Androgens Exert a Cysticidal Effect upon Taenia crassiceps by Disrupting Flame Cell Morphology and Function

The effects of testosterone (T₄) and dihydrotestosterone (DHT) on the survival of the helminth cestode parasite Taenia crassiceps, as well as their effects on actin, tubulin and myosin expression and their assembly into the excretory system of flame cells are described in this paper. In vitro evaluations on parasite viability, flow cytometry, confocal microscopy, video-microscopy of live flame cells, and docking experiments of androgens interacting with actin, tubulin, and myosin were conducted. Our results show that T₄ and DHT reduce T. crassiceps viability in a dose- and time-dependent fashion, reaching 90% of mortality at the highest dose used (40 ng/ml) and time exposed (10 days) in culture. Androgen treatment does not induce differences in the specific expression pattern of actin, tubulin, and myosin isoforms as compared with control parasites. Confocal microscopy demonstrated a strong disruption of the parasite tegument, with reduced assembly, shape, and motion of flame cells. Docking experiments show that androgens are capable of affecting parasite survival and flame cell morphology by directly interacting with actin, tubulin and myosin without altering their protein expression pattern. We show that both T₄ and DHT are able to bind actin, tubulin, and myosin affecting their assembly and causing parasite intoxication due to impairment of flame cell function. Live flame cell video microscopy showing a reduced motion as well changes in the shape of flame cells are also shown. In summary, T₄ and DHT directly act on T. crassiceps cysticerci through altering parasite survival as well as the assembly and function of flame cells.     

Steroid hormone production by parasites: the case of Taenia crassiceps and Taenia solium cysticerci

Many examples of reciprocal endocrine interactions between parasites and hosts have been found in insects, arthropods and mammals. Cysticercosis produced by Taenia solium metacestodes is a widely distributed parasite infection that affects the human and the pig. Taenia crassiceps experimental murine cysticercosis has been used to explore the role of biological factors involved in host–parasite interactions. We had shown that T. crassiceps cysticercosis affects the serum concentration of steroid hormones and the reproduction behavior of the male mice host. In an effort to understand the biology of the parasite, we had investigated the parasite capacity to produce sex steroids. For this purpose, T. crassiceps cysticerci were incubated in the presence of different steroid precursors. TLC and recrystallization procedures showed that testosterone is produced from ³H-androstenedione in cysticerci. The conversion of ³H-testosterone to androstenedione, although present is much less significant. In addition, we had studied the production of testosterone by T. solium cysticerci. For this purpose, cysticerci were dissected from pork meat and incubated as above described. The results showed that T. solium cysticerci also produce testosterone. We have speculated about the importance of androgens in the growth of T. crassiceps cysticerci and found that the addition of the antiandrogen flutamide to the culture media of the parasites significantly decreased ³H-thymidine incorporation. We therefore hypothesized, that the ability of cysticerci to produce testosterone from steroid precursors might be important for the parasite growth and development.     

Parasiticidal effect of 16α-bromoepiandrosterone (EpiBr) in amoebiasis and cysticercosis

The effect of the dehydroepiandrosterone analog 16α-bromoepiandrosterone (EpiBr) was tested on the tapeworm Taenia crassiceps and the protist Entamoeba histolytica, both in vivo and in vitro. Administration of EpiBr prior to infection with cysticerci in mice reduced the parasite load by 50% compared with controls. EpiBr treatment induced 20% reduction on the development of amoebic liver abscesses in hamsters. In vitro treatment of T. crassiceps and E. histolytica cultures with EpiBr, reduced reproduction, motility and viability in a dose- and time-dependent fashion. These results leave open the possibility of assessing the potential of this hormonal analog as a possible anti-parasite drug, including cysticercosis and amoebiasis.     

Taenia crassiceps antigens induce a Th2 immune response and attenuate injuries experimentally induced by neurotoxoplasmosis in BALB/c mice

Toxoplasma gondii is a pathogenic agent responsible for causing both systemic and local disease which elicits a typically pro-inflammatory, Th1 immune response. Taenia crassiceps antigen induces a Th2 immune response that immunomodulates Th1 based infections. Therefore the aim of this study was to evaluate whether T. crassiceps cysticerci antigens are able to modulate the inflammatory response triggered in experimental neurotoxoplasmosis (NT). BALB/c mice were inoculated with T. gondii cysts and/or cysticerci antigens and euthanized at 60 and 90 days after inoculation (DAI). The histopathology of the brains and cytokines produced by spleen cells culture were performed. The animals from the NT group, 90DAI (NT90), presented greater intensity of lesions such as vasculitis, meningitis and microgliosis and cytokines from Th1 profile characterized by high levels of IFN-gamma. While in the T. crassiceps antigens group, 60DAI, there were more discrete lesions and high levels of IL-4, a Th2 cytokine. In the NT co-inoculated with cysticerci antigens group the parenchyma lesions were more discrete with lower levels of IFN-gamma and higher levels of IL-4 when compared to NT90. Therefore the inoculation of T. crassiceps antigens attenuated the brain lesions caused by T. gondii inducing a Th2 immune response.     

Taenia crassiceps: chloride currents expressed in Xenopus oocytes upon injection of mRNA of cysticerci (WFU strain) isolated from mice

To study the properties of ion channels of the tapeworm Taenia crassiceps, mRNA was isolated from cysticerci and injected into mature oocytes of the frog Xenopus laevis and ion currents were recorded four days after injection with the two-electrode voltage clamp technique. Oocytes injected with mRNA of T. crassiceps expressed outward currents (I_TC) that activated instantly after onset of the test pulse, followed by a slow inactivation at potentials over +40 mV, with a reversal potential of −23.2 ± 5 mV. They were not affected by changes on monovalent cationic composition of external media, but replacement of external chloride by gluconate shifted significantly the reversal potential, suggesting that I_TC are anion currents, with a permeability sequence of . These currents were sensitive to changes of external pH but not to hypotonic challenges. They were significantly inhibited by DIDS, NPPB and Niflumic acid, but not by 9-anthracene. These results suggest that I_TC are the result of expression of anion channels from the tapeworm T. crassiceps.     

Taenia crassiceps: energetic and respiratory metabolism from cysticerci exposed to praziquantel and albendazole in vitro

Praziquantel prevents glucose uptake, influencing energy metabolism, while albendazole selectively inhibits the uptake of glucose, leading to glycogen storage depletion in the parasite. The objective of this study was to determine the concentrations of glucose and organic acids related to energy and respiratory metabolisms in in vitroTaenia crassiceps cysticerci in the initial, larval and final stages exposed to sub lethal dosages of anti-helminthic drugs. Spectrophotometric and chromatographic analysis were performed to detect glucose, lactate, oxaloacetate, citrate, malate, fumarate and succinate secreted/excreted by in vitro cysticerci with 0.03 and 0.06 μg/mL of praziquantel and 0.05 and 0.075 μg/mL of albendazole. The anti-helminthic drugs decreased the excretion of lactate and induced aerobic energy pathways. Concentrations of glucose remained unaltered confirming blockage of its uptake.     

Immunological response of the rat to infection with Taenia taeniaeformis: protective antibody response to implanted parasites.

Intraperitoneally implanted metacestodes of either T. taeniaeformis or T. crassiceps in rats provoked a high degree of resistance to oral challenge with eggs of T. taeniaeformis. This resistance was passively transferred to normal recipients with serum. Immunoglobulin fractions of immune serum containing IgG₁ or IgM were most effective in passive transfer and little activity was associated with IgG₂ antibodies. No skin-sensitizing antibodies were detectable in immune sera. These findings are in sharp contrast to previous observations involving protective immunoglobulins and reaginic antibodies in serum from rats with hepatic cysticerci of T. taeniaeformis. Possible reasons for this are discussed. Cysticerci implanted into normal rats survived for at least 21 days with no sign of host rejection, whereas those implanted into rats with hepatic infections with T. taeniaeformis were killed and encapsulated. Similar results were obtained by implanting cysticerci in normal rats given inoculations of complete Freund's adjuvant. Repeated inoculations of immune serum had no effect on the survival of implanted cysticerci, and it was concluded that exposure to infection by oncospheres provokes cellular defense mechanisms which can be effective against cysticerci in abnormal sites. Why these mechanisms are inoperative against hepatic cysticerci remains unclear.     

Genetic variation in the Cytb gene of human cerebral Taenia solium cysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishment of larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity.     

Plasmodium falciparum Dynein Light Chain 1 Interacts with Actin/Myosin during Blood Stage Development

Dynein light chain 1 (LC1), a member of the leucine-rich repeat protein family, has been shown to be engaged in controlling flagellar motility in Chlamydomonas reinhardtii and Trypanosoma brucei via its interaction with the dynein γ heavy chain. In Plasmodium falciparum, we have identified the LC1 ortholog, designated Pfdlc1. Negative attempts to disrupt the dlc1 gene by reverse genetic approaches in both P. falciparum and P. berghei suggest either its essentiality for parasite survival or the inaccessibility of its locus. Expression studies revealed high levels of DLC1 protein in late trophozoites and schizonts, pointing to an unexpected role of this protein in blood-stage parasites as they do not have flagella. Interactions studies and co-immunoprecipitation experiments revealed that PfDLC1 was able to bind to P. falciparum myosin A and actin 1. The PfDLC1 interacting domains present in P. falciparum myosin A and actin 1 were mapped to sequences containing SDIE and/or EEMKT motifs present in the upper 50-kDa segment of the myosin A head domain and in the subdomain IV of actin 1, respectively. Detection of PfDLC1 by fluorescence tagging and immunofluorescence staining using specific antibodies showed a cytoplasmic location similar to actin and immunofluorescence studies showed a co-localization of PfDLC1 and myosin A. Taken together, these findings suggest that PfDLC1 might play an important role in P. falciparum erythrocytic stages by its interaction with myosin A and actin 1, known to be essential for parasite development.     

Early removal of alternatively activated macrophages leads to Taenia crassiceps cysticercosis clearance in vivo

To determine the role of alternatively activated macrophages in modulating the outcome of experimental cysticercosis caused by Taenia crassiceps, we investigated the effect of removal of alternatively activated macrophage by injecting clodronate-loaded liposomes into susceptible BALB/c mice. Following T. crassiceps infection, mice receiving PBS-loaded liposomes developed a dominant Th2-type response associated with the presence of alternatively activated macrophages together with antigen-specific hyporesponsiveness and high parasite burden. In contrast, similarly infected mice treated with clodronate-loaded liposomes mounted a mixed Th1/Th2-type response, reversed antigen-specific hyporesponsiveness and did not carry notable alternatively activated macrophage populations. These factors were associated with increased resistance to T. crassiceps cysticercosis. Interestingly, early AAM? depletion was enough to limit parasite growth. However, if macrophages were depleted late in the infection, no effect on parasite burden was observed. These findings demonstrate that alternatively activated macrophages play a critical role in mediating susceptibility to experimental cysticercosis in which their early recruitment may favor parasite survival.     

Protococcidian Eleutheroschizon duboscqi,an Unusual Apicomplexan Interconnecting Gregarines and Cryptosporidia

This study focused on the attachment strategy, cell structure and the host-parasite interactions of the protococcidian Eleutheroschizon duboscqi, parasitising the polychaete Scoloplos armiger. The attached trophozoites and gamonts of E. duboscqi were detected at different development stages. The parasite develops epicellularly, covered by a host cell-derived, two-membrane parasitophorous sac forming a caudal tipped appendage. Staining with Evans blue suggests that this tail is protein-rich, supported by the presence of a fibrous substance in this area. Despite the ultrastructural evidence for long filaments in the tail, it stained only weakly for F-actin, while spectrin seemed to accumulate in this area. The attachment apparatus consists of lobes arranged in one (trophozoites) or two (gamonts) circles, crowned by a ring of filamentous fascicles. During trophozoite maturation, the internal space between the parasitophorous sac and parasite turns translucent, the parasite trilaminar pellicle seems to reorganise and is covered by a dense fibrous glycocalyx. The parasite surface is organised in broad folds with grooves in between. Micropores are situated at the bottom of the grooves. A layer of filaments organised in bands, underlying the folds and ending above the attachment fascicles, was detected just beneath the pellicle. Confocal microscopy, along with the application of cytoskeletal drugs (jasplakinolide, cytochalasin D, oryzalin) confirmed the presence of actin and tubulin polymerised forms in both the parasitophorous sac and the parasite, while myosin labelling was restricted to the sac. Despite positive tubulin labelling, no microtubules were detected in mature stages. The attachment strategy of E. duboscqi shares features with that of cryptosporidia and gregarines, i.e. the parasite itself conspicuously resembles an epicellularly located gregarine, while the parasitophorous sac develops in a similar manner to that in cryptosporidia. This study provides a re-evaluation of epicellular development in other apicomplexans and directly compares their niche with that of E. duboscqi.     

Taenia saginata and T. hydatigena: intramuscular vaccination of calves with oncospheres

This is a preliminary attempt to immunize calves against bovine cysticercosis (Cestoda) by intramuscular injection of artificially hatched homologous or heterologous oncospheres. In the first two experiments five calves were vaccinated with the oncospheres of Taenia saginata. These calves, together with five controls, were subsequently challenged orally with the eggs of T. saginata. At autopsy all the vaccinated animals harbored a colony of living cysticerci at the site of injection. Three of these calves were completely protected against the oral challenge, and in the other two, only one, and two cysticerci, respectively were found in the sites of election. The five controls harbored 1154, 1680, 820, 960, and 1820 living cysticerci, respectively, in the sites of election.     

A Toxoplasma gondii Class XIV Myosin,Expressed in Sf9 Cells with a Parasite Co-chaperone,Requires Two Light Chains for Fast Motility

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.     

Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum

A myosin-like protein was purified from amoebae of the cellular slime mold Dictyostelium discoideum. The purification utilized newly discovered solubility properties of actomyosin in sucrose. The amoebae were extracted with a 30% sucrose solution containing 0.1 m-KCl, and actomyosin was selectively precipitated from this crude extract by removal of the sucrose. The myosin and actin were then solubilized in a buffer containing KI and separated by gel filtration.The purified Dictyostelium myosin bears a very close resemblance to muscle myosin. The amoeba protein contains two heavy chains, about 210,000 molecular weight each, and two classes of light chains, 16,000 and 18,000 molecular weight. Dictyostelium myosin is insoluble at low ionic strength and forms bipolar thick filaments. The myosin possesses ATPase activity that is activated by Ca²⁺ but not EDTA, and is inhibited by Mg²⁺; under optimal conditions the specific activity of the enzyme is 0.09 μmol P₁/min per mg myosin.Dictyostelium myosin interacts with Dictyostelium actin or muscle actin, as shown by electron microscopy and by measurements of enzymatic activity. The ATPase activity of Dictyostelium myosin, in the presence of Mg²⁺ at low ionic strength, exhibits an average ninefold activation when actin is added.     

Taenia crassiceps in the rat: transfer of immunity and immunocompetence with lymph node cells.

Using T. crassiceps infections of young AS₂ rats as a model system, it was shown that immunity can be transferred adoptively with lymph node cells, whereas serum from the same donors was ineffective. Normal adult rat lymph node cells also conferred immunocompetence on neonatal recipients. There was no correlation between antibody and elimination or persistence of metacestodes in infected rats. It is suggested that susceptibility of neonates to infection with this parasite is the result of functional immaturity of thymus-derived cells involved in cell-mediated immune responses rather than T-cell facilitation of antibody production.     

Phosphate and ADP Differently Inhibit Coordinated Smooth Muscle Myosin Groups

Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo—suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (P_i, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [P_i] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (f_mot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased f_mot. We introduced specific P_i and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [P_i] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [P_i] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state.     

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP₂ and PIP₃ to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.