Toxoplasma gondii-vertebrate cell interactions. I. The influence of bicarbonate ion, CO2, pH and host cell culture age on the invasion of vertebrate cells in vitro.

A controlled-environment culture system was used to show that both physical and biologic parameters can influence the penetration of vertebrate cells by Toxoplasma gondii. The optimum bicarbonate ion concentration for the penetration of bovine embryo skeletal muscle (BESM) cells is 36.25 mM. Higher or lower bicarbonate ion concentrations are increasingly inhibitory to penetration. As CO2 increases in the range from 0.5-3.7 mM, penetration is progressively inhibited. No relationship was found between penetration and pH in the pH range of 6.949-7.765. The culture age of the BESM cells directly influenced the ability of the parasites to penetrate the cells. Older BESM cells were more refractory to penetration than younger cells.     

Toxoplasma gondii-Vertebrate Cell Interactions. I. The Influence of Bicarbonate Ion, CO2, pH and Host Cell Culture Age on the Invasion of Vertebrate Cells In Vitro

SYNOPSIS A controlled-environment culture system was used to show that both physical and biologic parameters can influence the penetration of vertebrate cells by Toxoplasma gondii. The optimum bicarbonate ion concentration for the penetration of bovine embryo skeletal muscle (BESM) cells is 36.25 mM. Higher or lower bicarbonate ion concentrations are increasingly inhibitory to penetration. As CO₂ increases in the range from 0.5–3.7 mM, penetration is progressively inhibited. No relationship was found between penetration and pH in the pH range of 6.949–7.765. The culture age of the BESM cells directly influenced the ability of the parasites to penetrate the cells. Older BESM cells were more refractory to penetration than younger cells.     

Factors Enhancing the Host-Cell Penetration of Toxoplasma gondii

The penetration into HeLa cells of Toxoplasma gondii was studied with a cell culture technique. The influence on the rate of penetration and the number of penetrating Toxoplasma parasites was tested by use of preparations of disintegrated parasites mixed with test parasites. These preparations were found to contain factors enhancing the penetrating rate of the parasites. This effect was demonstrable by use of untreated parasites as well as parasites lacking active motility owing to a previous exposure to Formalin. The preparations of disintegrated parasites contained, in addition, components inhibitory to the penetration-enhancing factors. These inhibitory components were able to reduce the penetrating capacity of normal Toxoplasma parasites, suggesting that the studied enhancing factors may play a role in the natural process of penetration. The efficacy of various techniques for disintegration of Toxoplasma parasites was investigated for release of penetration-enhancing factors from Toxoplasma parasites. The methods used resemble those used for liberation of lysosomal enzymes. Reduced osmotic pressure was obviously not adequate for release of enhancing factors, whereas the freezing and thawing procedure, sonic treatment, and irradiation produced high yields. It was difficult to evaluate the effect of incubation at acid pH on release of enhancing activity, because the penetration-promoting factors seemed unstable on both the acid and the alkaline sides of pH 7.6.     

Dynamin inhibitor impairs Toxoplasma gondii invasion

The protozoan parasite Toxoplasma gondii infects its host cells through an active mechanism. In this work, we obtained evidence that host cells also play a fundamental role during the infection process. We found that previous incubation of the host cells, but not the parasites, with Dynasore, a small molecule that inhibits dynamin GTPase activity, markedly reduced the penetration of T. gondii tachyzoites into LLC-MK2 cells. In contrast, parasite adhesion to the host cell surface increased, as observed both by light and electron microscopy. Intriguingly, the few parasites internalized by Dynasore-treated cells remained in vacuoles located at the periphery of the cell, in contrast to the perinuclear localization seen in the control.     

Parasiticidal activity of Haemaphysalis longicornis longicin P4 peptide against Toxoplasma gondii

The Haemaphysalis longicornis longicin P4 peptide is an active part peptide produced by longicin which displays bactericidal activity against both Gram-negative and Gram-positive bacteria and other microorganisms. In the present study, the effect of the longicin P4 peptide on the infectivity of Toxoplasma gondii parasites was examined in vitro. Tachyzoites of T. gondii incubated with longicin P4 had induced aggregation and lost the trypan blue dye exclusion activity and the invasion ability into the mouse embryonal cell line (NIH/3T3). Longicin P4 bound to T. gondii tachyzoites, as demonstrated by fluoresce microscopic analysis. An electron microscopic analysis and a fluorescence propidium iodide exclusion assay of tachyzoites exposed to longicin P4 revealed pore formation in the cellular membrane, membrane disorganization, and hollowing as well as cytoplasmic vacuolization. The number of tachyzoites proliferated in mouse macrophage cell line (J774A.1) was significantly decreased by incubation with longicin P4. These findings suggested that longicin P4 conceivably impaired parasite membranes, leading to the destruction of Toxoplasma parasites in J774A.1 cells. Thus, longicin P4 is an interesting candidate for antitoxoplasmosis drug design that causes severe toxicity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that longicin P4 causes aggregation and membrane injury of parasites, leading to Toxoplasma tachyzoite destruction.     

Penetration of maturating red blood cells by Toxoplasma gondii

Penetration of Toxoplasma gondii tachyzoites was studied in vitro using murine erythroid cells at different stages of development. Toxoplasma gondii penetrated nucleated erythroblasts and macroreticulocytes from foetal mouse liver and the circulating erythrocytes of foetal, neonatal or severely anaemic adult mice. Immature reticulocytes were more susceptible to penetration than mature ones, indicating that some change in their membrane properties occurred during maturation. The present results confirmed our previous finding that the major erythrocyte membrane-specific proteins do not prevent erythrocyte penetration since these proteins are known to be present in the reticulocyte membrane.     

中药大蒜素体外抗弓形虫效应及其机制的研究

目的 观察大蒜提取物体外抗弓形虫的作用效果及其机制.方法 将弓形虫RH株速殖子与兔肾细胞共同培养,加入不同浓度的大蒜素(实验组)和磺胺嘧啶(阳性对照组),培养不同时间后取出细胞,固定染色后观察细胞感染率及每个纳虫泡中弓形虫速殖子的数量;采用MTT比色法观察大蒜素对弓形虫速殖子侵袭细胞及其正常细胞增殖的影响;采用台盼兰着色法观察大蒜素对弓形虫速殖子活性的影响;采用TUNEL末端标记法检测弓形虫速殖子凋亡率,对药物的效应和机制进行探讨.结果 (1)10～80 μg/ml的大蒜素能抗弓形虫的感染,呈现剂量依赖性,与时间无明显的相关性.(2)40 μg/ml、80 μg/ml的大蒜素不能抑制细胞的增殖,对细胞无明显的毒副作用;160 μg/ml的大蒜素对细胞有明显的毒副作用.(3)大蒜素80 μg/ml时,台盼兰着色率最高,弓形虫的活力最低.(4)大蒜素80 μg/ml的浓度时,弓形虫速殖子凋亡率最高.结论 大蒜素可以抑制弓形虫速殖子的活力、对宿主细胞的感染力、在细胞内的增殖,无明显的毒副作用,最适宜浓度为80 μg/ml.大蒜素是一种良好的抗弓形虫药物,诱导弓形虫速殖子凋亡是其抗弓形虫机制之一.     

Toxoplasma gondii: perfecting an intracellular life style

Toxoplasma gondii is a widespread protozoan parasite that infects all nucleated cell types of warm-blooded vertebrates. Parasite motility is regulated by polymerization of new actin filaments that provide a substrate for the small myosin TgMyoA. Interaction between the cytoplasmic tails of parasite adhesins and the actin-binding protein aldolase links these cell surface proteins with the cytoskeleton. Translocation of adhesins coupled to extracellular receptors allows the parasite to glide across the substrate. This conserved system is important for active penetration into host cells and tissue migration by T. gondii . Entry into the host cell is accompanied by dramatic remodeling of the intracellular vacuole that the parasite resides in. This compartment resists fusion with host cell endocytic organelles, yet recruits mitochondria and endoplasmic reticulum in order to gain access to host cell nutrients. The combined abilities to actively penetrate host cells and control the fate of the parasite-containing vacuole contributes to the remarkable success of T. gondii as an intracellular parasite.     

Toxoplasma gondii secretes a calcium-independent phospholipase A(2)

Phospholipases A(2) (PLA(2)) play an important role in Toxoplasma gondii host cell penetration. They are also key enzymes in the host cell response to the parasite invasion. PLA(2) hydrolyse cellular phospholipids, releasing multiple inflammatory lipidic mediators. We have investigated the biochemical characterisation of T. gondii PLA(2) activity in a mouse-cultured tachyzoite homogenate and in the peritoneal exudate from infected mice, using the hydrolysis of a fluorescent phosphatidylglycerol labelled at the sn-2 position. Spectrofluorimetry and thin-layer chromatography showed a PLA(2) activity (about 0.5-2 nmol/min per mg), calcium-independent, secreted into infected mice peritoneal exudate, with a broad pH activity ranging between 6.5 and 9.5 and resistant to a great number of potential PLA(2) inhibitors except dithio-nitrobenzoic acid (1 mM). An associated phospholipase A(1) activity was also displayed. These results suggest that Toxoplasma gondii displays specific phospholipases different from host enzymes and probably involved at critical steps of infectious cycle.     

Review: Toxoplasma gondii cellular invasion.

Toxoplasma gondii, the etiologic agent of toxoplasmosis, is a ubiquitous protozoan parasite that requires an intracellular site for growth and replication. The invasive process involves six steps: a) cellular recognition, b) parasite movements by means of a subpellicular microtubule cytoskeleton, c) cell to cell adhesion, d) rhoptry secretion of penetrating enhancing factor (PEF) with Ca++ and Ca++ activated ATPase dependence, e) conoid penetration, f) induction of a parasitophorous vacuole, a protective and exchange site, interiorization of the parasite. The invasion is an active, oriented and specific process depending on chemical factors as energy sources, cations, as well as microviscosity and membrane structures. Toxoplasma gondii stimulates T cell subsets and induces lymphokine (IFN gamma, IL2) release.     

Endotoxin increases hepatic susceptibility to lipid peroxidation: a possible role of iron

The purpose of this study was to investigate the possible mechanism by which endotoxin enhances peroxidative damage to membrane lipids. Male B6C3 mice were treated with endotoxin intraperitoneally 0 or 20 mg/kg body weight for 24 h. Freshly prepared liver homogenate was incubated with either 1-5 mM of reduced glutathione (GSH), glucose, H(2)O(2), ascorbic acid (AA), FeSO(4), FeCl(3), EDTA, FeCl(3) plus AA, AA plus EDTA or EDTA plus FeCl(3) in phosphate-buffered saline (PBS), pH 7.0, or PBS, at 37 degrees C for 60 min. The levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), were significantly higher in the liver of endotoxin-treated mice, and the values were markedly increased following incubation. Compared to PBS, incubation with H(2)O(2), FeCl(3), FeSO(4), and AA, but not glucose, significantly enhanced TBAR formation. The greatest increase of TBAR was found when AA and FeCl(3) were added together. On the other hand, EDTA and GSH inhibited the formation of TBAR during incubation. When added before AA, EDTA completely inhibited the peroxidative effect of AA or FeSO4, and when added subsequent to AA, EDTA partially prevented the adverse effect of AA. The results obtained suggest that ionic iron plays an important role in initiating endotoxin-induced peroxidative damage to membrane lipids, and that AA may be involved in releasing iron from its protein complex and/or maintaining ionic iron in a reduced or catalytic state.     

A soluble phospholipase of Toxoplasma gondii associated with host cell penetration

We previously reported that phospholipase increases host cell penetration by Toxoplasma gondii. Here we show that calcium-dependent phospholipase A (PLA) activity is found in the supernatant of sonically disrupted T. gondii. When fractions of disrupted T. gondii were incubated with host cells, the release of fatty acids and lysolipids was detected. Fractions of sonically disrupted T. gondii with PLA activity increased T. gondii host cell penetration in a bioassay. In addition, a protein of approximately 20 kDa was detected by immunoblot of T. gondii antigens with horse antiserum to snake venom, the major antibody of which recognizes PLA2. Incubation of T. gondii with exogenous PLA2 resulted in increased solubility of a rhoptry protein. This protein, which we previously characterized as involved with enhanced parasite invasion of host cells and which is recognized by monoclonal antibody Tg49, was detected in increased amounts in supernatant fractions of extracellular parasites treated with PLA2. Whereas without PLA2 treatment, it is only slightly soluble under physiological conditions. This raises the possibility that PLA may be implicated in the release of rhoptry proteins.     

Parasiticidal activity of bovine lactoperoxidase against Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasitic protozoan transmitted via the ingestion of raw, infected meat that causes congenital infections. In a cell-free environment, virulent Toxoplasma was strikingly resistant to H2O2. The activity of H2O2 or H2O2 generated by glucose-glucose oxidase against the resistant tachyzoite stage of pathogenic T. gondii was enhanced by adding KI and bovine lactoperoxidase (bLPO), referred to here as the bLPO system. Replacing bLPO (heme content, 90%) with recombinant bLPO (heme content, 6%) did not enhance the parasiticidal activity with KI and H2O2. These results indicated that heme contributed to the enzyme activity and resulted in the killing of tachyzoites of T. gondii. Tachyzoites treated with the bLPO system also lost the ability to penetrate the mouse fibroblast cell line (NIH/3T3), and could be killed intracellularly after exposure by bLPO to a mouse macrophage cell line (J774A.1). These findings suggested that toxicity was mediated through small amounts of H2O2 generated by phagocytic events in naive macrophages, and by the peroxidative activity of bLPO. Our observations suggest that the bLPO system could help prevent the development of Toxoplasmosis in humans after ingesting raw, infected meat.     

A Soluble Phospholipase of Toxoplasma gondii Associated with Host Cell Penetration

We previously reported that phospholipase increases host cell penetration by Toxoplasma gondii . Here we show that calcium-dependent phospholipase A (PLA) activity is found in the supernatant of sonically disrupted T. gondii . When fractions of disrupted T. gondii were incubated with host cells, the release of fatty acids and lysolipids was detected. Fractions of sonically disrupted T. gondii with PLA activity increased T. gondii host cell penetration in a bioassay. In addition, a protein of approximately 20 kDa was detected by immunoblot of T. gondii antigens with horse antiserum to snake venom, the major antibody of which recognizes PLA₂. Incubation of T. gondii with exogenous PLA₂ resulted in increased solubility of a rhoptry protein. This protein, which we previously characterized as involved with enhanced parasite invasion of host cells and which is recognized by monoclonal antibody Tg49, was detected in increased amounts in supernatant fractions of extracellular parasites treated with PLA₂. Whereas without PLA₂ treatment, it is only slightly soluble under physiological conditions. This raises the possibility that PLA may be implicated in the release of rhoptry proteins.     

Attachment and invasion of host cells by Toxoplasma gondii

Recent studies indicate that Toxoplasma gondii attachment is mediated via a parasite ligand-host cell receptor interaction. Lloyd Kosper and Jose Mineo here survey factors involved in the attachment to and penetration and invasion of host cells by T. gondii.     

Importance of glycolysable substrates for in vitro capacitation of human spermatozoa

To investigate the importance of glycolysable substrate for supporting the ability of human sperm to capacitate and penetrate oocytes in vitro, washed spermatozoa were incubated with or without various sugars in BWW culture medium containing pyruvate and lactate. Sperm penetration was assayed using zona-free hamster oocytes. After an 18-h preincubation, glucose (1 mg/ml) supported higher penetration of sperm into oocytes than either mannose or fructose (60.7% vs. 28.2% or 21.5%, respectively) at the same concentration. Penetration was even lower when medium contained the nonmetabolizable sugar galactose (2.1% at 1 mg/ml). On the other hand, higher concentrations (5 or 10 mg/ml) of glucose, but not fructose, suppressed penetration, provided the glucose was present throughout the 18-h preincubation. When caffeine, a stimulant of glycolysis in human sperm, was present along with glucose, sperm penetration was enhanced, but only after 6 h of sperm preincubation. This effect was not observed in glucose-free medium, however, where penetration remained low over a 10-h incubation period. In these experiments, the percentage of motile sperm was unaffected by treatment, but the quality of motility was diminished in the absence of glucose. We conclude that stimulation of glycolysis may promote capacitation of human spermatozoa in vitro and that optimization of penetrating ability of sperm is dependent upon both the type and concentration of glycolysable sugar present.     

Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation

Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.     

弓形虫GRA6蛋白和P30蛋白的融合表达、纯化及抗原性分析

利用基因工程技术制备抗原性好的弓形虫GRA6蛋白和P30蛋白的融合蛋白,并用作抗原检测弓形虫抗体。根据弓形虫GRA6蛋白和P30蛋白的氨基酸序列,通过计算机分析,筛选出其中较强的抗原决定簇。用PCR方法分别扩增含抗原决定簇的基因片段。将这两个基因片段克隆至同一质粒pET28a(+)内,表达一个融合蛋白。将重组质粒转化大肠杆菌BL21(DE3),筛选表达该融合蛋白的工程菌。纯化表达的融合蛋白,用已知的6份抗弓形虫IgM阳性血清和大量正常人血清,ELISA法检测纯化融合蛋白的抗原性和特异性。获得了高效表达含弓形虫GRA6蛋白和P30蛋白抗原表位的工程菌,表达的融合蛋白约占菌体蛋白总量的25%。纯化获得了表达的融合蛋白,该蛋白有较好的抗原性和特异性。表达的弓形虫GRA6和P30融合蛋白可用做抗原检测弓形虫抗体,用于临床及孕妇检测,对优生优育有较大意义。     

Massive excystation of Toxoplasma gondii sporozoites

In the present study, several stimuli were tested to liberate Toxoplasma gondii sporozoites from oocysts. Incubation in Na(2)CO(3)-CO(2) buffer at 40 degrees C with ultrasound, followed by incubation at 40 degrees C with bile or deoxycholic acid, was the only method that resulted in 95% liberation of motile infective sporozoites. A protocol of reproducibly high efficiency is provided, that compares favorably with any of the methods previously published.     

Enhancement of human natural killer cell activity by subcellular components of Toxoplasma gondii

The ability of sonicates and subcellular fractions of the intracellular parasite Toxoplasma gondii to enhance in vitro human natural killer (NK) cell activity was examined. Incubation of nylon-wool-non-adherent human peripheral blood lymphocytes (PBL) with sonicates of T. gondii for 18-72 hr resulted in increased NK activity against an NK-sensitive, as well as an insensitive, target cell. Single-cell assays revealed that augmentation of NK activity was not due to an increased binding of K562 target cells to effector cells. Differential centrifugation studies indicated that NK-augmenting activity was distributed in membrane-enriched and cytoplasmic fractions. This activity was found to be resistant to treatment with ribonuclease (RNase) and deoxyribonuclease (DNase), but susceptible to proteolysis. Antibodies present in the serum of humans infected with Toxoplasma blocked the NK cell-augmenting effect of the membrane-enriched fractions. Enhancement of NK activity by PBL incubated with Toxoplasma sonicate was accompanied by a concomitant increase in interferon (IFN), but not of interleukin 2 (IL-2), levels in supernatants of the cell cultures.