Trypanosoma brucei vacuolar protein sorting 41 (VPS41) is required for intracellular iron utilization and maintenance of normal cellular morphology.

Procyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in DeltaScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of DeltaScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in DeltaScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.

A role of mast cell glycosaminoglycans for the immunological expulsion of intestinal nematode, Strongyloides venezuelensis.

We examined effects of mast cell glycosaminoglycans on the establishment of the intestinal nematode, Strongyloides venezuelensis, in the mouse small intestine. When intestinal mastocytosis occurred, surgically implanted adult worms could not invade and establish in the intestinal mucosa. In mast cell-deficient W/Wv mice, inhibition of adult worm invasion was not evident as compared with littermate +/+ control mice. Mucosal mastocytosis and inhibition of S. venezuelensis adult worm mucosal invasion was tightly correlated. To determine effector molecules for the invasion inhibition, adult worms were implanted with various sulfated carbohydrates including mast cell glycosaminoglycans. Among sulfated carbohydrates tested, chondroitin sulfate (ChS)-A, ChS-E, heparin, and dextran sulfate inhibited invasion of adult worms into intestinal mucosa in vivo. No significant inhibition was observed with ChS-C, desulfated chondroitin, and dextran. ChS-E, heparin, and dextran sulfate inhibited adhesion of S. venezuelensis adult worms to plastic surfaces in vitro. Furthermore, binding of intestinal epithelial cells to adhesion substances of S. venezuelensis, which have been implicated in mucosal invasion, was inhibited by ChS-E, heparin, and dextran sulfate. Because adult worms of S. venezuelensis were actively moving in the intestinal mucosa, probably exiting and reentering during infection, the possible expulsion mechanism for S. venezuelensis is inhibition by mast cell glycosaminoglycans of attachment and subsequent invasion of adult worms into intestinal epithelium.

Cloning and molecular characterization of calpain, a calcium-activated neutral proteinase, from different strains of Schistosoma japonicum.

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.

Immune response against protozoal and nematodal infection in mice with underlying Schistosoma mansoni infection.

Schistosoma mansoni infection induces T helper (Th) 2-dominant immune response in mice not only to S. mansoni itself but also to other coexisting antigens. In the present study, we challenged S. mansoni-infected mice with the intestinal nematode, Strongyloides venezuelensis, and the intracellular protozoa, Leishmania major to see whether such Th2-dominant immune responses alter susceptibility of the host to other concomitant parasitic infections. The recovery of S. venezuelensis adult worms from the small intestine was significantly decreased by S. mansoni infection, and the protection to S. venezuelensis appeared to act on migrating larvae. Antibodies elicited by S. mansoni infection showed cross-binding to third-stage larvae antigen of S. venezuelensis. On the other hand, S. mansoni infection did not affect the outcome of L. major infection in both susceptible BALB/c and resistant C57BL/6 mice. Popliteal lymph node cells of BALB/c mice expressed mRNA for interleukin (IL)-10 rather than IL-4, regardless of S. mansoni infection, and those of C57BL/6 mice expressed IFN-gamma mRNA upon L. major antigen stimulation, even in S. mansoni-infected mice. Our findings suggest that Th2-dominant immune response induced by S. mansoni protects mice from intestinal helminthic infections, whereas they do not always modulate protozoal infections.

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli.

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.

Formation of reactive oxygen intermediates might be involved in the trypanocidal activity of gallic acid.

We investigated the mechanism of the trypanocidal activity of gallic acid (GA). GA-induced trypanocidal activity was significantly reduced by pretreatment with superoxide dismutase (SOD) and/or catalase. The ESR technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent revealed that a DMPO-OH adduct was detected in culture medium containing GA. The intensity of ESR signals of the DMPO-OH adduct was increased in a time dependent manner. SOD also inhibited the formation of GA-induced DMPO-OH adducts. Furthermore, GA enhanced DNA single-strand breaks induced by Fenton reagent. These results suggest the possibility that GA acts as pro-oxidant for trypanocidal activity.

Trypanocidal effects of curcumin in vitro.

During the course of study searching for the antiparasitic agents from natural compounds, we found that curcumin showed cytotoxicity against African trypanosomes in vitro. The LD50 values of curcumin were 4.77 +/- 0.91 microM for bloodstream forms and 46.52 +/- 4.94 microM for procyclic forms of Trypanosoma brucei brucei (GUTat 3.1 clone).

Trypanocidal effects of gallic acid and related compounds.

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally abundant plant phenolic compound and it is well known as a component of hydrolyzable tannins. We report here that gallic acid and related compounds have trypanocidal activity against Trypanosoma brucei brucei (GUTat 3.1) in both the long slender bloodstream forms and the procyclic forms, in vitro. LD50 values of gallic acid are 46.96 +/- 1.28 microM for bloodstream forms and 30.02 +/- 3.49 for procyclic forms, respectively. A study of structurally related compounds suggested that the pyrogallol moiety could be responsible for this activity.

Continuous growth of bloodstream forms of Trypanosoma brucei brucei in an axenic culture system containing a low concentration of serum.

An effective axenic culture system for bloodstream forms of Trypanosoma brucei brucei GUT at 3.1 containing a low concentration of serum is described. Bloodstream forms routinely maintained in Iscove's modification of Dulbecco's medium supplemented with 100 microM hypoxanthine, 30 microM thymidine, 40 microM adenosine, 1 mM sodium pyruvate, 50 microM L-glutamine, 100 microM 2-mercaptoethanol and 20% FBS for more than one year were grown in the same medium supplemented with 5% FBS without reducing their growth rate. Then culture adapted trypanosomes in the culture medium containing 5% FBS were transferred into the modified medium supplemented with 0.5% FBS. For the constant growth of bloodstream forms in the medium containing 0.5% FBS, the culture medium was further supplemented with 200 microM L-alanine, 100 microM glycine, 10 microM L-oruithine hydrochloride and 10 microM L-citrullin. The trypanosomes propagated in this culture system for one year retained their infectivity for mice. This culture system was also shown to be useful for cloning of T.b. brucei GUT at 3.1 which is important for separation of mutants.

Antitrypanosomal effects of traditional Chinese herbal medicines on bloodstream forms of Trypanosoma brucei rhodesiense in vitro.

The antitrypanosomal activity of traditional Chinese herbal medicines and these crude drug ingredients were determined using axenic cultured bloodstream forms of Trypanosoma b. rhodesiense which is one of the two causative agents of African sleeping sickness in man. The drugs tested were 8 traditional Chinese herbal medicines and these 14 crude drug ingredients. Of these traditional Chinese medicines examined, san'o-shasin-to and oren-gedoku-to showed most potent antitrypanosomal effect. The minimal effective concentration (MEC) which killed all bloodstream form populations within 24 hours of both drug exposure was 125 microg/ml. The 50% effective concentration (EC50) of san'o-shashin-to and oren-gedoku-to was 63 and 74 microg/ml, respectively. In the crude drug ingredients tested, Scutellaria baicalensis G. and Coptis japonica M. which are the main components of san'o-shasin-to and oren-gedoku-to, showed the most powerful antitrypanosomal activity. The MEC and EC50 value of these crude drug ingredients were 30 and 60 microg/ml, and 20 and 36 microg/ml.

An antibiotic, ascofuranone, specifically inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma brucei brucei.

Ascofuranone, a prenylphenol antibiotic isolated from a phytopathogenic fungus, Ascochyta visiae, strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O2 consumption of long slender bloodstream forms of Trypanosoma brucei brucei. This inhibition was suggested to be due to inhibition of the mitochondrial electron-transport system, composed of glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) and plant-like alternative oxidase. Ascofuranone noncompetitively inhibited the reduced coenzyme Q1-dependent O2 uptake of the mitochondria with respect to ubiquinol (Ki = 2.38 nM). Therefore, the susceptible site is deduced to be the ubiquinone redox machinery which links the two enzyme activities. Further, ascofuranone in combination with glycerol completely blocked energy production, and potently inhibited the in vitro growth of the parasite. Our findings suggest that ascofuranone might be a promising candidate for the chemotherapeutic agents of African trypanosomiasis.

An antibiotic, ascofuranone, specifically inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma brucei brucei.

Ascofuranone, a prenylphenol antibiotic isolated from a phytopathogenic fungus, Ascochyta visiae, strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O2 consumption of long slender bloodstream forms of Trypanosoma brucei brucei. This inhibition was suggested to be due to inhibition of the mitochondriai electron-transport system, composed of glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) and plant-like alternative oxidase. Ascofuranone noncompetitively inhibited the reduced coenzyme Q1-dependent O2 uptake of the mitochondria with respect to ubiquinol (Ki = 2.38 nM). Therefore, the susceptible site is deduced to be the ubiquinone redox machinery which links the two enzyme activities. Further, ascofuranone in combination with glycerol completely blocked energy production, and potently inhibited the in vitro growth of the parasite. Our findings suggest that ascofuranone might be a promising candidate for the chemotherapeutic agents of African trypanosomiasis.

Antioxidant, gallic acid, induces apoptosis in HL-60RG cells.

Gallic acid, a naturally occurring plant phenol with antioxidative activity, was found to induce cell death in promyelocytic leukemia HL-60RG cells, although many antioxidants are well known to protect the cell from oxidative stress. Morphological and biochemical studies indicated that the gallic acid-induced cell death is apoptosis. Flow cytometric analysis revealed that the apoptosis was not triggered at a specific phase of the cell cycle and that 2 h exposure of gallic acid to HL-60RG cells was enough to induce apoptosis. The inhibitory assay suggested that gallic acid-induced cell death was mediated by reactive oxygen species such as hydrogen peroxide, superoxide anion in addition to Ca2+ ion, calmodulin-dependent enzymes. Structure-activity analysis suggests that gallic acid induces apoptosis in HL-60RG cells, depending on its distinctive feature derived from the structure but not on its antioxidative activity.

An axenic culture system for the transformation of bloodstream forms to procyclic forms of Trypanosoma brucei brucei in vitro.

An axenic culture system for continuous cultivation of bloodstream forms of Trypanosoma brucei GUT at 3.1 and subsequent transformation of bloodstream forms to procyclic forms is described. Bloodstream forms were continuously grown at 37 degrees C in Iscove's modification of Dulbecco's medium (M-DMEM, with bovine serum albumin, transferrin and soybean lecithin supplemented with 100 microM hypoxanthine, 30 microns thymidine, 40 microM adenosine, 1mM sodium pyruvate, 50 microM L-glutamine, 100 microM 2-mercaptoethanol and 20% (v/v) heat-inactivated fetal bovine serum. In this system, 2-mercaptoethanol (2-ME) was essential and in the absence of 2-ME, 100 microM L-cysteine and 10 microM bathocuproine sulfonate could not be substituted for 2-ME. This culture system was useful for long-term culture of bloodstream forms of this clone. Axenic cultivation of bloodstream forms at 27 degrees C resulted in transformation to procyclic forms within 5 days in the same medium supplemented with 5 mM L-proline, 8 micrograms/ml hemin and 4 micrograms/ml hematin, respectively and, instead of FBS, 20% (v/v) hemoglobin-poor fraction of fetal bovine serum.

Inhibition of IgM antibody-mediated aggregation of Trypanosoma gambiense in the presence of complement.

This paper deals with the immune reaction between Trypanosoma gambiense and monoclonal IgM mouse antibody at equivalence with or without rabbit complement. Antibody-mediated trypanosome clumps formed in the absence of complement, and were readily dissociated by complement to become free. In the presence of complement, on the other hand, T. gambiense were not aggregated by the antibody. Free parasites adhered readily to cultured peritoneal macrophages. Complement-mediated dissociation of the clumped trypanosomes in the equivalence area released a large number of previously bound surface antigens. These antigens were capable of binding again to fresh IgM antibody. Experimental results further indicated that the complement system caused a functional alteration, changing the multivalent nature of the IgM antibody in the immune complex into a univalent one. This phenomenon is of great advantage to the infected host in clearing pathogens in vivo, as it allows more antibodies to attach to trypanosomes and subsequently initiate complement activity.

Immunological activities of monoclonal IgG1 antibody against Trypanosoma gambiense.

The present paper deals with the immune reaction between a monoclonal IgG1 antibody and Trypanosoma gambiense. The aggregation of trypanosomes, immune adherence to macrophages and protection against infection are associated with the antibody. IgG1-mediated clumping of trypanosomes is readily dissociated by the addition of complement. Dissociation of the clumped trypanosomes in the equivalence area released approximately fifty percent of previously bound surface antigens. These antigens were capable of binding again to new IgG1 antibody. Complement deposition rendered bivalent IgG1 antibody in the immune complex functionally univalent. Such an event in the presence of complement is of great advantage to the infected host in killing pathogens in vivo, as it allows more antibodies to attach to surface antigens and subsequently to initiate complement activity.

Diversity of complement-mediated immune reaction to Trypanosoma gambiense.

Intact and papain-treated Trypanosoma gambiense clone populations, each expressing special antigens on their cell surfaces, were mixed with rabbit antiserum in the presence of complement. Two distinct types of immune reaction between trypanosomes and antisera were observed: clumping followed by dissociation (CD) and inhibition of aggregation (IA). Special antigens on the cell surface of trypanosomes exposed after papain digestion are implicated in both types of immune reaction. IA was considered to be more effective as an immunological response which would allow the infected host to clear the pathogen without any obstruction of capillaries and impairment of blood flow caused by clumping masses of trypanosomes.

Immune mechanism facilitating clearance of Trypanosoma gambiense by IgG3 antibody from infected host.

The aggregation of trypansomes, immune adherence to macrophage and protection against infection of Trypanosoma gambiense are associated with IgG3 antibody. IgG3-mediated clumping trypanosomes are readily dissociated by the aid of complement to become free. Dissociation of the clumped trypanosomes in the equivalence area released approximately fifty percent of previous bound surface antigens. These antigens were capable of binding again to new IgG3 antibody. Experiments indicated that complement deposition altered functionally bivalent IgG3 antibody in the immune complex into a univalent one. Such event in the presence of complement is of great advantage to the infected host in killing pathogens in vivo, as it allows more antibodies to attach to surface antigens and subsequently initiate complement activity.

Dissociation of IgG antibody-mediated clumps of Trypanosoma brucei gambiense by complement.

Aggregated trypanosome masses that had formed in the region of equivalence in the absence of complement were readily dissociated by complement. Complement-mediated dissociation of the clumped trypanosomes in the area of equivalence released approximately 50% of the previously bound surface antigens. These antigens were capable of rebinding to new IgG antibody. Experiments indicated that complement deposition altered the bivalent IgG antibody in the immune complex into a univalent form. The occurrence of such an event in the presence of complement provides a great advantage to the infected host in the killing of pathogens in vivo, as it enables a greater number of antibodies to attach to surface antigens and subsequently initiate complement activity.