Effect of metalloprotease inhibitors on invasion of red blood cell by Plasmodium falciparum

For successful invasion, the malaria merozoite needs to attach to the red blood cell membrane, undergo reorientation, form a junction of the apical end with the host membrane, and internalize. Malaria proteases have been implicated in the invasion process, but their specific cellular functions remain unclear. To demonstrate the involvement of metalloprotease in the process of Plasmodium falciparum merozoite entry into host red blood cell, schizont-infected red blood cells and parasitophorous vacuolar membrane-enclosed merozoite structures were treated with 1,10-phenanthroline, a metal chelator, resulting in a reduction of invasion with IC50 value of 25 and 29 microM, respectively. Absence of an accumulation of schizont stages after treatment with 1,10-phenanthroline indicated that the inhibitory effect was not due to suppression of merozoite release from red blood cells, but on the invasion step. Although treatment with GM6001, a well-known inhibitor of the mammalian matrix and disintegrin metalloprotease family, was less effective, nevertheless this study points to the importance of metal-requiring protease in the process of invasion of host red blood cell by the malaria parasite.     

鼠疟原虫裂殖子进入红细胞后的早期超微结构演化

为了探讨疟原虫红内早期超微结构的演化，用透射电镜观察了伯氏和约氏疟原虫。裂殖子入侵后补围于内陷红细胞膜所构成的纳虫泡中，起初停留于浅层红细胞浆内，造成该处表面隆起。虫体浑圆，顶端结构很快消失，球形体与核和线粒体分离并萎缩。分离了的线粒体略显舒展，内质网扩大。核变长弯形，核周隙有部分地扩大。双层内膜渐与外膜分离，断裂、卷曲、以至消失。最后变成单层质膜的滋养体。早期滋养体的组织结构仍较精细，以后密度逐渐减低。胞浆内有个别多角形的大内质网池，质膜下出现小食物泡。然后虫体变扁平，边缘卷起，包围红细胞浆，终至封口融合，形成一个食物泡；接着在其旁出现消化泡和色素粒。长成更大的中期滋养体。     

Rhoptry secretion of membranous whorls by Plasmodium falciparum merozoites

Multilamellar membranous whorls were localized, by electron microscopy, in elements of the rhoptry-microneme complex from glutaraldehyde-tannic acid (TA)-fixed merozoites of the human malarial parasite, Plasmodium falciparum. These multilaminate structures, which have a dark line-to-dark line periodicity of approximately 5 nm, were also found in the nuclear envelope and closely apposed to the external surface of merozoites. Segmented schizonts, which contain intracellular merozoites, often showed membranous whorls within their parasitophorous vacuoles and closely apposed to the external surface of the parasitophorous vacuole membrane. Whorls were not found in trophozoites, immature schizonts, and uninfected erythrocytes. Most rhoptries in merozoites fixed in glutaraldehyde-TA were electron-lucent whereas rhoptries fixed in glutaraldehyde alone were electron-dense. Some merozoites fixed in glutaraldehyde-TA had both an electron-dense and an electron-lucent rhoptry. These findings suggest that TA induces the premature extrusion of rhoptry materials. Our findings support previous suggestions in the literature that phospholipid materials secreted from merozoite rhoptries are involved in merozoite interaction with host erythrocytes.     

Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum

Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non-receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP(2)) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP(2), although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection.     

Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore.

Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 +/- 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 microns2). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.     

隐孢子虫病实验与临床研究——Ⅲ.实验小鼠隐孢子虫病透射电镜观察

描述对免疫抑制小鼠肠上皮细胞寄生的隐孢子虫滋养体、裂殖体和大配子的超微结构及肠上皮细胞改变的透射电镜观察结果。虫体在肠上皮细胞附着处可见一明显电子致密带,并被肠上皮细胞来源的纳虫空泡包围。虫体在附着处其胞浆膜形成复杂的膜性皱褶并和肠上皮细胞质膜紧密接触。在裂殖子内可见电子致密颗粒和棒状体样结构,其意义尚不明瞭。     

The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve.

The obligate intracellular protozoan parasite Toxoplasma gondii creates and enters into a unique membrane-bounded cytoplasmic compartment, the parasitophorous vacuole, when invading mammalian host cells. By microinjecting polar fluorescent molecules into individual T. gondii-infected fibroblasts, we show here that the parasitophorous vacuole membrane (PVM) surrounding the parasite functions as a molecular sieve. Lucifer yellow (457 Da) displayed free bidirectional flux across the PVM and distinctly outlined the parasites, which did not take up the dye, within the vacuole. This dye movement was not appreciably delayed by pretreatment of cells with 5 mM probenecid or chilling the monolayer to 5 degrees C, suggesting that dye movement was due to passive permeation through a membrane pore rather than active transport. Calcein, fluo-3, and a series of fluorescein isothiocyanate-labeled peptides up to 1291 Da crossed the PVM in a size-restricted fashion. A labeled peptide of 1926 Da and labeled dextrans and proteins (> or = 3000 Da) failed to transit the PVM. This putative channel in the PVM therefore allows exchange of molecules up to 1300-1900 Da between the host cell cytoplasm and the parasitophorous vacuolar space.     

Metabolic fate of fatty acids in type II cells isolated from adult rat lung

The present study is concerned with the metabolic fate of palmitate, oleate and linoleate in isolated rat lung type II cells. The cells readily oxidize the exogenously supplied fatty acids to CO₂ and incorporate them into lipids. The distribution between the pathways of oxidation and esterification is similar for saturated and unsaturated fatty acids. The majority of the fatty acids taken up by the cells is utilized for lipid synthesis. The fatty acids are incorporated preferentially into phospholipids, particularly into phosphatidylcholine. Addition of unsaturated fatty acids decreases the utilization of palmitate by type II cells. The distribution of palmitate between oxidation and esterification is not altered in the presence of unsaturated fatty acids. Addition of carnitine stimulates the fatty acid oxidation and decreases the esterification of fatty acids.     

Phospholipid transfer between plasma and platelets in vitro

Washed rabbit platelets were resuspended in plasma in which all of the major phospholipids had been isotopically labeled by injection of 32PO4 into rabbits. At certain time intervals during a 6-hr incubation at 37 degrees C, aliquots were removed from the incubation mixture and the platelets were isolated and subjected to lipid extraction and phospholipid analysis. A continuous rise in platelet non-lipid-bound and lipid-bound radioactivity was observed through-out the incubation period. Two platelet phospholipids, lecithin and lysolecithin, were significantly labeled, whereas little or no labeling of the other phospholipids was found. There was no detectable change in total or individual platelet phospholipid content. At 6 hr, 4% of total platelet phospholipid, 43% of platelet lysolecithin, and 7% of platelet lecithin were labeled. Platelets incubated in plasma from rabbits with diet- induced hyperlipidemia took up and incorporated significantly more label into their phospholipids than did platelets in normal plasma. Labeling of both platelet lysolecithin and lecithin could be due to uptake and metabolism of plasma lysolecithin by platelets. However, labeling of platelet lecithin could at least in part be the result of direct exchange of this phospholipid with the plasma. Uptake and incorporation of endogenous plasma lysolecithin by platelets and, possibly, direct exchanged of platelet lecithin may be important mechanisms in the modification by plasma lipids of platelet membrane phospholipid fatty acid composition and platelet function.     

An ultrastructural study of the exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi in Macaca mulatta

Exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi were examined by electron microscopy in biopsy samples of primate livers. With maturity the parasitophorous vacuole membrane becomes highly sculptured by the addition of a discontinuous dense thickening, the distribution of which can be a distinguishing character between these two species. The parasitophorous vacuole membrane follows the contours of the parasite faithfully with a minimal surrounding vacuole. The marked destruction of the cytoplasm of the host hepatocyte by most of the parasites studied however gave the distinct, but erroneous, appearance of a large parasitophorous vacuole at the light microscope level. The mature parasite often exhibited a highly invaginated surface contour with the result that the cytoplasm of the host cell and parasite became intimately interdigitated, this interweaving is unlikely to be recognized in light microscopic studies.     

Pseudo-angiomatous growth pattern in recurrent plasma cell myeloma

Erythrocytes are remarkably dynamic structures, possessing multiple and complex pathways for regulating cell membrane properties to compensate for the absence of a nucleus and internal membranes. Unlike the invasion strategies of many viruses and bacteria into their eukaryotic hosts, however, the accepted model for malaria parasite entry into human erythrocytes casts the host cell in a largely passive role. This is in contrast to mounting evidence for a suite of dynamic alterations that the erythrocyte membrane undergoes during the rapid process of invasion by the blood stage malaria parasite – the merozoite. Here we review the cellular and molecular basis for merozoite invasion of the erythrocyte and explore the idea that radical changes in the erythrocyte membrane protein and lipid architecture probably accompany this key step in the establishment of human malaria disease.     

Differences in free fatty acid and glucose metabolism of human blood neutrophils and lymphocytes.

Comparison of isolated human neutrophils and lymphocytes in short-term tissue culture revealed marked differences in their rates of lipid biosynthesis. Ficoll-Hypaque gradients were used to separate lymphocytes and neutrophils from the blood of normal subjects. Neutrophils incorporated more palmitate into cell lipids (151.0 +/- 16.6 nmole/hr/10(8) cells) than lymphocytes (41.6 +/- 4.1). By contrast, the lymphocytes oxidized more palmitate (8.3 +/- 0.5 nmole/hr/10(8) cells) as compared to neutrophils (1.1 +/- 0.1). The greater fatty acid uptake by the neutrophils was due to a sixfold greater rate of incoporation of palmitate into their triglyceride fraction. Triglyceride synthesis by neutrophils increased as the molar ratio of free fatty acid to albumin was raised, whereas incorporation into phospholipids remained relatively constant; there was preferential labeling of neutrophil triglycerides throughout the physiologic range. Studies using linoleate and oleate gave similar results. The distribution of radioactivity into various phospholipids determined by thin-layer chromatography was similar for the two cell types. When labeled glucose was used as a substrate to measure incorporation primarily into the glycerol backbone of the cell lipids, neutrophils incorporated more radioactivity into total lipids and triglycerides than lymphocytes. These results indicate that neutrophils take up much more fatty acid than lymphocytes primarily because they synthesize much larger quantities of triglycerides, a storage form. Since cellular triglycerides may act as a source of fatty acid for lecithin synthesis during phagocytosis, the greater rate of fatty acid incorporation in the neutrophil may reflect a metabolic pattern that permits efficient phagocytosis.     

Turnover of phospholipids in rat aortic smooth muscle cells in culture

Smooth muscle cells derived from rat aortic media were explanted and grown in culture for 14 to 60 days. During that time they formed a confluent multilayer and deposited extracellular material resembling newly formed elastin. The lipid composition of the cells in culture differed slightly from the parent cells in the intact aorta with respect to a higher phospholipid/DNA ratio and a higher lecithin content. The cholesterol content resembled that of parent cells. After incubation with labeled precursor the cultured cells show an active lipid synthesis; choline is incorporated mainly into lecithin, whereas glycerol and palmitate appear in phospholipids and to a lesser extent in neutral lipids. After a 2 hour pulse and up to 96 hour chase there is a linear fall in the specific activity of lecithin with a half-time of 28 to 30 hours. The rate of fall in specific activity of glycerol- or choline-labeled lecithin was found to be similar, indicating that choline does not turn over by an exchange reaction and is a suitable marker for studying phospholipid turnover in cultured cells. The results provide a basis for investigation of the effect of increasing cellular cholesterol content on phospholipid turnover.     

The interaction between the malaria parasite and the red cell membrane

The malaria parasite intimately interacts with the host red cell membrane throughout the cycle of invasion and intracellular development. Direct interaction between the merozoite surface and the red cell membrane involves specific binding between the surface components of both cells, which leads to the subsequent endocytotic process still incompletely understood. Intracellular development of the parasite is accompanied by various changes in the structure and function of red cell membrane components. Some changes may benefit parasite survival while others trigger host immune response. An understanding of both the direct interaction between the surface components of the parasite and the red cell during invasion, and the subsequent changes in the red cell membrane following invasion, should lead to better ways of controlling malaria.     

Effects of vitamin A and ethanol on liver plasma membrane fluidity

To study possible mechanisms whereby vitamin A and ethanol may affect liver plasma membranes, rats were fed liquid diets containing either 6 international units of vitamin A per kcal or 5 times more, with or without ethanol (36% of total energy as isocaloric substitution for carbohydrate). Vitamin A supplementation resulted in 2- to 3-fold increases of liver plasma membrane free retinol (p less than 0.005) and retinyl esters (p less than 0.001), particularly esters of palmitate and oleate, whereas cholesterol esters did not change. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene revealed decreased fluidity as measured by an increase in fluorescence polarization which correlated significantly with retinyl palmitate plus oleate content in membranes. In rats fed ethanol chronically, we first verified our previous observation of a decrease in liver plasma membrane fluorescence polarization. We now find this effect to be associated with (and possibly due to) an increase of cholesterol ester content. In linear regression analysis, the change in fluorescence polarization correlated positively with vitamin A (p less than 0.02) and negatively with cholesterol ester contents (p less than 0.001). Ethanol feeding partially offset the effect of vitamin A supplementation on fluorescence polarization. We conclude from these observations that liver plasma membranes contain a significant amount of vitamin A, that vitamin A supplementation increases membrane fluorescence polarization and that chronic ethanol administration can interfere with this effect.     

Role of Phospholipase A2 in Cholesterol Gallstone Formation Is Associated with Biliary Phospholipid Species Selection at the Site of Hepatic Excretion

Phospholipase A₂ plays a role in cholesterol gallstone development by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. Lysolecithin and polyunsaturated free fatty acids are known to stimulate the synthesis and/or secretion of gallbladder mucin via a prostanoid pathway, leading to enhancing cholesterol crystal nucleation and growth, and therefore, the action of phospholipase A₂ is associated, in part, with bile phospholipid fatty acid. To clarify this hypothesis, we evaluated the effect on bile lipid metastability in vitro of replacing phospholipids with lysolecithin and various free fatty acids. Supersaturated model biles were created with an identical composition (cholesterol saturation index, 1.8; egg yolk lecithin, 34 mM; taurocholate, 120 mM; cholesterol, 25 mM) except for 5%, 10%, or 20% replacement of egg yolk lecithin with a combination of palmitoyl–lysolecithin and a free fatty acid (palmitate, stearate, oleate, linoleate, or arachidonate), followed by time-sequentially monitoring of vesicles and cholesterol crystals using spectrophotometer and video-enhanced differential contrast microscopy. Replacement with hydrophilic fatty acids (linoleate and arachidonate) reduced vesicle formation and promoted cholesterol crystallization, whereas an enhanced cholesterol-holding capacity was evident after replacement with hydrophobic fatty acids (palmitate and stearate). These results indicate that the effect of phospholipase A₂ on bile lithogenecity is modulated by the fatty acid species in bile phospholipids, and therefore, that the role of phospholipase A₂ in cholesterol gallstone formation is dependent, in part, on biliary phospholipid species selection at the site of hepatic excretion.     

Altered fatty acid composition in the plasma, platelets, and aorta of the streptozotocin-induced diabetic rat

Decreased arachidonate levels have been described in various tissues of the streptozotocin-induced diabetic rat. However, reported arachidonate changes in platelets from diabetic patients have been variable. In this communication, we describe experiments that indicate that in the short-term streptozotocin diabetic rat (2 to 3 weeks), the fatty acid composition of plasma and red blood cell lipids was altered but remained unchanged in platelet and aorta phospholipids. The altered fatty acid composition of the diabetic red blood cells and plasma cholesterol esters and phospholipids was similar to that previously found in the diabetic liver. However, in long-term diabetes (6 weeks), the phospholipid fatty acid composition of the platelet and aorta became significantly altered. Thus, in the 6-week diabetic platelet, there were increases of linoleate, dihomo-gamma-linolenate, docosapentaenoate (C22:5n-3), and docosahexaenoate, and decreases of oleate, arachidonate, and docosatetraenoate. In the aorta, there were increases of linoleate, eicosapentaenoate, and docosahexaenoate, and decreases of arachidonate, docosatetraenoate, and docosapentaenoate (C22:5n-6). Results from these experiments indicate that the fatty acid composition of plasma and red blood cell lipids was altered in short-term diabetes (2 to 3 weeks), but that of platelet and aorta phospholipids was not changed until more prolonged diabetes was present. Insulin treatment of the diabetic rat increased the levels of palmitoleate and oleate and decreased the levels of linoleate in platelet and aorta lipids from insulin-treated diabetic rats, suggesting an overcorrection of diminished delta 9 and delta 6 fatty acid desaturation as compared with the nondiabetic control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trafficking of the major virulence factor to the surface of transfected P. falciparum-infected erythrocytes

After invading human red blood cells (RBCs) the malaria parasite Plasmodium falciparum remodels the host cell by trafficking proteins to the RBC compartment. The virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) is responsible for cytoadherence of infected cells to host endothelial receptors. This protein is exported across the parasite plasma membrane and parasitophorous vacuole membrane and inserted into the RBC membrane. We have used green fluorescent protein chimeras and fluorescence photobleaching experiments to follow PfEMP1 export through the infected RBC. Our data show that a knob-associated histidine-rich protein (KAHRP) N-terminal protein export element appended to the PfEMP1 transmembrane and C-terminal domains was sufficient for efficient trafficking of protein domains to the outside of the P. falciparum-infected RBC. The physical state of the exported proteins suggests trafficking as a complex rather than in vesicles and supports the hypothesis that endogenous PfEMP1 is trafficked in a similar manner. This study identifies the sequences required for expression of proteins to the outside of the P. falciparum-infected RBC membrane.     

Enzymology of lipid A palmitoylation in bacterial outer membranes

The enzymology of palmitate addition to lipid A can be traced to the early discovery of monosaccharide lipid A precursors, but the functional importance of lipid A palmitoylation in bacterial resistance to the host immune response has emerged only recently. Lipid A palmitoylation in enterobacteria is determined by a PhoP/PhoQ-activated gene pagP, which encodes an unusual outer membrane enzyme of lipid A biosynthesis. PagP structure and dynamics have now been elucidated by both NMR spectroscopy and X-ray crystallography. PagP is an 8-stranded antiparallel beta-barrel preceded by an N-terminal amphipathic alpha-helix. The PagP barrel axis is uniquely tilted by 30 degrees with respect to the membrane normal. An interior hydrophobic pocket in the upper half of the molecule functions as a hydrocarbon ruler, which allows the enzyme to distinguish palmitate from other acyl chains found in phospholipids. Internalization of a phospholipid palmitoyl group within the barrel appears to occur by lateral diffusion from the outer leaflet through non-hydrogen bonded regions between beta-strands. The MsbA-dependent trafficking of lipids from the inner membrane to the outer membrane outer leaflet is necessary for lipid A palmitoylation in vivo. Efforts to determine the PagP catalytic mechanism may lead to the development of inhibitors for the treatment of infections.     

Plasma and erythrocyte lipids in sickle cell anaemia

Summary. The content and composition of plasma and erythrocyte lipids from individuals having sickle cell anaemia has been determined. The plasma contains a significantly reduced content of both cholesterol and phospholipid while the ratio of cholesteryl ester to total cholesterol, the distribution of phospholipids classes, triglyceride concentration and the fatty acid content were similar to normal values. However, the free glycerol content of the plasma was eight-fold higher than normal. Erythrocyte lipids contain an elevated level of cholesterol and control levels of phospholipid content, phospholipid distribution and fatty acid content. The plasma was separated by ultracentrifugation into VLDL (density <1.006), LDL (1.006< d <1.063) and HDL (1.063< d< 1.21). Significant decreases in cholesterol and phospholipid were observed in LDL and HDL.