Yoshisada Yabu

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.

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.

The efficacy of ascofuranone in a consecutive treatment on Trypanosoma brucei brucei in mice.

Consecutive administration of ascofuranone without glycerol was found to have therapeutic efficacy against Trypanosoma brucei brucei infection in mice. A suspension of ascofuranone (25-100 mg/kg) was administrated intraperitoneally every 24 h for 1-4 consecutive days to trypanosome-infected mice and efficacy was compared with oral treatment. With intraperitoneal administration, all mice treated with 100 mg/kg ascofuranone for 4 consecutive days were cured. On contrary, with oral treatment a higher dose of ascofuranone (400 mg/kg) was needed for 8 consecutive days to cure the mice. With intraperitoneal treatment, parasitemia was strongly suppressed, with almost all long slender bloodstream forms of the parasite changed to short stumpy forms by day 3 and the parasites have been eliminated 4 days after the start of treatment. These ascofuranone-induced short stumpy forms were morphologically analogous to the stumpy forms 2 days after peak parasitemia of pleomorphic clone of T. b. brucei GUTat 3.1. However, the properties of ubiquinol oxidase activity, which is the target of ascofuranone, in mitochondria isolated from before and after treatment, were almost same. The enzymatic activities of ubiquinol oxidase were only decreased to approximately 30% within a day after treatment, and then kept at nearly the same level. In the present study, we have improved regimen for administration of ascofuranone without glycerol, and demonstrated that consecutively administrated ascofuranone showed trypanocidal effects in T. b. brucei infected mice. Our present results strongly suggest that consecutive administration of ascofuranone may be an effective chemotherapy for African trypanosomiasis.

Erratum to ``An antibiotic, ascofuranone, specifically inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma brucei brucei'': [Mol. Biochem. Parasitol. 81 (1996) 127–136]1

Abstract Ascofuranone, a prenylphenol antibiotic isolated from a phytopathogenic fungus, Ascochyta visiae , strongly inhibited both glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O 2 consumption of long slender bloodstream forms of Trypanosoma brucei brucei . This inhibition was suggested to be due to inhibition of the mitochondria 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 Q 1 -dependent O 2 uptake of the mitochondria with respect to ubiquinol (K i =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.

IMMUNE RESPONSES 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-γ 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.

Protective Effect of Vaccination with Toxoplasma Lysate Antigen and CpG as an Adjuvant against Toxoplasma gondii in Susceptible C57BL/6 Mice

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems to both humans and livestock and of great economic impact worldwide. Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote Th1 responses, an adjuvant activity that is desirable for vaccination against intracellular pathogens. We investigated the feasibility of using CpG as an adjuvant combined with Toxoplasma lysate antigen (TLA) as a vaccine against toxoplasmosis. Genetically susceptible C57BL/6 mice were vaccinated with TLA with or without CpG ODN as an adjuvant and then challenged with 85 cysts of the moderately virulent RRA (Beverley) strain of T gondii. Prior to challenge infection, immunization with TLA plus CpG ODN directed cellular and humoral immunity toward a Th1 pattern, characterized by enhanced INF7 production by splenic cells in response to TLA, and enhanced production of toxoplasma‐specific IgG and IgG2a antibodies. Consequently, CpG/TLA‐treated mice showed prolonged survival and 64% reduction in brain parasite burden compared to non‐CpG/TLA treated group. Our results suggest that CpG ODN would provide a stable and effective adjuvant for use in vaccination against toxoplasmosis.

Purification of active recombinant trypanosome alternative oxidase

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain in long slender bloodstream forms of African trypanosomes. TAO is a cytochrome‐independent, cyanide‐insensitive quinol oxidase. These characteristics are distinct from those of the bacterial quinol oxidases, proteins that belong to the heme‐copper terminal oxidase superfamily. The inability to purify stable TAO has severely hampered biochemical studies of the alternative oxidase family. In the present study, we were able to purify recombinant TAO to homogeneity from Escherichia coli membranes using the detergent digitonin. Kinetic analysis of the purified TAO revealed that the specific inhibitor ascofuranone is a competitive inhibitor of ubiquinol oxidase activity.

Oral and Intraperitoneal treatment of Trypanosoma brucei brucei with a combination of ascofuranone and glycerol in mice

Abstract On the basis of our previous report of ascofuranone, an antibiotic isolated from Ascochyta visiae , which strongly inhibited both the mitochondrial O 2 consumption in mitochondrial preparation and growth of in vitro cultured bloodstream forms of Trypanosoma brucei brucei in combination with glycerol, we investigated the chemotherapeutic efficacy of ascofuranone on experimental African trypanosomiasis in mice. A suspension of ascofuranone (6–200 mg/kg) was given and then glycerol (1 g/kg) was administered orally or intraperitoneally at 30-min intervals to heavily parasitemic mice. Both orally (100 mg/kg) and intraperitoneally (25 mg/kg) administered ascofuranone combined with a total dose of 3 g/kg glycerol showed potent antitrypanosomal activity in infected mice. The trypanocidal activity of ascofuranone was very powerful and all trypanosomes disappeared within 30 and 180 min after final intraperitoneal and oral treatment, respectively. This combination treatment showed high efficacy and low toxicity. Our results strongly suggest that ascofuranone in combination with glycerol may be an effective tool in chemotherapy for African trypanosomiasis.

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338microM and V(max) of 601micromol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.

Alternative Oxidase (AOX) Genes of African Trypanosomes: Phylogeny and Evolution of AOX and Plastid Terminal Oxidase Families

Abstract. To clarify evolution and phylogenetic relationships of trypanosome alternative oxidase (AOX) molecules, AOX genes (cDNAs) of the African trypanosomes, Trypanosoma congolense and Trypanosoma evansi, were cloned by PCR. Both AOXs possess conserved consensus motifs (‐E‐, ‐EXXH‐). The putative amino acid sequence of the AOX of T. evansi was exactly the same as that of T. brucei. A protein phylogeny of trypanosome AOXs revealed that three genetically and pathogenically distinct strains of T. congolense are closely related to each other. When all known AOX sequences collected from current databases were analyzed, the common ancestor of these three Trypanosoma species shared a sister‐group position to T. brucei/T. evansi. Monophyly of Trypanosoma spp. was clearly supported (100% bootstrap value) with Trypanosoma vivax placed at the most basal position of the Trypanosoma clade. Monophyly of other eukaryotic lineages, terrestrial plants + red algae, Metazoa, diatoms, Alveolata, oomycetes, green algae, and Fungi, was reconstructed in the best AOX tree obtained from maximum likelihood analysis, although some of these clades were not strongly supported. The terrestrial plants + red algae clade showed the closest affinity with an α‐proteobacterium, Novosphingobium aromaticivorans, and the common ancestor of these lineages, was separated from other eukaryotes. Although the root of the AOX subtree was not clearly determined, subsequent phylogenetic analysis of the composite tree for AOX and plastid terminal oxidase (PTOX) demonstrated that PTOX and related cyanobacterial sequences are of a monophyletic origin and their common ancestor is linked to AOX sequences.