RESUMO
The respective life histories of human subjects and mice are well defined and describe a unique story of evolutionary conservation extending from sequence identity within the genome to the underpinnings of biochemical, cellular, and physiologic pathways. As a consequence, the hematopoietic lineages of both species are invariantly maintained, each with identifiable eosinophils. This canonical presence nonetheless does not preclude disparities between human and mouse eosinophils, their effector functions, or both. Indeed, many books and reviews dogmatically highlight differences, providing a rationale to discount the use of mouse models of human eosinophilic diseases. We suggest that this perspective is parochial and ignores the wealth of available studies and the consensus of the literature that overwhelming similarities (and not differences) exist between human and mouse eosinophils. The goal of this review is to summarize this literature and in some cases provide experimental details comparing and contrasting eosinophils and eosinophil effector functions in human subjects versus mice. In particular, our review will provide a summation and an easy-to-use reference guide to important studies demonstrating that although differences exist, more often than not, their consequences are unknown and do not necessarily reflect inherent disparities in eosinophil function but instead species-specific variations. The conclusion from this overview is that despite nominal differences, the vast similarities between human and mouse eosinophils provide important insights as to their roles in health and disease and, in turn, demonstrate the unique utility of mouse-based studies with an expectation of valid extrapolation to the understanding and treatment of patients.
Assuntos
Eosinófilos/fisiologia , Animais , Degranulação Celular , Proteína Catiônica de Eosinófilo/fisiologia , Peroxidase de Eosinófilo/fisiologia , Evolução Molecular , Glicoproteínas/fisiologia , Hematopoese , Humanos , Lisofosfolipase/fisiologia , CamundongosRESUMO
PURPOSE: To determine the virulence of extracellular phospholipase B (PLB) of Candida albicans in keratomycosis. METHODS: A model of keratomycosis was established in 48 New Zealand albino rabbits covered with contact lenses. Effects of PLB-deficient mutant strain of C. albicans and its isogenic parental strain on the keratomycosis were compared. In vitro, these two strains were incubated with corneal stromal cells respectively (37 degrees C, 5% CO(2)). The influence of the strains on monolayer keratocytes was detected by scanning electron microscopy, enzyme-linked immunosorbent assay, and flow cytometry with Annexin-V/propidium iodide. RESULTS: Fungal hyphae grew perpendicularly to the corneal stromal lamellae. The difference of the two strains in hyphal invasion was significant at two days after inoculation (p = 0.002) but not significant at the other timepoints. Severity of inflammation in rabbits with the parental strain was the same as that with the PLB null strain (p > 0.05). The morphogenesis and number of adherent germ tubes of the two strains were similar (p > 0.05), but the number of germ tubes penetrating cell monolayer was significantly different (p = 0.009). More prostaglandin E(2) was detected in the culture supernatants of the parental strain group than the null strain group. The percentages of cells with damaged cellular membrane were 3.02%, 2.04%, and 0.12% in the parental group, the PLB null group, and the control group, respectively. Apoptosis cells accounted for 33.17%, 27.56%, and 1.46%, and living cells accounted for 63.81%, 70.40%, and 98.41% in the three groups, respectively. CONCLUSION: PLB can play a role as a virulent factor in triggering fungal invasion in corneas immediately after fungal adherence by decomposing membrane phospholipids and leading to cell lysis. However, its virulent effect does not appear to be as critical as in the hematogenous model of disseminated candidiasis.
Assuntos
Candida albicans/enzimologia , Candida albicans/patogenicidade , Candidíase/microbiologia , Doenças da Córnea/microbiologia , Infecções Oculares Fúngicas/microbiologia , Lisofosfolipase/fisiologia , Animais , Candidíase/patologia , Células Cultivadas , Doenças da Córnea/patologia , Substância Própria/microbiologia , Substância Própria/ultraestrutura , DNA Fúngico/análise , Dinoprostona/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Infecções Oculares Fúngicas/patologia , Citometria de Fluxo , Hifas/fisiologia , Hifas/ultraestrutura , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase , Coelhos , VirulênciaRESUMO
Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing beta-1,6-linked glucan was released from H99 (wild-type strain) cell walls by beta-1,3 glucanase, consistent with covalent attachment of Plb1 via beta-1,6-linked glucans to beta-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained beta-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, DeltaPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of DeltaPLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.
Assuntos
Parede Celular/metabolismo , Cryptococcus neoformans/enzimologia , Lisofosfolipase/fisiologia , Motivos de Aminoácidos , Transporte Biológico , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Lisofosfolipase/química , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Frações Subcelulares , Superóxido Dismutase/metabolismo , TemperaturaRESUMO
BACKGROUND: Surfactant dysfunction is implicated in small airway closure in asthma. Increased activity of secretory phospholipase A(2) (sPLA(2)) in the airways is associated with asthma exacerbations. Phosphatidylcholine, the principal component of pulmonary surfactant that maintains small airway patency, is hydrolyzed by sPLA(2). The lysophosphatidylcholine product is the substrate for eosinophil lysophospholipases. OBJECTIVE: To determine whether surfactant phospholipid hydrolysis by the combined activities of sPLA(2)s and eosinophil lysophospholipases induces surfactant dysfunction. METHODS: The effect of these enzymes on surfactant function was determined by capillary surfactometry. Thin layer chromatography was used to correlate enzyme-induced changes in surfactant phospholipid composition and function. Phosphatidylcholine and its hydrolytic products were measured by using mass spectrometry. RESULTS: Eosinophils express a 25-kd lysophospholipase and group IIA sPLA(2). Phospholipase A(2) alone induced only a small decrease in surfactant function, and 25-kd lysophospholipase alone degraded lysophosphatidylcholine but had no effect on surfactant function. The combined actions of sPLA(2) and lysophospholipase produced dose-dependent and time-dependent losses of surfactant function, concomitant with hydrolysis of phosphatidylcholine and lysophosphatidylcholine. Lysates of AML14.3D10 eosinophils induced surfactant dysfunction, indicating these cells express all the necessary lipolytic activities. In contrast, lysates of blood eosinophils required exogenous phospholipase A(2) to induce maximal surfactant dysfunction. CONCLUSION: The combined activities of sPLA(2)s and eosinophil lysophospholipases are necessary to degrade surfactant phospholipids sufficiently to induce functional losses in surfactant activity as reported in asthma. CLINICAL IMPLICATIONS: The phospholipases and lysophospholipases expressed by eosinophils or other airway cells may represent novel therapeutic targets for blocking surfactant degradation, dysfunction, and peripheral airway closure in asthma.
Assuntos
Eosinófilos/enzimologia , Glicoproteínas/metabolismo , Lisofosfolipase/metabolismo , Fosfolipases A/metabolismo , Fosfolipídeos/metabolismo , Surfactantes Pulmonares/antagonistas & inibidores , Surfactantes Pulmonares/metabolismo , Animais , Catálise , Linhagem Celular Tumoral , Células Cultivadas , Sinergismo Farmacológico , Ativação Enzimática/fisiologia , Eosinófilos/metabolismo , Glicoproteínas/fisiologia , Fosfolipases A2 do Grupo II , Humanos , Hidrólise , Lisofosfolipase/fisiologia , Camundongos , Fosfolipases A/fisiologia , Fosfolipídeos/fisiologiaRESUMO
Cryptococci survive and replicate within macrophages and can use exogenous arachidonic acid for the production of eicosanoids. Phospholipase B1 (PLB1) has a putative, but uninvestigated, role in these processes. We have shown that uptake and esterification of radiolabeled arachidonic, palmitic, and oleic acids by the Cryptococcus neoformans var. grubii H99 wild-type strain and its PLB1 deletion mutant strain (the Deltaplb1 strain) are independent of PLB1, except under hyperosmolar stress. Similarly, PLB1 was required for metabolism of 1-palmitoyl lysophosphatidylcholine (LysoPC), which is toxic to eukaryotic cell membranes, under hyperosmolar conditions. During both logarithmic and stationary phases of growth, the physiologically relevant phospholipids, dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylcholine, were taken up and metabolized via PLB1. Exogenous DPPC did not enhance growth in the presence of glucose as a carbon source but could support it for at least 24 h in glucose-free medium. Detoxification of LysoPC by reacylation occurred in both the H99 wild-type and the Deltaplb1 strains in the presence of glucose, but PLB1 was required when LysoPC was the sole carbon source. This indicates that both energy-independent (via PLB1) and energy-dependent transacylation pathways are active in cryptococci. Phospholipase A(1) activity was identified by PLB1-independent degradation of 1-palmitoyl-2-arachidonoyl phosphatidylcholine, but the arachidonoyl LysoPC formed was not detoxified by reacylation. Using the human macrophage-like cell line THP-1, we demonstrated the PLB1-dependent incorporation of macrophage-derived arachidonic acid into cryptococcal lipids during cryptococcus-phagocyte interaction. This pool of arachidonate can be sequestered for eicosanoid production by the fungus and/or suppression of host phagocytic activity, thus diminishing the immune response.
Assuntos
Cryptococcus/metabolismo , Metabolismo dos Lipídeos , Lisofosfolipase/fisiologia , Macrófagos/metabolismo , Ácido Araquidônico/farmacocinética , Linhagem Celular , Criptococose/enzimologia , Criptococose/etiologia , Cryptococcus/enzimologia , Cryptococcus/crescimento & desenvolvimento , Humanos , Lisofosfolipase/metabolismo , Macrófagos/enzimologia , Macrófagos/microbiologia , Ácido Oleico/farmacocinética , Osmose , Ácido Palmítico/farmacocinética , Fagocitose , Fosfolipídeos/farmacocinética , Internalização do VírusRESUMO
Secreted phospholipase B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection by Cryptococcus neoformans and for dissemination from the lung via the lymphatics and blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (deltaplb1), and a reconstituted strain (deltaplb1rec). Adhesion of H99 and deltaplb1rec was approximately 69% greater than deltaplb1 at 4 h. Adhesion of deltaplb1 significantly increased after killing by chemicals or heat, and Fourier-transformed analysis by FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. Inhibition by specific PLB1 antibodies, or inhibitors of phospholipase B (PLB), but not lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activities decreased the adhesion of H99 and deltaplb1rec by 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased both PLB secretion and subsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplb1 were observed in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the binding of C. neoformans to host lung epithelial cells, possibly due to production of fatty acids from plasma membranes and/or surfactant by PLB activity.
Assuntos
Aderência Bacteriana , Criptococose/microbiologia , Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/fisiologia , Lisofosfolipase/fisiologia , Linhagem Celular Tumoral , Cryptococcus neoformans/enzimologia , Células Epiteliais/microbiologia , Proteínas Fúngicas/genética , Humanos , Pulmão , Lisofosfolipase/genéticaRESUMO
Human LCAT-like lysophospholipase (LLPL), or lysophospholipase 3, was first identified in vitro, in foam cells derived from THP-1 cells. We demonstrated that LLPL was present in foam cells in the severe atherosclerotic lesions that develop in apolipoprotein E-null (apoE(-/-)) mice. This indicated that LLPL might affect lipid metabolisms in foam cells and, therefore, atherogenesis. Accordingly, we created LLPL-knockout mice by gene targeting and crossed them with apoE(-/-) mice. We showed that the absence of LLPL increased lesion formation markedly in apoE(-/-) mice but had little effect on the plasma-lipid profile. In addition, LLPL-deficient peritoneal macrophages were more sensitive to apoptosis induced by exposure to oxidized low-density lipoprotein. LLPL might provide a link between apoptosis in macrophages and atherogenesis. Our data demonstrate that LLPL activity is anti-atherogenic and indicate that the regulation of this enzyme might be a novel drug target for the treatment of atherosclerosis.
Assuntos
Apolipoproteínas E/fisiologia , Arteriosclerose/genética , Lisofosfolipase/fisiologia , Acilação , Animais , Apolipoproteínas E/genética , Apoptose , Lipoproteínas LDL/metabolismo , Lisofosfolipase/genética , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
OBJECTIVE: Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU toxin being delivered directly into the eukaryotic cell cytosol through the type III secretion system. The amino-acid sequence of ExoU has a potato patatin-like phospholipase domain, similar to the sequence of mammalian Ca-independent phospholipase A2. We examined whether the acute lung injury caused by cytotoxic P. aeruginosa was dependent on the patatin-like phospholipase domain of ExoU. DESIGN: Laboratory investigation using an established mouse model for P. aeruginosa pneumonia with quantitative measurements of acute lung injury and mortality. SETTING: University experimental research laboratory. SUBJECTS: Balb/c mice. INTERVENTIONS: First, a site-directional mutation was introduced in the predicted catalytically active site of the patatin-like phospholipase domain of recombinant ExoU protein. The effect of the mutation on the catalytic activity of ExoU was tested by the in vitro lysophospholipase A assay. Second, the same site-directional mutation was introduced into the exoU gene of P. aeruginosa PA103. Mice were intratracheally infected with either a wild-type P. aeruginosa strain PA103 or an isogenic mutant containing the mutation in exoU. Acute epithelial lung injury, lung edema, bacteremia, and mortality were evaluated quantitatively. MEASUREMENTS AND MAIN RESULTS: Recombinant ExoU had lysophospholipase A activity. Site-directional mutations in the predicted catalytic site of ExoU caused a loss of the lysophospholipase A activity. Whereas the airspace instillation of PA103 caused acute lung injury and death of the infected mice, the airspace instillation of isogenic mutants secreting catalytically inactive ExoU were noncytotoxic and did not cause acute lung injury or death of the infected mice. CONCLUSION: Virulent P. aeruginosa causes acute lung injury and death by the cytotoxic activity derived from the patatin-like phospholipase domain of ExoU.
Assuntos
Proteínas de Bactérias/fisiologia , Modelos Animais de Doenças , Lisofosfolipase/fisiologia , Pneumonia Bacteriana/complicações , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/fisiologia , Síndrome do Desconforto Respiratório/microbiologia , Doença Aguda , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/antagonistas & inibidores , Domínio Catalítico/fisiologia , Citosol/microbiologia , Citosol/fisiologia , Células Eucarióticas/microbiologia , Células Eucarióticas/fisiologia , Immunoblotting , Inflamação , Lisofosfolipase/análise , Lisofosfolipase/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/fisiologia , Pneumonia Bacteriana/enzimologia , Mutação Puntual , Infecções por Pseudomonas/enzimologia , Pseudomonas aeruginosa/patogenicidade , Síndrome do Desconforto Respiratório/mortalidade , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/prevenção & controle , Alinhamento de Sequência , Taxa de SobrevidaRESUMO
Neuropathy target esterase (NTE) is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants, and plasticizers, leading to OP-induced delayed neuropathy in people (>30,000 cases of human paralysis) and hens (the best animal model for this demyelinating disease). The active site region of NTE as a recombinant protein preferentially hydrolyzes lysolecithin, suggesting that this enzyme may be a type of lysophospholipase (LysoPLA) with lysolecithin as its physiological substrate. This hypothesis is tested here in mouse brain by replacing the phenyl valerate substrate of the standard NTE assay with lysolecithin for an "NTE-LysoPLA" assay with four important findings. First, NTE-LysoPLA activity, as the NTE activity, is 41-45% lower in Nte-haploinsufficient transgenic mice than in their wild-type littermates. Second, the potency of six delayed neurotoxicants or toxicants as in vitro inhibitors varies from IC50 0.02 to 13,000 nM and is essentially the same for NTE-LysoPLA and NTE (r2 = 0.98). Third, the same six delayed toxicants administered i.p. to mice at multiple doses inhibit brain NTE-LysoPLA and NTE to the same extent (r2 = 0.90). Finally, their in vivo inhibition of brain NTE-LysoPLA generally correlates with delayed toxicity. Therefore, OP-induced delayed toxicity in mice, and possibly the hyperactivity associated with NTE deficiency, may be due to NTE-LysoPLA inhibition, leading to localized accumulation of lysolecithin, a known demyelinating agent and receptor-mediated signal transducer. This mouse model has some features in common with OP-induced delayed neuropathy in hens and people but differs in the neuropathological signs and apparently the requirement for NTE aging.
Assuntos
Encéfalo/enzimologia , Hidrolases de Éster Carboxílico/fisiologia , Lisofosfolipase/fisiologia , Animais , Encéfalo/metabolismo , Hidrolases de Éster Carboxílico/química , Doenças Desmielinizantes , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Lisofosfatidilcolinas/química , Lisofosfolipase/química , Masculino , Camundongos , Camundongos Transgênicos , Modelos Químicos , Doenças do Sistema Nervoso/induzido quimicamente , Compostos Organofosforados/efeitos adversos , Praguicidas/efeitos adversos , Proteínas Recombinantes/química , Transdução de SinaisRESUMO
Biogenesis of an active ribosome complement and a dynamic cell surface complement are two major determinants of cellular growth. In yeast, the 60S ribosomal subunit protein RpL10p/Grc5p functions during successive stages in ribosome biogenesis, specifically rRNA processing, nucle(ol)ar preribosomal subunit assembly, nucleo-cytoplasmic transport and cytoplasmic maturation of ribosomes. Here, we report that a two-hybrid screen identified yeast genes SED1, ACS2 and PLB3 as encoding proteins physically interacting with both ribosomal RpL10p/Grc5p and its human homologue hRpL10p/QMp. SED1 encodes a differentially expressed cell wall protein which is proposed to be first transiently secreted to the plasma membrane as a GPI (glycosylated derivative of phosphoinositol)-anchored form and to be then transferred to the glucan layer of the cell wall. Ectopic expression of SED1 rescues both the aberrant growth phenotype and the translation defect of grc5-1(ts) temperature-sensitive cells. Furthermore, we report that Sed1p associates with translating ribosomes suggesting a novel, cytoplasmic role for Sed1p. ACS2 encodes one of the two yeast acetyl-CoA synthases and represents a key enzyme in one of several metabolic routes to produce acetyl-CoA, which in turn is indispensable for lipid biosynthesis. PLB3 encodes a phospholipase, which is active in the breakdown of membrane lipids. Our results support the view that Grc5p/RpL10p links ribosome function to membrane turnover and cell surface biogenesis.
Assuntos
Glicoproteínas de Membrana/fisiologia , Biossíntese de Proteínas/fisiologia , Proteínas Ribossômicas/fisiologia , Ribossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Leveduras/fisiologia , Sequência de Bases , Parede Celular/genética , Parede Celular/fisiologia , Coenzima A Ligases/genética , Coenzima A Ligases/fisiologia , Teste de Complementação Genética , Lisofosfolipase/genética , Lisofosfolipase/fisiologia , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Biossíntese de Proteínas/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genéticaRESUMO
Cryptococcal phospholipase (PLB1) is a secreted enzyme with lysophospholipase hydrolase and lysophospholipase transacylase activities. To investigate the role of PLB1 in the evasion of host immune responses, we characterized pulmonary immune responses to the parental (H99), the plb1 mutant, and the plb1(rec) reconstituted mutant strains of Cryptococcus neoformans in mice. PLB1 was required for virulence during infection acquired via the respiratory tract. Mice infected with either H99 or the plb1(rec) strain generated a nonprotective inflammatory response with subsequent eosinophilia, while mice infected with the plb1 mutant generated a protective immune response that controlled the infection. Because PLB1 is believed to facilitate virulence through host cell lysis, we examined the interaction of these strains with macrophages. The plb1(rec) mutant exhibited decreased survival during coculture with macrophages. One factor which may be involved in the survival of yeast in the presence of macrophages is fungal eicosanoid production. Host eicosanoids have been shown to down-modulate macrophage functions. plb1 exhibited a defect in eicosanoid production derived from exogenous arachidonoyl-phosphatidylcholine, suggesting that PLB1 is required for the release of arachidonic acid from phospholipids. These data suggest that cryptococcal PLB1 may act as a virulence factor by enhancing the ability to survive macrophage antifungal defenses, possibly by facilitating fungal eicosanoid production.
Assuntos
Criptococose/etiologia , Cryptococcus neoformans/enzimologia , Eicosanoides/biossíntese , Proteínas Fúngicas/fisiologia , Inflamação/etiologia , Pneumopatias Fúngicas/etiologia , Lisofosfolipase/fisiologia , Animais , Ácido Araquidônico/metabolismo , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Feminino , Leucotrienos/biossíntese , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos CBA , Prostaglandinas/biossíntese , VirulênciaRESUMO
We previously showed that Legionella pneumophila secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids. In this study, we identified and characterized plaA, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids. The plaA gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL. In Escherichia coli, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol. An L. pneumophila plaA mutant was generated by allelic exchange. Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of L. pneumophila. The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of plaA. Overexpression of plaA completely protected L. pneumophila from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids. The plaA mutant grew like the wild type in U937 cell macrophages and Hartmannella vermiformis amoebae, indicating that PlaA is not essential for intracellular infection of L. pneumophila. In the course of characterizing plaA, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by L. pneumophila.
Assuntos
Genes Bacterianos , Legionella pneumophila/genética , Lisofosfatidilcolinas/metabolismo , Lisofosfolipase/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Escherichia coli/genética , Humanos , Legionella pneumophila/efeitos dos fármacos , Legionella pneumophila/enzimologia , Lipólise , Lisofosfatidilcolinas/farmacologia , Lisofosfolipase/química , Lisofosfolipase/fisiologia , Metaloendopeptidases/fisiologia , Dados de Sequência Molecular , Mutação , Células U937RESUMO
Dietary retinyl esters are hydrolyzed in the intestine by the pancreatic enzyme, pancreatic triglyceride lipase (PTL), and intestinal brush border enzyme, phospholipase B. Recent work on the carboxylester lipase (CEL) knockout mouse suggests that CEL may not be involved in dietary retinyl ester digestion. The possible roles of the pancreatic lipase-related proteins (PLRP) 1 and 2 and other enzymes require further investigation. Unesterified retinol is taken up by the enterocytes, perhaps involving both diffusion and protein-mediated facilitated transport. Once in the cell, retinol is complexed with cellular retinol-binding protein type 2 (CRBP2) and the complex serves as a substrate for reesterification of the retinol by the enzyme lecithin:retinol acyltransferase (LRAT). Retinol not bound to CRBP2 is esterified by acyl-CoA acyltransferase (ARAT). The retinyl esters are incorporated into chylomicrons, intestinal lipoproteins that transport other dietary lipids such as triglycerides, phospholipids, and cholesterol. Chylomicrons containing newly absorbed retinyl esters are then secreted into the lymph.
Assuntos
Digestão/fisiologia , Vitamina A , Animais , Dieta , Humanos , Hidrólise , Absorção Intestinal , Lisofosfolipase/fisiologia , Camundongos , Suco Pancreático/fisiologia , Vitamina A/metabolismo , Vitamina A/farmacocinéticaRESUMO
Cell walls (0.1-0.5 microm in thickness) of dermatophytes, at least Trichophyton mentagrophytes and Epidermophyton floccosum, are built of microfibrils (20 nm in diameter) and matrix embedding the fibrils. These fibrils are composed of chitin (70-80%) and a small amount of glucans, and the matrix is composed of beta-1-3, beta1-6 glucan, glucomannan, galactomannan and peptides. Another characteristic structure is the outermost layer (20-50 nm in thickness) of the cell wall, which consists of hydrophobic protein rodlets. Lipids are thought to play important roles in the regulation of dimorphism and virulence in pathogenic fungus. Generally, the ratio of phospholipid/ergosterol is less than 1 in yeast form and 2-20 in mycelial form cells in Candida albicans and Sporothrix schenckii. During the transition from yeast to mycelial forms, phosphatidylinositol and phosphatidylserine are reduced, whereas phosphatidylcholine increases. Phospho-lipase D is activated on this transition. Phospholipase B is now known to be a virulence factor in C. albicans. Polyene antifungal agents bind to ergosterol in membrane to form complexes, which generate pores and destroy the structures and functions of membrane. Azole antifungal agents inhibit the synthesis of ergosterol leading to deficiency in ergosterol content in membrane, and impair the function of membranes in fungal cells. We show the effects of polyenes on the ultrastructure of fungal plasma membrane and impairment of ionomycin-induced calcium influx in T. mentagrophytes, so that we can compare the differences in mode of actions between these two groups of agents.
Assuntos
Antifúngicos/farmacologia , Arthrodermataceae/citologia , Azóis/farmacologia , Parede Celular/ultraestrutura , Lipídeos de Membrana/fisiologia , Polienos/farmacologia , Arthrodermataceae/efeitos dos fármacos , Arthrodermataceae/patogenicidade , Cálcio/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitina/metabolismo , Ergosterol/biossíntese , Ergosterol/metabolismo , Glucanos/metabolismo , Lisofosfolipase/metabolismo , Lisofosfolipase/fisiologia , Lipídeos de Membrana/metabolismo , Polienos/metabolismo , VirulênciaRESUMO
Listeria monocytogenes (LM) is a common pathogen of cerebral infections. Experimental studies in mice have revealed that epithelial cells of the choroid plexus, ependymal cells, macrophages/microglia, and neurons are the target cells of LM. For the intracerebral pathogenesis of LM cell-to-cell spread via phospholipase C was particularly important. However, phospholipase C-deficient LM were not completely attenuated and, therefore, other virulence factors may also contribute to the intracerebral spread of LM. In general, all mice suffering from cerebral listeriosis rapidly succumbed to the disease. Active systemic immunization prior to intracerebral infection reduced the mortality rate to 40%. The favorable prognosis of immunized mice correlated with a reduced intracerebral bacterial load, an increased recruitment of protective CD4+ and CD8+ T cells as well as an upregulated mRNA production of protective cytokines.
Assuntos
Encéfalo/imunologia , Encéfalo/microbiologia , Infecções Bacterianas do Sistema Nervoso Central/microbiologia , Listeriose/imunologia , Animais , Proteínas de Bactérias/fisiologia , Encéfalo/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções Bacterianas do Sistema Nervoso Central/prevenção & controle , Modelos Animais de Doenças , Humanos , Lisofosfolipase/fisiologia , Macrófagos/microbiologia , Proteínas de Membrana/fisiologia , Camundongos , Infiltração de Neutrófilos , Fosfolipases Tipo C/fisiologiaAssuntos
Grânulos Citoplasmáticos/química , Eosinófilos/metabolismo , Ribonucleases , Animais , Proteínas Sanguíneas/fisiologia , Citotoxicidade Imunológica , Proteínas Granulares de Eosinófilos , Peroxidase de Eosinófilo , Neurotoxina Derivada de Eosinófilo , Eosinófilos/ultraestrutura , Feminino , Humanos , Lisofosfolipase/fisiologia , Masculino , Peroxidases/fisiologia , GravidezRESUMO
Lysophospholipids are generated during the turnover and breakdown of membrane phospholipids. We have identified and partially characterized three enzymes involved in the metabolism of lysophospholipids in human brain, namely, lysophospholipase, lysophospholipid:acyl-CoA acyltransferase (acyltransferase), and lysophospholipid:lysophospholipid transacylase (transacylase). Each enzyme displayed comparable levels of activity in biopsied and autopsied human brain, although in all cases the activity was somewhat lower in human than that in rat brain. All three enzymes were localized predominantly in the particulate fraction, with lysophospholipase possessing the greatest activity followed by acyltransferase and transacylase. Lysophosphatidylcholine possessed a Km in the micromolar range for lysophospholipase and transacylase, and in the millimolar range for acyltransferase, whereas arachidonyl-CoA displayed a Km in the micromolar range for acyltransferase. The three enzymes differed in their pH optima, with lysophospholipase being most active at pH 8.0, transacylase at pH 7.5, and acyltransferase at pH 6.0. Both bromophenacyl bromide and N-ethylmaleimide inhibited lysophospholipase activity and, to a lesser extent, that of acyltransferase and transacylase. None of the enzyme activities were affected by the presence of dithiothreitol or EDTA, although particulate lysophospholipase was activated approximately two-fold by the addition of 5 mM MgCl2 or CaCl2 but not KCl. Transacylating activity was stimulated by CoA, the EC50 of activation being 6.8 microM. Acyltransferase displayed an approximately threefold preference for arachidonyl-CoA over palmitoyl-CoA, whereas the acylation rate of different lysophospholipids was in the order lysophosphatidylinositol > 1-palmitoyl lysophosphatidylcholine > 1-oleoyl lysophosphatidylcholine >> lysophosphatidylserine > lysophosphatidylethanolamine. This, and the preference of human brain phospholipase A2 for phosphatidylinositol, suggests that this phospholipid may possess a higher turnover rate than the other phospholipid classes examined. Human brain homogenates also possessed the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylethanolamine. In addition, we also present evidence that diacylglycerophospholipids can act as acyl donors for the transacylation of lysophospholipids. We have therefore demonstrated the presence of, and partially characterized, three enzymes that are involved in the metabolism of lysophospholipids in human brain. Our results suggest that lysophospholipase may be the major route by which lysophospholipids are removed from the cell membrane in human brain. However, all three enzymes likely play an important role in the remodeling of membrane composition and thereby contribute to the overall functioning of membrane-associated processes.
Assuntos
Aciltransferases/análise , Encéfalo/enzimologia , Lisofosfolipase/análise , Lisofosfolipídeos/metabolismo , Complexos Multienzimáticos/análise , Acetofenonas/farmacologia , Aciltransferases/metabolismo , Aciltransferases/fisiologia , Adulto , Idoso , Encéfalo/metabolismo , Ácido Edético/farmacologia , Etilmaleimida/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Lisofosfolipase/metabolismo , Lisofosfolipase/fisiologia , Pessoa de Meia-Idade , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/fisiologia , Fosfolipases/antagonistas & inibidoresRESUMO
The direct techniques of 1H spin-echo and 31P-NMR spectroscopy made it possible to monitor the release of glycerophosphocholine from lysophosphatidylcholine in lysates from human red blood cells. Thus, the existence of a lysophospholipase in human erythrocytes was confirmed using a new more direct method. No evidence for a phospholipase A2 activity in the haemolysates was found with the same approach; since this enzyme is present in leukocytes, the absence of activity helped verify the purity of the erythrocyte preparation. The lysophospholipase may constitute, with the earlier described glycerophosphocholine phosphodiesterase activity, a metabolic unit for the removal of haemolytic lysophosphatidylcholine which is formed in the erythrocyte membranes as well as taken up from the plasma.