Multi-membrane-bound structures of Apicomplexa: I. the architecture of the Toxoplasma gondii apicoplast

Apicomplexan parasites carry a plastid-like organelle termed apicoplast. The previous documentation of four membranes bordering the Toxoplasma gondii apicoplast suggested a secondary endosymbiotic ancestry of this organelle. However, a four-membraned apicoplast wall could not be confirmed for all Apicomplexa including the malarial agents. The latter reportedly possesses a mostly tri-laminar plastid wall but also displays two multi-laminar wall partitions. Since these sectors apparently evolved from regional wall membrane infoldings, the malarial plastid could have lost one secondary wall membrane in the course of evolution. Such wall construction was however not unambiguously resolved. To examine whether the wall of the T. gondii apicoplast is comparably complex, serial ultra-thin sections of tachyzoites were analyzed. This investigation revealed a single pocket-like invagination within a four-laminar wall segment but also disclosed that four individual membranes do not surround the entire T. gondii apicoplast. Instead, this organelle possesses an extensive sector that is bordered by two membranes. Such heterogeneous wall construction could be explained if the inner two membranes of a formerly four-membraned endosymbiont are partially lost. However, our findings are more consistent with an essentially dual-membraned organelle that creates four-laminar wall sectors by expansive infoldings of its interior border. Given this architecture, the T. gondii apicoplast depicts a residual primary plastid not a secondary one as presently proposed.     

Subversion of innate and adaptive immune responses by <Emphasis Type="Italic">Toxoplasma Gondii</Emphasis>

The intracellular apicomplexan parasite Toxoplasma gondii is able to survive and persist in immunocompetent intermediate hosts for the host’s life span. This is despite the induction of a vigorous humoral and—more importantly—cell-mediated immune response during infection. In order to establish and maintain such chronic infections, however, T. gondii has evolved multiple strategies to avoid or to interfere with potentially efficient anti-parasitic immune responses of the host. Such immune evasion includes (1) indirect mechanisms by altering the expression and secretion of immunomodulatory cytokines or by altering the viability of immune cells and (2) direct mechanisms by establishing a lifestyle within a suitable intracellular niche and by interference with intracellular signaling cascades, thereby abolishing a number of antimicrobial effector mechanisms of the host. Despite the parasite’s ability to interfere successfully with the host’s efforts to eradicate the infection, the immune response is, however, not completely abrogated but is rather partially diminished after infection. T. gondii thus keeps a delicate balance between induction and suppression of the host’s immune response in order to guarantee the survival of the host as a safe harbor for parasite development and to allow its transmission to the definitive host.     

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by <Emphasis Type="Italic">Neospora caninum</Emphasis> tachyzoites

Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4^c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4^c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.     

A cytochemistry study of the inner membrane complex of the pellicle of tachyzoites of Toxoplasma gondii

Confocal laser scanning microscopy and transmission electron microscopy were used to study the inner membrane complex of tachyzoites of Toxoplasma gondii. DiOC₆, a lipophilic cationic fluorescent dye used to visualize the endoplasmic reticulum of eukaryotic cells, labeled cytoplasmic structures in a reticulated pattern and the periphery of the nucleus of the host cell. Intracellular and extracellular tachyzoites were stained. Observation of several focal planes showed labeling of the most peripheral region of the protozoan. Reaction product was observed in the outer nuclear membrane, in profiles of the endoplasmic reticulum, and in the inner membrane complex of tachyzoites subjected to the KI-OsO₄ technique. Taken together, these observations suggest that the inner membrane complex may represent a specialized region of the endoplasmic reticulum of tachyzoites of T. gondii. Received: 20 May 1996/Accepted: 6 September 1996     

Evaluation of drug effects on Toxoplasma gondii nuclear and plastid DNA replication using real-time PCR

Toxoplasma gondii Nicolle and Manceaux, 1908 is a unicellular protozoan that can infect a broad spectrum of organisms including humans. In addition to a nuclear genome, it also carries a circular DNA within a plastid-like organelle (apicoplast) and a linear genome within its mitochondria. The plastid organelle has been shown to be the target of various anti-parasitic drugs or antibiotics. To evaluate the effects of agents on the DNA replication of T. gondii, we tested six drugs (ciprofloxacin, acetylspiramycin, clindamycin, azithromycin, artemether, and sulfadiazine) on the parasite cultured in Hela cells. After drug treatment for 48 h, the parasite growth and DNA replication were evaluated and quantitated using TaqMan real-time quantitative PCR with oligonucleotide primers synthesized based on a gene from the apicoplast genome (ycf24, Genbank accession no. U87145) and a gene from the nuclear genome (uprt, Genbank accession no. U10246). Our results showed that ciprofloxacin was the most effective in inhibiting the replication of the plastid DNA after 48 h drug treatment, with a reduction of 22% in the copy number of the plastid DNA. Artemether was the most effective drug in suppressing the proliferation of tachyzoites. This study also demonstrates that real-time quantitative PCR is a simple and useful technique for monitoring parasite growth and DNA replication.     

Transplacental transmission of Toxoplasma gondii in reinfected pregnant female canines

Twelve pregnant female canines, naturally infected with Toxoplasma gondii, were reinfected with T. gondii: three (GI) received tachyzoites subcutaneously (1.0 × 107), three (GII) were orally inoculated with oocysts (1.5 × 104), and six (GIII) were kept as a nonreinfected control group. All the reinfected female canines (GI and GII) miscarried or presented fetal death, while only one GIII female presented a stillborn in a litter of four pups (P < 0.01). Fever, lymphoadenopathy, miscarriage, and fetal death were the main clinical alterations observed. The highest serological titers detected through the indirect fluorescence antibody test (IFAT) were 1,024 (GI) and 4,096 (GII). In group III, the titers ranged between 64 and 256. By bioassays in mice, T. gondii was isolated in 17 organs of the reinfected adult canines, in 11 of the control group, and in 20 of the neonates. Positive immunostaining of cysts and/or tachyzoites were observed in 26 canine tissues (14 from GI and GII and ten from GIII). The agent was detected by immunohistochemistry in the encephalon of a neonate and in the spinal cord of a stillborn, thus, confirming that T. gondii infected canine fetuses, provoking miscarriages, even in bitches that presented primoinfection.     

Seroprevalence of Neospora caninum and Toxoplasma gondii in camels (Camelus dromedarius) in Mashhad, Iran

One hundred twenty camels were blood-sampled and used to evaluate serological screening for Neospora caninum and Toxoplasma gondii infection by indirect fluorescent antibody test (IFAT) in Mashhad, Iran, during years 2004–2005. Of the 120 camels, antibodies to N. caninum were found in three in titers of 1:20 and in four in titers of 1:40 using whole N. caninum tachyzoites as IFAT slide (VMRD Inc., Pullman, WA 99163, USA). Antibodies to T. gondii were found in three camels in titers 1:20 and in two camels in titers 1:40 using whole T. gondii tachyzoites as IFAT slide (BIOGENE, Iran).     

The effect of prolactin (PRL) on the growth of Toxoplasma gondii tachyzoites in vitro

During the development and effector phases of the anti-Toxoplasma response, the immunological system of a host is involved in several complex interactions with the endocrine system, and prolactin (PRL) is one of the most important hormones involved in immunoregulation. In this work, the influence of the recombinant human prolactin (rhPRL) on the viability, penetration, and intensity of intracellular proliferation of Toxoplasma gondii BK strain in vitro was evaluated. Using one murine (L929) and two human cell lines (Hs27 and HeLa), no toxic effect of the rhPRL on host cells was found (by determining cellular viability using MTT assay). A similar lack of rhPRL cytotoxic activity was found in the case of the extracellular tachyzoites of T. gondii BK. Replication of parasites in the presence of rhPRL was analyzed first by simultaneous addition of the hormone and the parasites into a microculture of the host cells (treatment during infection). No statistically significant changes in the intensity of parasite proliferation in all used host cells were found for a wide range of the hormone concentrations. However, pre-incubation of the tachyzoites with rhPRL resulted in a significant reduction (up to 36.15%) in the replication abilities of the parasite. Further experiments revealed that in fact, the inhibition of replication was caused by a limited capacity of the parasites to penetrate host’s cells as demonstrated by the reduced number of infected cells.     

Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain

In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2⁺ neurons, GFAP⁺ astrocytes, F4/80⁺ microglia, and O1⁺ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection. Received: 25 March 1997 / Accepted: 16 April 1997     

Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion

Apicomplexa are primarily obligate intracellular protozoa that have evolved complex developmental stages important for pathogenesis and transmission. Toxoplasma gondii, responsible for the disease toxoplasmosis, has the broadest host range of the Apicomplexa as it infects virtually any warm-blooded vertebrate host. Key to T. gondii’s pathogenesis is its ability to differentiate from a rapidly replicating tachyzoite stage during acute infection to a relatively non-immunogenic, dormant bradyzoite stage contained in tissue cysts. These bradyzoite cysts can reconvert back to tachyzoites years later causing serious pathology and death if a person becomes immune-compromised. Like the sexual stage sporozoites, bradyzoites are also orally infectious and a major contributor to transmission. Because of the critical role of stage conversion to pathogenesis and transmission, a major research focus is aimed at identifying molecular mediators and pathways that regulate differentiation. Tachyzoite to bradyzoite development can occur spontaneously in vitro and be induced in response to exogenous stress including but not limited to host immunity. The purpose of this review is to explore the potential contributors to stage differentiation in infection and how a determination is made by the parasite to differentiate from tachyzoites to bradyzoites.     

Cryopreservation of Toxoplasma gondii in infected murine tissues

Laboratory maintenance of the RH strain of Toxoplasma gondii is generally done by passage in mice, in vitro propagation in fibroblasts, or cryopreservation of peritoneal exudates from mice infected with T. gondii. To explore alternative techniques for preserving laboratory T. gondii tachyzoites, we propose a new method of freezing tissues from infected mice. The effect of storage of T. gondii tissue tachyzoites in two different cryoprotectant combinations and at two different temperatures was studied. The liver and spleen tissues, and peritoneal exudates from mice infected with RH-GFP strain of T. gondii, suspended in RPMI 1640 medium supplemented with 12 % glycerol plus 20 % calf serum, or 12 % dimethyl sulfoxide (DMSO) plus 20 % calf serum, were stored for 3 months at ?20 °C in an ordinary refrigerator or at ?80 °C in a deep freezer, respectively. The viability of tissue T. gondii tachyzoites was determined by animal inoculation method, which was assessed by monitoring survival and tissue parasitemia in recipient mice. Our data showed that toxoplasma tachyzoites in the above tissues remained viable after cryopreservation in 12 % DMSO plus 20 % calf serum at ?80 °C, the infectivity of tachyzoites from the tissues and peritoneal fluids was demonstrated in inoculated murine tissues. Our data indicate that freezing infected murine tissues at ?80 °C provides a simple and appropriate method for preservation of T. gondii tachyzoites in laboratory without the need for costly liquid nitrogen preservation procedures.     

Calmodulin expression during <Emphasis Type="Italic">Giardia intestinalis</Emphasis> differentiation and identification of calmodulin-binding proteins during the trophozoite stage

Calmodulin (CaM) is the primary sensor for calcium in the cell. It modulates various functions by activating CaM-binding proteins (CaMBPs). This study examined the calcium/CaM-dependent system in the ancient eukaryote Giardia intestinalis. A specific antibody against the parasite’s CaM was developed; this protein’s expression and location during different stages of the parasite’s life cycle were analyzed. The results showed that it is a housekeeping protein which is possibly involved in the parasite’s motility. No CaMBP has been identified in G. intestinalis to date. Pull-down assays were used for isolating proteins which specifically bind to CaM in a calcium-dependent way. Three of them were identified through mass spectrometry; they were GASP180, α-tubulin, and pyruvate phosphate dikinase (PPDK).The first two are cytoskeleton proteins, and the last one is an essential enzyme for glycolysis. The presence of binding sites was analyzed through bioinformatics in each protein sequence. This is the first report of a CaMBP in this organism; it is considered to be a very interesting differentiation model, indicating that CaM is involved at least in two vital processes: G. intestinalis motility and energetic metabolism     

Histopathology of the reproductive system of male sheep experimentally infected with Toxoplasma gondii

The aim of this study was to investigate the histopathological changes in reproductive system (testicles, epididymis, seminal vesicles, and prostate) of small male ruminants after Toxoplasma gondii infection. Eight sheep were inoculated with T. gondii: group I, four sheep (2.0 × 10⁵ P-strain oocysts); group II, four sheep (1.0 × 10⁶ RH-strain tachyzoites); and group III, two uninfected sheep maintained as control. Infection with T. gondii was confirmed by seroconversion (indirect fluorescent antibody test-IgG) in all the infected animals beginning on post-inoculation day (PID) 7. On PID 70, all the animals were euthanized and tissue samples (testicles, epididymis, seminal vesicles, and prostate) were collected and processed for histological analysis. The main changes detected were a focal mononuclear interstitial inflammatory infiltrate in the prostate and seminal vesicles; diffuse testicular degeneration associated with calcification foci and a multifocal mononuclear interstitial inflammatory infiltrate; and a mononuclear interstitial infiltrate and focal necrotic areas of the muscle fibers surrounding the seminal vesicles. The histopathological findings of this work, along with the detection of T. gondii in the examined parenchyma tissues (immunohistochemistry) and the results obtained by other authors examining different tissues, suggest that histological changes diagnosed in the reproductive system of rams infected with T. gondii are strongly suggestive of toxoplasmatic infection.     

Cell-Culture System for Continuous Production of Toxoplasma gondii Tachyzoites

 The aim of this study was to identify a sustainable cell line and culture method that could continuously provide a sufficient quantity of Toxoplasma gondii tachyzoites to serve the needs of a general hospital laboratory. Three continuous cell lines (HeLa, LLC and Vero) and three cell-culture methods (culture in conventional flasks, culture in membrane-based flasks and an automated culture system) were investigated. In multiplicity-of-infection and time-course experiments, HeLa was the cell line of choice. Harvests from HeLa cells had significantly higher tachyzoite yields than those from LLC cells (P<0.00005) or Vero cells (P<0.05). Membrane-based flasks gave higher yields (6.15×10⁶ tachyzoites/ml) than conventional flasks (1–2×10⁶ tachyzoites/ml) initially, but these were not sustained. The automated cell-culture system was unsuitable for parasite culture. Continuous passage in 25 cm² flasks was successful, yielding 1×10⁶ tachyzoites/ml; viability exceeded 90% after 96–120 h of infection throughout 38 passes, during which time the viability improved and the time to harvest became more consistent. Toxoplasma gondii grown in continuous culture in HeLa cells can provide a regular supply of viable tachyzoites. Demonstration that HeLa-derived tachyzoites could be used for the dye test confirms the potential of this in vitro system for use in general hospital laboratories.     

Toxoplasma gondii: localization of purine nucleoside phosphorylase activity in vitro and in vivo by electron microscopy

Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) activity was revealed by enzyme histochemistry in Toxoplasma gondii ME49 strain isolated from murine cerebral cysts and from in vitro cultivation. The activity of the enzyme was revealed by an insoluble electron-opaque precipitate of lead phosphate at the site of the reaction. In bradyzoites and tachyzoites of T. gondii, the enzyme activity could be observed only in the cytoplasm. In bradyzoites, one or two foci of important PNP activity were detected near the nucleus. In tachyzoites, an important PNP activity underlined the plasma membrane. For both bradyzoites and tachyzoites, localization neither in the nucleus nor in cytoplasmic organelles could be detected.     

Psychosis may be associated with toxoplasmosis

Many parasites induce characteristic changes in their host. The effect of Toxoplasma gondii infection on the cerebrum and neuropsychiatric patients has been increasingly emphasized in recent years. T. gondii has a high affinity for brain tissue where tachyzoites may form tissue cysts and persist for a life long time. Some psychiatric symptoms such as schizophrenia and mental retardation may be induced by the infection of T. gondii. Furthermore, experiments demonstrated that some antipsychotics and mood stabilizers used to treat psychosis displayed the function of inhibiting T. gondii replication. Investigations from various regions in China in psychotic patients support the hypotheses that psychosis may be linked to T. gondii infection.     

Improved methods for examination of Toxoplasma gondii cytoskeleton at ultrastructural level

To evaluate an improved method for identifying the presence of the structural elements of the cytoskeleton of Toxoplasma gondii and their influence on invasion of the parasite in host cells, copper grids coated with plastic film were used for adhesion of whole parasites. Tachyzoites were incubated with 0.5% Triton X-100 in PHEM buffer containing protease inhibitors, post-fixed in 1% glutaraldehyde, stained with uranyl acetate and submitted to critical point drying. In order to analyze the presence of the structural elements of the cytoskeleton, immunolocalization was carried out using colloidal gold. Invasion of the parasite was examined on cell culture after treatment of tachyzoites with cytochalasin B (CB). In order to observe this effect, an immunocytochemical assay using alkaline phosphatase was carried out. A very well conserved extraction of the cytoskeleton elements of T. gondii, such as conoid and microtubules, as well as the rhoptries, was observed. By immunolocalization with colloidal gold, it was possible to detect the actin in its globular form. In the assay of invasion of the parasite on the host cell, after treatment of the T. gondii tachyzoites with CB, the invasion process was totally inhibited. Received: 18 April 2000 / Accepted: 19 August 2000     

Toxoplasma gondii in Capybara (Hydrochaeris hydrochaeris) antibodies and DNA detected by IFAT and PCR

Toxoplasmosis is considered nowadays as one of the most important foodborne diseases in the world. One of the emerging risks in acquiring infection with Toxoplasma gondii is the increasing popularity of wild animals and game meat. Capybara (Hydrochaeris hydrochaeris) is the world’s largest extant rodent and is used for human consumption in many areas of South America, and in case it carries T. gondii cysts, it may act as a source of infection. In the present study, we detected infection with T. gondii in capybaras from the south of Brazil. Antibodies to T. gondii were assayed in the serum of capybaras using the indirect fluorescent antibody test (IFAT ≥ 1:16). Blood, liver, heart, lymph nodes, and spleen tissues were collected and tested by polymerase chain reaction (PCR) for B1 gene and ITS1 region. The results showed that 61.5% (16/26) capybaras were seropositive to T. gondii. Titers of specific antibodies to T. gondii ranged from 1:16 to 1:512. Among the feral rodents studied, 7.7% (2/26) were PCR positive for B1 gene assay and 11.5% (3/26) were positive for ITS1 PCR assay; for both test, the prevalence was 15.4%. Liver, heart, and blood tissues were those which tested positive for the apicomplexan. Our findings show a high percentage of infection with T. gondii in asymptomatic capybaras. Based on those data, we hypothesize that the consumption of raw or undercooked capybara meat could be a source of infection for humans.     

Functional and biophysical analyses of the class XIV Toxoplasma gondii Myosin D

Summary The obligate intracellular parasite Toxoplasma gondii uses gliding motility to migrate across the biological barriers of the host and to invade cells. This unique form of locomotion requires an intact actin cytoskeleton and involves at least one motor protein (TgMyoA) that belongs to the class XIV of the myosin superfamily. TgMyoA is anchored in the inner membrane complex and is essential for the gliding motion, host cell invasion and egress of T. gondii tachyzoites. TgMyoD is the smallest T. gondii myosin and is structurally very closely related to TgMyoA. We show here that TgMyoD exhibits similar transient kinetic properties as the fast single-headed TgMyoA. To determine if TgMyoD also contributes to parasite gliding motility, the TgMyoD gene was disrupted by double homologous recombination. In contrast to TgMyoA, TgMyoD gene is dispensable for tachyzoite propagation and motility. Parasites lacking TgMyoD glide normally and their virulence is not compromised in mice. The fact that TgMyoD is predominantly expressed in bradyzoites explains the absence of a phenotype observed with myodko in tachyzoites and does not exclude a role of this motor in gliding that would be restricted to the cyst forming but nevertheless motile stage of the parasite.Both authors contributed equally to the work.