Catabolism of Tryptophan by Trypanosoma evansi

ABSTRACT. Trypanosoma brucei gambiense , which causes human African trypanosomiasis, catabolizes the aromatic amino acid tryptophan via an initial aminotransferase catalyzed reaction to form several indole end products, which have been suggested to contribute to the pathogenesis of trypanosomiasis. To determine if this same pathway exists in T. evansi , the closely related trypanosome pathogen of domestic animals, tryptophan catabolism was examined in vitro and in vivo. As is the case with human African trypanosomes, T. evansi catabolized tryptophan to form indole-3-pyruvic acid and smaller amounts of indole-3-acetic acid and indole-3-lactic acid. Large concentrations of indole-3-pyruvic acid are excreted in urine of trypanosome-infected mice. However, indole-3-ethanol could not be detected in incubates of T. evansi or T. b. gambiense , even though the latter species had previously been reported to form this neutral metabolite. A new, previously unreported tryptophan metabolite was isolated and partially characterized from incubates of T. evansi and T. b. gambiense. Although the functional significance of tryptophan catabolism to trypanosomatids remains obscure, the pathway is quantitatively significant in all species examined thus far.     

Expression of alternative oxidase inhibits programmed cell death-like phenomenon in bloodstream form of Trypanosoma brucei rhodesiense

Trypanosoma brucei rhodesiense is one of the causative agents of African Trypanosomiasis. Programmed cell death (PCD) is fundamental in the development, homeostasis and immune mechanisms of multicellular organisms. It has been shown that, other than occurring in multicellular organisms, the PCD phenomenon also takes place in unicellular organisms. In the present study, we have found that under high-density axenic culture conditions, bloodstream form of T. b. rhodesiense depicts a PCD-like phenomenon. We investigated the association of the PCD-like phenomenon with expression of trypanosome alternative oxidase (TAO) under low-temperature stress conditions. We observed that bloodstream form of T. b. rhodesiense did not show any PCD but had up-regulated expression of TAO. Inhibition of TAO by the addition of ascofranone caused the development of PCD in bloodstream T. b. rhodesiense under low-temperature stress, implying that expression of TAO may contribute to the inhibition of PCD.     

Quantitative Ultrastructural Differences between Strains of the Trypanosoma brucei Subgroup During Transformation in Blood*

SYNOPSIS. Differences in the relative and absolute cell organization between strains of the Trypanosoma brucei subgroup were studied during the transformation from slender to stumpy bloodforms. Two pleomorphic and 1 monomorphic T. b. brucei, and 1 pleomorphic T. b. rhodesiense strains were investigated. Volume densities, surface densities and surface to volume ratios showed barely significant differences between the 2 pleomorphic T. b. brucei strains; absolute parameters, however, differ markedly between all the strains investigated. Only the relative parameters of the mitochondrion show notable differences between T. b. brucei and T. b. rhodesiense examined here. During the transformation from slender to stumpy forms the enlargement of the mitochondrial volume in T. b. brucei is achieved by an increase in width of the mitochondrial tube and in T. b. rhodesiense by the formation of a more elaborate network. The ratio of the inner mitochondrial membrane surface area to the mitochondrial matrix volume showed no significant change in all 3 pleomorphic strains examined. Because of their morphometric similarity to slender forms of pleomorphic T. b. brucei strains, it can be assumed that the monomorphic trypanosomes correspond morphologically to slender trypanosomes. Neither pleomorphism nor strain specificity have a significant influence on the relative amount of “vesicles” and lipid inclusions.     

Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N′-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.     

Quantitative Ultrastructural Investigations of the Life Cycle of Trypanosoma brucei: A Morphometric Analysis*

SYNOPSIS. The quantitative ultrastructure of the developmental stages of Trypanosoma brucei brucei in its vector Glossina morsitans was studied by morphometric analysis. Values from ectoperitrophic midgut forms, proventricular forms, epimastigote and metacyclic forms in the salivary gland are compared with results from bloodstream forms, published previously. Significant differences in the volume densities of the trypanosome's single mitochondrion, of microbody-like organelles and in the surface densities of inner and outer mitochondrial membranes were found throughout the whole life cycle. A great increase in volume density of the mitochondrion was observed after transfer to the insect host; reduction took place during metacyclic development. Parallel to the biogenesis of the mitochondrion a reduction of microbodies was found in proventricular forms and there was a great increase in metacyclic forms concomitant with the regression of the mitochondrion. Metacyclic forms had a close quantitative morphologic similarity to bloodstream forms. The results are discussed in connection with changes in structure and in oxidative metabolism.     

Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal,cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosomacruzi and Trypanosoma brucei, as well as drug-resistantforms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic andgenotoxic properties, but has been largely used as a lead compound. Here, we comparedthe activity of 7 with its 4H-1,2,4-triazole bioisostere (8) inbloodstream forms of T. brucei and T. cruzi andevaluated their activation by T. brucei type I nitroreductase(TbNTR) enzyme. We also analysed the cytotoxic and genotoxiceffects of these compounds in whole human blood using Comet and fluoresceindiacetate/ethidium bromide assays. Although the only difference between 7 and 8 isthe substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazolein 8), the results indicated that 8 had poorer antiparasitic activity than 7 and wasnot genotoxic, whereas 7 presented this effect. The determination of Vmax indicatedthat although 8 was metabolised more rapidly than 7, it bounds to theTbNTR with better affinity, resulting in equivalent kcat/KMvalues. Docking assays of 7 and 8 performed within the active site of a homologymodel of the TbNTR indicating that 8 had greater affinity than7.     

Will the real Trypanosoma brucei rhodesiense please step forward?

The sleeping sickness trypanosomes Trypanosoma brucei rhodesiense and T. brucei gambiense are morphologically indistinguishable from each other and from T. brucei brucei, which does not infect humans. The relationships between these three subspecies have been controversial. Several years ago, the characterization of T. brucei gambiense was reviewed in an attempt to clarify and draw together the results, and to put them in the context of the biology of the organism. The discovery of a gene associated with human-serum resistance in T. brucei rhodesiense and the consequent reappraisal of the identity of this trypanosome prompt this companion article.     

The effect of Plasmodium berghei and Trypanosoma brucei infections on the immune expulsion of the nematode Trichuris muris from mice

The effects of concurrent P. berghei or T. brucei infections on the immune expulsion of primary and challenge infections of T. muris from CFLP strain mice have been examined. CFLP mice usually expel the nematode 18–21 days after a primary infection and within 4–6 days after a challenge infection. Both acute malaria and trypanosome infections initiated at the same time as the T. muris infection suppressed worm expulsion; when the protozoal infections were started 7 days after the T. muris infection worm expulsion was suppressed in a proportion of the mice. Acute trypanosome and malaria infections delayed the expulsion of a challenge infection from immune mice, but in the case of P. berghei the delay was short-lived.     

Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei

Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.     

以布氏锥虫亮氨酰tRNA合成酶为靶标的抗锥虫药物筛选系统的建立

锥虫是人的血液寄生虫,对热带乃至拉丁美洲贫困地区影响极大,但目前的传统治疗药物存在副作用大、有效性不断降低的问题。根据亮氨酰tRNA合成酶在微生物中可作为药物靶点的事实,在新型抗锥虫药物筛选中,通过对锥虫的亮氨酰tRNA合成酶的克隆、表达和纯化,以及该酶活性测定的优化,建立了该酶抑制物的筛选系统。筛选结果表明,这一以锥虫亮氨酰tRNA合成酶为靶标的抗锥虫药物筛选系统可以有效筛选抗锥虫化合物,选出的化合物有一定的抗锥虫特异性,并可以用于化合物的进一步优化和测定其半抑制浓度。使用这一系统筛选到了对锥虫有较好抑制作用,但对人类细胞毒性较小的一系列新型化合物,因而锥虫亮氨酰tRNA合成酶很可能成为开发有效抗锥虫药物的新靶标。     

The in vitro trypanocidal activity of N-substituted p-benzoquinone imines: assessment of biochemical structure-activity relationships using the Hansch approach

It has previously been found that naphthoquinones can potentiate the rate of hydrogen peroxide production by mitochondrial preparations of Trypanosoma brucei brucei and that organisms treated with naphthoquinones are more susceptible to lysis, especially in the presence of compounds such as heme, which promote the homolytic cleavage of hydrogen peroxide. We have evaluated the lytic effect of various N-substituted p-benzoquinone imines both in vitro and in vivo and have attempted to correlate their structure with trypanocidal activity using the Hansch approach. While none of the compounds tested proved to be active in vivo, all caused the lysis of trypanosomes in vitro. The parameters that correlated best with trypanocidal activity were the conditional redox potential, the lipophilicity of the substituent attached to the nitrogen atom and the number of active hydrogens on the quinonoid ring. These findings suggest two possible modes of action, which may in fact be related. Conjugate nucleophilic addition and/or oxidative damage could be responsible for lysis of the parasites. These same compounds were previously found to be active against the ascitic sarcoma 180 in mice. The strong correlation between antineoplastic activity in vivo and trypanocidal activity in vitro suggests a similar mode of action in both cases. Further studies aimed at developing a quinonelike compound that will be active against trypanosomes in vivo are now in progress.