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.     

Proteomic analysis of calcium-dependent secretion in Toxoplasma gondii

Toxoplasma gondii is an intracellular protozoan parasite that invades a wide range of nucleated cells. In the course of intracellular parasitism, the parasite releases a large variety of proteins from three secretory organelles, namely, micronemes, rhoptries and dense granules. Elevation of intracellular Ca(2+) in the parasite causes microneme discharge, and microneme secretion is essential for the invasion. In this study, we performed a proteomic analysis of the Ca(2+)-dependent secretion to evaluate the protein repertoire. We found that Ca(2+)-mobilising agents, such as thapsigargin, NH(4)Cl, ethanol and a Ca(2+) ionophore, A23187, promoted the secretion of the parasite proteins. The proteins, artificially secreted by A23187, were used in a comparative proteomic analysis by 2-DE followed by PMF analysis and/or N-terminal sequencing. Major known microneme proteins (MICs), such as MIC2, MIC4, MIC6 and MIC10 and apical membrane antigen 1 (AMA1), were identified, indicating that the proteomic analysis worked accurately. Interestingly, new members of secretory proteins, namely rhoptry protein 9 (ROP9) and Toxoplasma SPATR (TgSPATR), which was a homologue of a Plasmodium secreted protein with an altered thrombospondin repeat (SPATR), were detected in Ca(2+)-dependent secretion. Thus, we succeeded in detecting Ca(2+)-dependent secretory proteins in T. gondii, which contained novel secretory proteins.     

Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii

Toxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle‐free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with the identifier PXD004895.     

弓形虫感染实验动物模型的特点及建模方案的选择

弓形虫感染对人类生活和畜牧业发展构成严重威胁。弓形虫感染实验动物模型是进行弓形虫学相关研究的基础条件之一。在实际研究工作中,根据不同的实验目的、选取不同的实验动物和以不同的实验方法所建立的实验动物模型,呈现出复杂和多变的特点。这一方面可以满足不同实验的需要,但同时也在实验结果的评价上导致一定程度的不足。根据弓形虫感染实验动物模型的不同特点,针对特定的实验目的,选择适合方法建立适合的实验动物模型,是进行相关弓形虫学研究的有效基础和前提。     

Construction of Toxoplasma gondii bradyzoite expressing the green fluorescent protein

The bradyzoite stage of Toxoplasma gondii is a key step in the parasite life cycle. For a better understanding of this stage, a sensitive system to detect the tissue cysts would be required. In this study, we generated the T. gondii cyst-forming strain PLK expressing green fluorescent protein (GFP) under control of the dense granule protein 1 promoter, which works at both the tachyzoite and the bradyzoite stages. The bradyzoites with GFP fluorescence within both small and large cysts were detectable in the brain of mice infected with the recombinant PLK. Indeed, the bradyzoites expressing GFP had infectivity to mice. This study shows that transfection of the cyst-forming strain with GFP gene under control of the GRA1 promoter could be a useful approach for the study of the bradyzoite stage of T. gondii.     

Regulation of Toxoplasma gondii multiplication in BeWo trophoblast cells: cross-regulation of nitric oxide production and polyamine biosynthesis

Materno-foetal transmission causes one of the most severe forms of infection with the protozoan parasite Toxoplasma gondii. Several studies have shown T. gondii in placental trophoblast cells, which form the barrier between maternal blood circulation and foetal tissue. Parasite multiplication in trophoblast cells is thus a critical step leading to infection of the foetus. Here, we show that multiplication of T. gondii tachyzoites was slow in BeWo trophoblast cells, compared with MRC-5 fibroblast cells. However, unlike MRC-5 cells, even combined stimulation with interferon-γ and tumor necrosis factor- did not reduce T. gondii replication in BeWo cells. This was associated with a lack of indoleamine-2,3-dioxygenase induction by these cytokines. Neither low availability of iron salts, nor an immunosuppressive action of cyclooxygenase-2 could be attributed to the low T. gondii multiplication rate in BeWo cells. However, treatment with the nitric oxide synthesis inhibitor N^G-methyl-l-arginine and addition of ornithine enhanced the proliferation rate of the intracellular pathogen. Despite detection of inducible nitric oxide synthase-II mRNA in BeWo cells, nitric oxide production could not be detected during cell culture. Thus, inhibition of arginase activity by nitric oxide synthesis may be partially responsible for the lower multiplication rate in BeWo cells.     

载体介导的刚地弓形虫MIC疫苗的研制现状

刚地弓形虫引起的弓形虫病是一种严重危害人类健康的人兽共患寄生虫病,采用疫苗防治该病是当前研究的热点领域之一。MIC抗原是一种有效的疫苗候选分子,本研究综述了鼠伤寒沙门菌、卡介苗、格登链球菌、腺病毒血清型5和假狂犬病毒等载体介导的刚地弓形虫MIC疫苗的研制现状。     

Membrane fluidity of Toxoplasma gondii: a fluorescence polarization study

Toxoplasma gondii membrane fluidity was investigated by fluorescence polarization. We used 1,6-diphenyl 1,3,5-hexatriene (DPH) as a fluorescent hydrophobic probe. Fluorescence anisotropy (r) and degree of order (s) showed high fluidity properties. Chemical analysis was performed on this parasite. We found a low cholesterol/phospholipid ratio, many unsaturated fatty acids chains, and high phosphatidylcholine and low sphingomyelin amounts. These results were in good agreement with the observed high fluidity. This may be related to the great adaptability of Toxoplasma gondii in infesting a wide variety of host cells.     

Experimental tissue cyst induced Toxoplasma gondii infections in dogs

Tissue cyst induced Toxoplasma gondii infections were examined in 2 beagle dogs orally inoculated with tissue cysts. Neither dog developed clinical signs of toxoplasmosis. Both dogs developed low antibody titers to T. gondii. The MAT and IFAT were superior to the LAT and IHT tests for detecting antibodies to T. gondii.     

Development of an in vitro model of Toxoplasma gondii cyst formation

Abstract Toxoplasma gondii tissue cyst reactivation is a major pathogenic mechanism in ocular toxoplasmosis, disease associated with AIDS and organ transplantation. The mechanisms associated with cyst formation and reactivation have not been elucidated. The complexity of studying these issues in animal models has led to the development of in vitro tissue culture strategies for cyst formation. In the present study we have adopted the human embryonic lung fibroblast (HEL) as the host cell and have compared the cyst forming abilities of eight clinical isolates. We describe by transmission electron microscopy and quantitative light microscopy the development of cysts in vitro. The numbers of in vitro cysts increased with time for all isolates. Cyst cultures were stabilised by manipulation of the free parasite load, an observation not previously recorded. Thus, in this paper we describe a viable model for the analysis of the mechanisms of Toxoplasma cyst development.     

Toxoplasma gondii: cross-immunity against the enteric cycle

Eight of nine cats inoculated with strain ME-49 and challenged with three different strains of Toxoplasma were immune to oocyst shedding, as ascertained with bioassays of their feces. In a second experiment, only toxoplasma asexual stages were seen in H&E stained gut sections of cats treated with suppressive doses of sulfamerazine and pyrimethamine starting 2 days after oral inoculation with cysts of the strain ME-49 and killed 6 days later. In a third experiment, four cats were similarly inoculated and treated for 20 days. Six weeks later, the cats received an oral homologous challenge with cysts, and none shed toxoplasma oocysts. An acceptable level of cross-protection was achieved with strain ME-49, and therefore, it can be used as a candidate strain from which antigens could be tested for enteric protection.     

Peritoneal macrophages from C57BL/6 mice orally immunized with Toxoplasma gondii antigens in association with cholera toxin possess an enhanced ability to inhibit parasite multiplication

Abstract Gamma-interferon (IFN-γ) has been reported to be a major mediator of resistance to toxoplasma infection, mainly through macrophage activation. Cholera toxin used as oral adjuvant induces enhanced protection. Following oral immunization of C57BL/6 mice with a Toxoplasma gondii sonicate (TSo), in association with either cholera toxin (CT) or its B subunit (CTB), the ability of primed sensitized peritoneal macrophages (PMgF) to prevent T. gondii intracellular proliferation in vitro was examined both with and without rIFN-γ activation. Under these conditions, the inhibition of T. gondii multiplication was greatly enhanced in PMgF from mice immunized with a TSo and CT as an oral adjuvant. In contrast, PMgF from mice immunized with a TSo in association with CTB showed a decrease in their microbiostatic activity towards T. gondii . This negative effect on IFN-γ-treated PMgF was cancelled out by the addition of a small amount of CT in association with TSo and CTB in the immunization regimen. These data suggest that CT could act as an oral adjuvant in vaccination against toxoplasmosis by increasing the microbiostatic activity of MgF activated with IFN-γ. Further studies, using intestinal effector cells such as enterocytes, are needed to confirm the value of CT for enhancing this major mechanism of protection against T. gondii infection.     

弓形虫上海株的棒状体蛋白ROP2的克隆表达及纯化

根据已发表基因序列(GenBank登录号为Z36906)设计引物,以弓形虫(Toxoplasma gondii)上海本地株的基因组DNA为模板,扩增编码ROP2(rhpotry protein2)蛋白的基因片段,定向克隆至表达质粒pET32a(+),重组质粒经限制性酶切鉴定后测序,结果表明插入片段长度为1044bp,与GenBank上登录的序列相比,同源性为96%-100%,其中与弓形虫RH株的rop2基因同源性为100%。重组原核表达质粒pET32a-rop2转化至大肠杆菌BL21(DE3),经诱导可表达分子量约60.9kD的融合蛋白,能被感染弓形虫RH株的绵羊阳性血清识别。     

Quantitative immunolocalization of four immunodominant antigens of Toxoplasma gondii

Ultrastructural localization of four immunodominant antigens of Toxoplasma gondii was investigated quantitatively on thin sections and replicas by an immunogold technique using four monoclonal antibodies (Mab). On immunoblot Mab IV47, GII9, II38 and IE10 identified proteins of 28, 30, 45 and 66-70 kDa, respectively. Use of digital image analyzer and a semi-automatic procedure developed by us, the patterns of label distribution were compared in three cell structures: cell surface, submembrane area and rhoptries. On the whole cell surface, protein P28 and P30 were 2.5 and 4 times more abundant than P66-70 respectively. The protein P28 was essentially concentrated in the submembrane area with a labeling of 195.4 +/- 46.7 gold particles/microns 2 that follows a decreasing gradient from this area to the cell centre. In the rhoptries, all four antigens were detected, P45 and P66-70 being major with a labeling of 97.1 +/- 31.1 gold particles/microns 2 and 155.1 +/- 39.3 gold particles/microns 2 respectively. The results support the hypothesis that rhoptries are the essential site for antigen storage.     

弓形虫（Toxoplasma gondii）的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30（SAG1）设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10^-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率（10％）稍高于单一PCR检测阳性率（8．67％）。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。     

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca²⁺) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca²⁺ signaling is essential for the cellular and developmental changes that support T. gondii parasitism.The characterization of the molecular players and pathways directly activated by Ca²⁺ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca²⁺ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca²⁺ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.     

Ultrastructure of Excystment of Toxoplasma gondii Oocysts*

SYNOPSIS. The excystation of sporozoites from intact Toxoplasma gondii oocysts or mechanically released sporocysts was studied by light and electron microscopy. Both intact oocysts and free sporocysts excysted in 5% bovine bile in 0.9% NaCl solution after 30–60 min incubation at 37 C. Sporozoites were first activated in either intact sporocysts or oocysts within 2–12 min of incubation in bile. Sporozoites escaped from sporocysts through 4 plate-like sutures in the sporocyst wall, and from the oocyst as the oocyst wall ruptured at one or more points.     

Detection of Toxoplasma gondii Infections using Virus-Like Particles Displaying T. gondii ROP4 Antigen

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.     

A Cell Culture System for Study of the Development of Toxoplasma gondii Bradyzoites

ABSTRACT. Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii . Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of γ-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.