity in the parent strain but not in two, independent phospholipase A mutants. A gene for phospholipase A, pldA (hereafter referred to as pla to avoid confusion with the gene for phospholipase D, pld), is present in all sequenced gonococcal strains. Fluid phase, hemolytic activity assays showed that 25 of 29 gonococcal strains tested had hemolytic activity greater than 50% of the positive control. In support of PLA as a gonococcal outer membrane protein, supernatants from 24-, 48-, and 72-h cultures of N. gonorrhoeae strain 1291 did not contain hemolysin activity, and a monoclonal antibody specific for gonococcal phospholipase A failed to detect the enzyme in these supernatants. The organism must be viable for lysis to occur, and the inclusion of EDTA in the media removes all activity. Our studies have shown that a phospholipase A mutant has significantly reduced survival in human neutrophils and primary human cervical epithelial cells compared to the parent gonococcal strain after 3 h of incubation. Collectively, our data demonstrate that gonococcal PLA lyses host cell membranes, which is important for intracellular survival.IMPORTANCEIntracellular survival is crucial to the success of Neisseria gonorrhoeae as a human pathogen. Multiple factors contribute to the intracellular survival of gonococci, including the ability to prohibit apoptosis of the epithelial cell the organism invades and mechanisms to evade host innate defense systems. The role of phospholipase A (PLA), an outer membrane protein, is important as it disrupts the host vacuolar and phagolysosomal membranes, preventing the effective delivery of innate immune factors that normally restrict organism growth within human cells. After cell entry, PLA disrupts the integrity of these host cell membranes, allowing the gonococcus to live free within disrupted vacuoles where it pilfers host cell nutrients that enable its survival and replication. A vaccine or drug that could neutralize PLA activity would disrupt the intracellular survival of the gonococcus.

Summary: Production of in planta haploid embryos that inherit chromosomes from only one parent can greatly increase breeding efficiency via quickly generating homozygous plants, called doubled haploid. One of the main players of in planta haploid induction is a pollen‐specific phospholipase A, which is able, when mutated, to induce in vivo haploid induction in numerous monocots. However, no functional orthologous gene has been identified in dicots plants.Here, we show that loss‐of‐function of gynoecium‐expressed phospholipase AII (pPLAIIγ) triggers maternal haploid plants in Arabidopsis, at an average rate of 1.07%. Reciprocal crosses demonstrate that haploid plants are triggered from the female side and not from the pollen, and the haploid plants carry the maternal genome.Promoter activity of pPLAIIγ shows enriched expression in the funiculus of flower development stages 13 and 18, and pPLAIIγ fused to yellow fluorescent protein reveals a plasma‐membrane localizationInterestingly, the polar localized PIN1 at the basal plasma membrane of the funiculus was all internalized in pplaIIγ mutants, suggesting that altered PIN1 localization in female organ could play a role in maternal haploid induction. [ABSTRACT FROM AUTHOR]

Summary: After polyploidization originated from one interspecific hybridization event in Gossypium, Gossypium barbadense evolved to produce extra‐long staple fibres than Gossypium hirsutum (Upland cotton), which produces a higher fibre yield. The genomic diversity between G. barbadense and G. hirsutum thus provides a genetic basis for fibre trait variation. Recently, rapid accumulation of gene disruption or deleterious mutation was reported in allotetraploid cotton genomes, with unknown impacts on fibre traits. Here, we identified gene disruptions in allotetraploid G. hirsutum (18.14%) and G. barbadense (17.38%) through comparison with their presumed diploid progenitors. Relative to conserved genes, these disrupted genes exhibited faster evolution rate, lower expression level and altered gene co‐expression networks. Within a module regulating fibre elongation, a hub gene experienced gene disruption in G. hirsutum after polyploidization, with a 2‐bp deletion in the coding region of GhNPLA1D introducing early termination of translation. This deletion was observed in all of the 34 G. hirsutum landraces and 36 G. hirsutum cultivars, but not in 96% of 57 G. barbadense accessions. Retrieving the disrupted gene GhNPLA1D using its homoeolog GhNPLA1A achieved longer fibre length in G. hirsutum. Further enzyme activity and lipids analysis confirmed that GhNPLA1A encodes a typical phospholipase A and promotes cotton fibre elongation via elevating intracellular levels of linolenic acid and 34:3 phosphatidylinositol. Our work opens a strategy for identifying disrupted genes and retrieving their functions in ways that can provide valuable resources for accelerating fibre trait enhancement in cotton breeding. [ABSTRACT FROM AUTHOR]

Contact-dependent hemolysins are virulence factors in a number of human pathogens, including Helicobacter pylori, Rickettsia typhi, Bartonella bacilliformis, Mycobacterium tuberculosis, entero-invasive Escherichia coli, and Shigella . Here we demonstrate that Neisseria gonorrhoeae produces an outer membrane protein, phospholipase A, that exhibits contact-dependent lytic activity on host cell membranes. This enzyme can lyse human erythrocytes over a 3-day period, whereas a phospholipase A mutant cannot. We demonstrated phospholipase A activity in the parent strain but not in two, independent phospholipase A mutants. A gene for phospholipase A, pldA (hereafter referred to as pla to avoid confusion with the gene for phospholipase D, pld ), is present in all sequenced gonococcal strains. Fluid phase, hemolytic activity assays showed that 25 of 29 gonococcal strains tested had hemolytic activity greater than 50% of the positive control. In support of PLA as a gonococcal outer membrane protein, supernatants from 24-, 48-, and 72-h cultures of N. gonorrhoeae strain 1291 did not contain hemolysin activity, and a monoclonal antibody specific for gonococcal phospholipase A failed to detect the enzyme in these supernatants. The organism must be viable for lysis to occur, and the inclusion of EDTA in the media removes all activity. Our studies have shown that a phospholipase A mutant has significantly reduced survival in human neutrophils and primary human cervical epithelial cells compared to the parent gonococcal strain after 3 h of incubation. Collectively, our data demonstrate that gonococcal PLA lyses host cell membranes, which is important for intracellular survival., Importance: Intracellular survival is crucial to the success of Neisseria gonorrhoeae as a human pathogen. Multiple factors contribute to the intracellular survival of gonococci, including the ability to prohibit apoptosis of the epithelial cell the organism invades and mechanisms to evade host innate defense systems. The role of phospholipase A (PLA), an outer membrane protein, is important as it disrupts the host vacuolar and phagolysosomal membranes, preventing the effective delivery of innate immune factors that normally restrict organism growth within human cells. After cell entry, PLA disrupts the integrity of these host cell membranes, allowing the gonococcus to live free within disrupted vacuoles where it pilfers host cell nutrients that enable its survival and replication. A vaccine or drug that could neutralize PLA activity would disrupt the intracellular survival of the gonococcus., Competing Interests: The authors declare no conflict of interest.

Tissue-specific cardiolipin fatty acyl profiles are achieved by remodeling of de novo synthesized cardiolipin, and four remodeling enzymes have thus far been identified. We studied the enzyme phospholipase A and acyltransferase 1 (PLAAT1), and we report the discovery that it has phosphatidylcholine (PC):monolysocardiolipin (MLCL) transacylase activity. Subcellular localization was analyzed by differential centrifugation and immunoblotting. Total levels of major phospholipids, and the fatty acyl profile of cardiolipin, were analyzed in HEK293 cells expressing murine PLAAT1 using gas chromatography. Apparent enzyme kinetics of affinity-purified PLAAT1 were calculated using radiochemical enzyme assays. This enzyme was found to localize predominantly to the endoplasmic reticulum (ER) but was detected at low levels in the mitochondria-associated ER matrix. Cells expressing PLAAT1 had higher levels of total cardiolipin, but not other phospholipids, and it was primarily enriched in the saturated fatty acids myristate, palmitate, and stearate, with quantitatively smaller increases in the n-3 polyunsaturated fatty acids linolenate, eicosatrienoate, and eicosapentanoate and the monounsaturated fatty acid erucate. Affinity-purified PLAAT1 did not catalyze the transacylation of MLCL using 1-palmitoyl-2-[14C]-linoleoyl-PC as an acyl donor. However, PLAAT1 had an apparent Vmax of 1.61 μmol/min/mg protein and Km of 126 μM using [9,10-3H]-distearoyl-PC as an acyl donor, and 0.61 μmol/min/mg protein and Km of 16 μM using [9,10-3H]-dioleoyl-PC. PLAAT1 is therefore a novel PC:MLCL transacylase. [ABSTRACT FROM AUTHOR]

Clubroot, caused by the obligate parasite Plasmodiophora brassicae , is one of the most devastating diseases of canola (Brassica napus) in Canada. The identification of novel genes that contribute to clubroot resistance is important for the sustainable management of clubroot, as these genes may be used in the development of resistant canola cultivars. Phospholipase As (PLAs) play important roles in plant defense signaling and stress tolerance, and thus are attractive targets for crop breeding. However, since canola is an allopolyploid and has multiple copies of each PLA gene, it is time-consuming to test the functions of PLAs directly in this crop. In contrast, the model plant Arabidopsis thaliana has a simpler genetic background and only one copy of each PLA. Therefore, it would be reasonable and faster to validate the potential utility of PLA genes in Arabidopsis first. In this study, we identified seven homozygous atpla knockout/knockdown mutants of Arabidopsis , and tested their performance following inoculation with P. brassicae. Four mutants (pla1-ii α, pla1-i γ 3 , pla1-iii , ppla-iii β, ppla-iii δ) developed more severe clubroot than the wild-type, suggesting increased susceptibility to P. brassicae. The homologs of these Arabidopsis PLAs (AtPLAs) in B. napus (BnPLAs) were identified through Blast searches and phylogenic analysis. Expression of the BnPLAs was subsequently examined in transcriptomic datasets generated from canola infected by P. brassicae , and promising candidates for further characterization identified. [ABSTRACT FROM AUTHOR]

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases of canola (Brassica napus) in Canada. The identification of novel genes that contribute to clubroot resistance is important for the sustainable management of clubroot, as these genes may be used in the development of resistant canola cultivars. Phospholipase As (PLAs) play important roles in plant defense signaling and stress tolerance, and thus are attractive targets for crop breeding. However, since canola is an allopolyploid and has multiple copies of each PLA gene, it is time-consuming to test the functions of PLAs directly in this crop. In contrast, the model plant Arabidopsis thaliana has a simpler genetic background and only one copy of each PLA. Therefore, it would be reasonable and faster to validate the potential utility of PLA genes in Arabidopsis first. In this study, we identified seven homozygous atpla knockout/knockdown mutants of Arabidopsis, and tested their performance following inoculation with P. brassicae. Four mutants (pla1-iiα, pla1-iγ3, pla1-iii, ppla-iiiβ, ppla-iiiδ) developed more severe clubroot than the wild-type, suggesting increased susceptibility to P. brassicae. The homologs of these Arabidopsis PLAs (AtPLAs) in B. napus (BnPLAs) were identified through Blast searches and phylogenic analysis. Expression of the BnPLAs was subsequently examined in transcriptomic datasets generated from canola infected by P. brassicae, and promising candidates for further characterization identified.

The aim of this work was to study the phospholipase A (PLA) enzyme activity of extracts from Trichoderma harzianum 1A and Trichoderma atroviride αCp8 culture media. Fermentation on submerged culture was performed and PLA activity of extracts was evaluated as a function of culture time. A turbidimetric method for continuous analysis of phospholipase activity was applied. By this, vesicles rather than soluble substrates are used resembling the in vivo environment. In Trichoderma's culture experiments, lecithin phospholipids as inductors promoted a PLA expression level of the same order to the corresponding strain control assay. T. harzianum developed the highest PLA activity and was considered as the most potent strain. Furthermore, the activity level was clearly greater when the culture medium included the plant pathogenic fungus Sclerotium rolfsii. The T. harzianum strain showed the highest PLA potential. Besides, both T. harzianum and T. atroviride, developed higher PLA activity at the first days of culture. In the present work, T. harzianum 1A and T. atroviride αCp8 at a lower level, showed an important PLA potential for S. rolfsii control. On the other side, a lower relative response was observed when both Trichoderma strains were assayed against other pathogens such as Macrophomina phaseolina and Rhizoctonia solani. The present work contributes to the scientific knowledge about PLA activity in Trichoderma and could be useful for technological purposes to prepare formulations for the biological control of S. rolfsii in agriculture. [ABSTRACT FROM AUTHOR]

Glucocorticoids (GCs) are known to play an important role in maintaining basal and stress-related homeostasis by interacting with endocrine mediators and prostaglandins (PGs). Although a growing body of evidence shows that GCs exert their regulatory action at a multitude of sites in the reproductive axis through corticosteroid receptors, little is known about the direct role of cortisol, an active form of GCs, in the equine endometrium. Thus, the study aimed to determine the effect of cortisol on PGF 2α synthesis in the endometrial tissue and cells in vitro. In Exp.1, the immunolocalization and the expression of the glucocorticoid receptor (GCR) in the endometrium throughout the estrous cycle were established. In Exp. 2 and 3, the effects of cortisol on PGF 2α secretion and transcripts associated with the arachidonic acid (AA) cascade in endometrial tissues, and cells were defined. Endometrial tissues obtained from the early, mid, and late luteal phases and the follicular phase of the estrous cycle were exposed to cortisol (100, 200, and 400 nM) for 24 h. Endometrial epithelial and stromal cells (early phase of estrous cycle) were exposed to cortisol (100 nM) for 24 h. Then, PGF 2α secretion and transcripts associated with the AA cascade (PLA2G2A, PLA2G4A, PTGS2, and PGFS) were assessed. GCR was expressed in the cytoplasm and the nucleus in the luminal and glandular epithelium as well as in the stroma. Endometrial GCR protein abundance was up-regulated at the late luteal phase compared to the mid-luteal phase of the estrous cycle. Cortisol dose-dependently decreased PGF 2α secretion, PLA2G2A and PLA2G4A transcripts in endometrial tissues. Additionally, cortisol treatment decreased PGF 2α secretion from endometrial epithelial and stromal cells. Moreover, it affected PLA2G2A , PLA2G4A , and PTGS2 transcripts in endometrial stromal cells. These findings suggest that cortisol suppresses the synthesis of PGF 2α by affecting the AA cascade in the equine endometrium during the estrous cycle. • GCR protein abundance was up-regulated at the late luteal phase. • Cortisol decreased PGF 2α secretion from endometrial tissue and cells. • Cortisol decreased PLA2G2A and PLA2G4A transcripts in endometrial tissues. [ABSTRACT FROM AUTHOR]

Tissue-specific cardiolipin fatty acyl profiles are achieved by remodeling of de novo synthesized cardiolipin, and four remodeling enzymes have thus far been identified. We studied the enzyme phospholipase A and acyltransferase 1 (PLAAT1), and we report the discovery that it has phosphatidylcholine (PC):monolysocardiolipin (MLCL) transacylase activity. Subcellular localization was analyzed by differential centrifugation and immunoblotting. Total levels of major phospholipids, and the fatty acyl profile of cardiolipin, were analyzed in HEK293 cells expressing murine PLAAT1 using gas chromatography. Apparent enzyme kinetics of affinity-purified PLAAT1 were calculated using radiochemical enzyme assays. This enzyme was found to localize predominantly to the endoplasmic reticulum (ER) but was detected at low levels in the mitochondria-associated ER matrix. Cells expressing PLAAT1 had higher levels of total cardiolipin, but not other phospholipids, and it was primarily enriched in the saturated fatty acids myristate, palmitate, and stearate, with quantitatively smaller increases in the n-3 polyunsaturated fatty acids linolenate, eicosatrienoate, and eicosapentanoate and the monounsaturated fatty acid erucate. Affinity-purified PLAAT1 did not catalyze the transacylation of MLCL using 1-palmitoyl-2-[14C]-linoleoyl-PC as an acyl donor. However, PLAAT1 had an apparent Vmax of 1.61 μmol/min/mg protein and Km of 126 μM using [9,10-3H]-distearoyl-PC as an acyl donor, and 0.61 μmol/min/mg protein and Km of 16 μM using [9,10-3H]-dioleoyl-PC. PLAAT1 is therefore a novel PC:MLCL transacylase.

SUMMARY: Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12‐HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn‐1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over‐expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase‐like PLAs (LCAT‐PLAs) in HFA biosynthesis were characterized. The LCAT‐PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2, and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT‐PLAs from the HFA‐accumulating plant species Physaria fendleri (PfLCAT‐PLA) and castor (RcLCAT‐PLA) could cleave acyl chains at both the sn‐1 and sn‐2 positions of PC, and displayed substrate selectivity towards sn‐2‐ricinoleoyl‐PC over sn‐2‐oleoyl‐PC. Furthermore, co‐expression of RcFAH12 with PfLCAT‐PLA or RcLCAT‐PLA, but not Arabidopsis AtLCAT‐PLA, resulted in increased occupation of HFA at the sn‐1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT‐PLA and RcLCAT‐PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops. Significance Statement: Plant phospholipase A has long been proposed to contribute to hydroxy fatty acid (HFA) enrichment, but the specifics of this process remain elusive. The current study showed that Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase‐like phospholipases are able to selectively cleave hydroxy acyl chains from phosphatidylcholine and may contribute to HFA turnover. [ABSTRACT FROM AUTHOR]

Abstract Background Many gram-negative bacteria produce an outer membrane phospholipase A (PldA) that plays an important role in outer membrane function and is associated with virulence. Results In the current study, we characterized a pldA mutant of Coxiella burnetii, an intracellular gram-negative pathogen and the agent of human Q fever. The C. burnetti pldA open reading frame directs synthesis of a protein with conserved PldA active site residues. A C. burnetii ΔpldA deletion mutant had a significant growth defect in THP-1 macrophages, but not axenic medium, that was rescued by complementation. Thin layer chromatography was employed to assess whether pldA plays a role in remodeling membrane lipids during C. burnetii morphological differentiation. Extracted lipids were analyzed from replicating, logarithmic phase large cell variants (LCVs), non-replicating, stationary phase small cell variants (SCVs), and a mixture of LCVs and SCVs. Similar to Escherichia coli, all three forms contained cardiolipin (CL), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). However, PE and PG were present in lower quantities in the SCV while three additional lipid species were present in higher quantities. Co-migration with standards tentatively identified two of the three SCV-enriched lipids as lyso-phosphatidylethanolamine, a breakdown product of PE, and free fatty acids, which are generally toxic to bacteria. Developmental form lipid modifications required the activity of PldA. Conclusions Collectively, these results indicate developmentally-regulated lipid synthesis by C. burnetii contributes to colonization of macrophages and may contribute to the environmental stability and the distinct biological properties of the SCV.

Legionella pneumophila encodes an extracellular secreted phospholipase A named PlaA that is translocated by the type II secretion system. It plays an essential role in maintaining the integrity of Legionella‐containing vacuoles in L. pneumophila pathogenesis. Here, it is shown that PlaA has a main lysophospholipase activity to hydrolyze fatty‐acyl groups in lysophospholipids. Although it has a very low phospholipase A activity to catalyze the hydrolysis of fatty‐acyl groups in phospholipids, PlaA can bind phospholipids such as 1,2‐dipalmitoylphosphatidylcholine with a dissociation constant of 11.1 µM. Sequence‐alignment analysis combined with activity assays revealed that PlaA contains a distinct substrate‐binding site among the known structures of the phospholipase A family, implying that PlaA may present a novel mechanism for substrate recognition. Native PlaA and its selenomethionine (SeMet)‐substituted form were purified and crystallized by vapour diffusion in hanging drops at 296 K. Diffraction data were collected to a resolution of 2.0 Å for native PlaA protein and to a resolution of 2.7 Å for SeMet‐substituted PlaA protein. The crystals of native PlaA belonged to the monoclinic space group P21, while the crystals of SeMet‐substituted PlaA belonged to the primitive orthorhombic space group P212121. Initial phases for PlaA were obtained from SeMet SAD data sets. [ABSTRACT FROM AUTHOR]

Key message: Castor patatin-like phospholipase A IIIβ facilitates the exclusion of hydroxy fatty acids from phosphatidylcholine in developing transgenic Arabidopsis seeds. Hydroxy fatty acids (HFAs) are industrial useful, but their major natural source castor contains toxic components. Although expressing a castor OLEATE 12-HYDROXYLASE in Arabidopsis thaliana leads to the synthesis of HFAs in seeds, a high proportion of the HFAs are retained in phosphatidylcholine (PC). Thus, the liberation of HFA from PC seems to be critical for obtaining HFA-enriched seed oils. Plant phospholipase A (PLA) catalyzes the hydrolysis of PC to release fatty acyl chains that can be subsequently channeled into triacylglycerol (TAG) synthesis or other metabolic pathways. To further our knowledge regarding the function of PLAs from HFA-producing plant species, two class III patatin-like PLA cDNAs (pPLAIIIβ or pPLAIIIδ) from castor or Physaria fendleri were overexpressed in a transgenic line of A. thaliana producing C18-HFA, respectively. Only the overexpression of RcpPLAIIIβ resulted in a significant reduction in seed HFA content with concomitant changes in fatty acid composition. Reductions in HFA content occurred in both PC and TAG indicating that HFAs released from PC were not incorporated into TAG. These results suggest that RcpPLAIIIβ may catalyze the removal of HFAs from PC in the developing seeds synthesizing these unusual fatty acids. [ABSTRACT FROM AUTHOR]

Crude oil degumming by phospholipid removal is crucial to guarantee oil quality. Phospholipase degumming could produce green vegetable oil by reducing energy consumption and protecting the environment. To develop a novel phospholipase for oil degumming, we cloned the Serratia marcescens outer membrane phospholipase A gene (OM-PLA1) and expressed its 33 KDa protein in engineered Escherichia coli BL21(DE3). OM-PLA1 activity reached 18.9 U mL−1 with the induction of 0.6 mM isopropyl β-D-1-thiogalactopyranoside for 4 h. The optimum temperature and pH were 50°C and 7.5, respectively. Mg2+, Ca2+, Co2+, and Mn2+ at 0.1 mM L−1 significantly increased OM-PLA1 activity. The kinetic equations of OM-PLA1 and Lecitase Ultra were y = 13.7x+0.74 (Km = 18.53 mM, Vmax = 1.35 mM min−1) and y = 24.42x+0.58 (Km = 42.1 mM, Vmax = 1.72 mM min−1), respectively. The phosphorus content decreased from 22.6 to 9.3 mg kg−1 with the addition of 15 units of free recombinant OM-PLA1 into 150 g of crude rapeseed oil. OM-PLA1 has the close degumming efficiency with Lecitase Ultra. The S. marcescens outer membrane phospholipase gene (OM-PLA1) possessed higher substrate affinity and catalytic efficiency than Lecitase Ultra. This study provides an alternative approach to achieve crude vegetable oil degumming with enzymatic technology.

Phospholipases (PLs) are lipid-hydrolyzing enzymes known to have diverse signaling roles during plant abiotic and biotic stress responses. They catalyze lipid remodeling, which is required to generate rapid responses of plants to environmental cues. Moreover, they produce second messenger molecules, such as phosphatidic acid (PA) and thus trigger or modulate signaling cascades that lead to changes in gene expression. The roles of phospholipases in plant abiotic and biotic stress responses have been intensively studied. Nevertheless, emerging evidence suggests that they also make significant contributions to plants’ cellular and developmental processes. In this mini review, we summarized recent advances in the study of the cellular and developmental roles of phospholipases in plants.

Phospholipases (PLs) are lipid-hydrolyzing enzymes known to have diverse signaling roles during plant abiotic and biotic stress responses. They catalyze lipid remodeling, which is required to generate rapid responses of plants to environmental cues. Moreover, they produce second messenger molecules, such as phosphatidic acid (PA) and thus trigger or modulate signaling cascades that lead to changes in gene expression. The roles of phospholipases in plant abiotic and biotic stress responses have been intensively studied. Nevertheless, emerging evidence suggests that they also make significant contributions to plants' cellular and developmental processes. In this mini review, we summarized recent advances in the study of the cellular and developmental roles of phospholipases in plants. [ABSTRACT FROM AUTHOR]

The concepts of phospholipase activity is often taught in undergraduate biology and biochemistry classes and reinforced in laboratory exercises. However, very rarely does the design of these exercises allow students to directly gain experience in the use of modern instruments such as digital imaging systems and fluorescence spectrophotometers. The laboratory exercise described here involves the use of fluorescent lipids to evaluate phospholipase activity. Students use thin layer chromatography (TLC) to understand how lipids change under different conditions (i.e. abiotic and biotic stress). They explore strategies to separate, visualize and quantify lipids by TLC, digital imaging, and fluorometry. They also have increased opportunities for hands‐on practise with experimental design, liposome sample preparation, and implementation of instrumentation commonly used by experienced researchers; all while learning and applying fundamental concepts about lipids. © 2018 International Union of Biochemistry and Molecular Biology, 47(1):100–105, 2018. [ABSTRACT FROM AUTHOR]

Doubled haploids offer a rapid method of producing homozygous lines for accelerated breeding of varieties and hybrids necessary to address the food demands of the next 2-3 decades. Further, doubled haploids are invaluable in basic genetic research such as gene discovery, genetic mapping, and genome sequencing, especially in perennial crops. Haploids are produced mainly through in vitro culture of anthers, microspores, or ovules. Some inter-specific, inter-generic crosses and crosses of different ploidy of the same species or different species also consistently give haploids through selective elimination of a set of chromosomes coming from one of the parents during zygotic divisions. Haploid inducer (HI) lines that yield haploids upon crossing have been commercially employed in breeding of maize, barley, and wheat. In recent years, novel ways of artificially synthesizing HI lines have been devised based on basic studies with Arabidopsis. The role of centromeric histone protein coding gene CENH3, which plays critical role in centromere specification and chromosome segregation, has been exploited to engineer HI lines in Arabidopsis and maize. Further, the discovery of the gene encoding pollen-specific phospholipase A (MTL/NLD/ZmPLA1) responsible for inducing haploids in maize has opened an additional way of constructing HI lines in crop species. We present here a brief overview of various in vivo haploid production methods and discuss the challenges in practical use of doubled haploid technology. [ABSTRACT FROM AUTHOR]

Phospholipids (PLs), as natural additives and nutritional supplements, are recovered from the gum phase during the degumming process of crude vegetable oils. The molecular species of phospholipids recovered from crude soybean (SO), flaxseed (FO) and peanut oil (PO) by different degumming processes, including water (WD), acid (AD), PLA 1 (EDA), PLC (EDC) and PLA 1 -PLC (EDAC) were investigated. According to the PLS-DA model, a total of 30, 38 and 21 characteristic markers were identified using UPLC-Q-TOF-MS in the phospholipids recovered from SO, FO and PO by different degumming processes, respectively. PC (18:2/18:2) and PE (18:2/18:2) were the main phospholipids for WD and AD, LPC (18:2) and LPE (18:2) were the main phospholipids for EDA and EDAC, and PI (16:0/18:2) and PI (16:0/18:1) were served as characteristic phospholipids for EDC. In addition, a lamellar liquid-crystalline phase was observed in the recovered phospholipids, which indicated that the microstructure of phospholipids recovered from crude vegetable oils were significantly influenced by different degumming processes. These differential molecular species of phospholipid products provide useful information to facilitate the potential utilization of phospholipid products as emulsifiers in food and as ingredients in the pharmaceutical industries. [Display omitted] • Molecules of phospholipid were affected by different degumming processes. • Profile of phospholipids recovered from crude flaxseed oil was first reported. • Differential phospholipids were screened as markers by degumming processes. • Microstructures of phospholipids were affected by different degumming processes. [ABSTRACT FROM AUTHOR]

Membrane protein and phospholipid (PL) composition changes in response to environmental cues and during infections. To achieve these, bacteria use adaptation mechanisms involving covalent modification and remodelling of the acyl chain length of PLs. However, little is known about bacterial pathways regulated by PLs. Here, we investigated proteomic changes in the biofilm of P. aeruginosa phospholipase mutant (∆ plaF) with altered membrane PL composition. The results revealed profound alterations in the abundance of many biofilm-related two-component systems (TCSs), including accumulation of PprAB, a key regulator of the transition to biofilm. Furthermore, a unique phosphorylation pattern of transcriptional regulators, transporters and metabolic enzymes, as well as differential production of several proteases, in ∆ plaF , indicate that PlaF-mediated virulence adaptation involves complex transcriptional and posttranscriptional response. Moreover, proteomics and biochemical assays revealed the depletion of pyoverdine-mediated iron uptake pathway proteins in ∆ plaF , while proteins from alternative iron-uptake systems were accumulated. These suggest that PlaF may function as a switch between different iron-acquisition pathways. The observation that PL-acyl chain modifying and PL synthesis enzymes were overproduced in ∆ plaF reveals the interconnection of degradation, synthesis and modification of PLs for proper membrane homeostasis. Although the precise mechanism by which PlaF simultaneously affects multiple pathways remains to be elucidated, we suggest that alteration of PL composition in ∆ plaF plays a role for the global adaptive response in P. aeruginosa mediated by TCSs and proteases. Our study revealed the global regulation of virulence and biofilm by PlaF and suggests that targeting this enzyme may have therapeutic potential. • Phospholipid remodelling phospholipase A, PlaF, is a biofilm regulator. • PlaF affects membrane PL homeostasis pathways. • PlaF affects two-component system-mediated sensing and transduction. • PlaF affects iron uptake and protein turnover. [ABSTRACT FROM AUTHOR]

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

The processes of free radical oxidation are shown to be activated at the expense of antioxidants depletion if tympanism of young sterlet is developing. At the same time the level of phospholipids decreases, the content of mono- and diacylglycerols increases considerably, and the processes of lipolysis are activated with simultaneous rising of the lisophosphatidylcholine and sphingomyelin levels and decreasing in phosphatidylserine.

The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity.

Human oral exposure to aflatoxin B1 (AFB1) and fumonisin B1 (FB1) is associated with increased hepatocellular carcinoma. Although evidence suggested interactive AFB1-FB1 hepatotoxicity, the underlying mechanisms remain mostly unidentified. This work was aimed at evaluating the possible AFB1-FB1 interplay to induce genetic and cell cycle toxicities in BRL-3A rat hepatocytes, reactive oxygen species (ROS) involvement, and the AFB1 metabolizing pathways cytochrome P450 (CYP) and arachidonic acid (ArAc) metabolism as ROS contributors. Flow cytometry of stained BRL-3A hepatocytes was used to study the cell cycle (propidium iodide), ROS intracellular production (DCFH-DA, HE, DAF-2 DA), and phospholipase A activity (staining with bis-BODIPY FL C11-PC). The CYP1A activity was assessed by the 7-ethoxyresorufin-O-deethylase (EROD) assay. Despite a 48-h exposure to FB1 (30 μM) not being genotoxic, the AFB1 (20 μM)-induced micronucleus frequency was overcome by the AFB1-FB1 mixture (MIX), presumably showing toxin interaction. The mycotoxins blocked G1/S-phase, but only MIX caused cell death. Overall, the oxidative stress led these alterations as the pretreatment with N-acetyl-l-cysteine reduced such toxic effects. While AFB1 had a major input to the MIX pro-oxidant activity, with CYP and ArAc metabolism being ROS contributors, these pathways were not involved in the FB1-elicited weak oxidative stress. The MIX-induced micronucleus frequency in N-acetyl- l-cysteine pretreated cells was greater than that caused by AFB1 without antioxidants, suggesting enhanced AFB1 direct genotoxicity probably owing to the higher CYP activity and ArAc metabolism found in MIX. The metabolic pathways modulation by AFB1-FB1 mixtures could raise its hepatocarcinogenic properties. [ABSTRACT FROM AUTHOR]

Russell's vipers are snakes of major medical importance in Asia. Russell's viper ( Daboia russelii) envenoming in Sri Lanka and South India leads to a unique, mild neuromuscular paralysis, not seen in other parts of the world where the snake is found. This study aimed to identify and pharmacologically characterise the major neurotoxic components of Sri Lankan Russell's viper venom. Venom was fractionated using size exclusion chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). In vitro neurotoxicities of the venoms, fractions and isolated toxins were measured using chick biventer and rat hemidiaphragm preparations. A phospholipase A (PLA) toxin, U1-viperitoxin-Dr1a (13.6 kDa), which constitutes 19.2 % of the crude venom, was isolated and purified using HPLC. U1-viperitoxin-Dr1a produced concentration-dependent in vitro neurotoxicity abolishing indirect twitches in the chick biventer nerve-muscle preparation, with a t of 55 ± 7 min only at 1 μM. The toxin did not abolish responses to acetylcholine and carbachol indicating pre-synaptic neurotoxicity. Venom, in the absence of U1-viperitoxin-Dr1a, did not induce in vitro neurotoxicity. Indian polyvalent antivenom, at the recommended concentration, only partially prevented the neurotoxic effects of U1-viperitoxin-Dr1a. Liquid chromatography mass spectrometry analysis confirmed that U1-viperitoxin-Dr1a was the basic S-type PLA toxin previously identified from this venom (NCBI-GI: 298351762; SwissProt: P86368). The present study demonstrates that neurotoxicity following Sri Lankan Russell's viper envenoming is primarily due to the pre-synaptic neurotoxin U1-viperitoxin-Dr1a. Mild neurotoxicity observed in severely envenomed Sri Lankan Russell's viper bites is most likely due to the low potency of U1-viperitoxin-Dr1a, despite its high relative abundance in the venom. [ABSTRACT FROM AUTHOR]

• Bacillus subtilis caused morphological distortions in Fusarium graminearum macroconidia and modified the mycelium's lipid content. • Bacillus subtilis and Fusarium graminearum modulate phospholipase A activity, proline synthesis, and the transcription of genes that code the metabolism of phosphatidylcholine in barley roots. • HvLPCAT and HvPLA were up-regulated in barley roots during the interaction with Fusarium macroconidia. • Bacillus subtilis and Fusarium graminearum evoke a differential expression of isochorismate synthase and phenylalanine ammonia-lyase. Barley (Hordeum vulgare), one of the main cereals cultivated around the world, is commonly affected by Fusarium head blight, a disease caused by fungal pathogen Fusarium graminearum. Although several control strategies have been developed, the lack of resistant cultivars and the risks of irritation posed by chemical fungicides mean that current efforts focus on biocontrol, a more sustainable and environmentally friendly approach. The aim of this research was to observe the alterations produced by a strain of Bacillus subtilis in the biology of Fusarium graminearum , and whether the response to the fungus was accordingly modified in barley roots. We found that Bacillus subtilis ATCC 6633 caused morphological distortions in Fusarium graminearum macroconidia and modified the mycelium's lipid content, so that fungal growth was significantly impaired even after exposure to the bacterium had stopped. In barley assays, interaction with the two microorganisms significantly affected the plant in terms of phospholipase A activity, proline synthesis, and the transcription of genes that code lysophosphatidylcholine acyltransferases (HvLPCAT) and phospholipase A (HvPLA), involved in the metabolism of phosphatidylcholine. The accumulation of reactive oxygen species, however, was not modulated. Analysis of other immunity events triggered by pathogen-associated molecular patterns indicated differential expression of the genes that code two key enzymes to synthesize salicylic acid (SA): an isochorismate synthase (ICS) and phenylalanine ammonia-lyase (PAL). Overall, these findings contribute to our understanding of Bacillus subtilis ATCC 6633′s biocontrol potential, with the ultimate aim of improving biocontrol schemes to protect barley from Fusarium disease [ABSTRACT FROM AUTHOR]

Cardiac resistance against acute ischemia/reperfusion (I/R) injury can be enhanced by adaptation to chronic intermittent hypoxia (CIH), but the changes at the molecular level associated with this adaptation are still not fully explored. Phospholipase A (PLA) plays an important role in phospholipid metabolism and may contribute to membrane destruction under conditions of energy deprivation during I/R. The aim of this study was to determine the effect of CIH (7000 m, 8 h/day, 5 weeks) on the expression of cytosolic PLAα (cPLAα) and its phosphorylated form (p-cPLAα), as well as other related signaling proteins in the left ventricular myocardium of adult male Wistar rats. Adaptation to CIH increased the total content of cPLAα by 14 % in myocardial homogenate, and enhanced the association of p-cPLAα with the nuclear membrane by 85 %. The total number of β-adrenoceptors (β-ARs) did not change but the β/β ratio markedly increased due to the elevation of β-ARs and drop in β-ARs. In parallel, the amount of adenylyl cyclase decreased by 49 % and Gα proteins increased by about 50 %. Besides that, cyclooxygenase 2 (COX-2) and prostaglandin E (PGE) increased by 36 and 84 %, respectively. In parallel, we detected increased phosphorylation of protein kinase Cα, ERK1/2 and p38 (by 12, 48 and 19 %, respectively). These data suggest that adaptive changes induced in the myocardium by CIH may include activation of cPLAα and COX-2 via β-AR/G-mediated stimulation of the ERK/p38 pathway. [ABSTRACT FROM AUTHOR]

Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.