Kinetoplast maxicircle DNA replication in Crithidia fasciculata and Trypanosoma brucei.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.     

The Cytostome of Trypanosoma cruzi and T. conorhini*

SYNOPSIS. A cytostome is described in culture forms and in intracellular stages of Trypanosoma cruzi and in culture forms of T. conorhini. The organelle opens into the flagellar pocket or close to it and runs deeply into the cell.     

Effect of Temperature and Osmolarity on Growth of Crithidia fasciculata,C. hutneri,C. luciliae thermophila,and Herpetomonas samuelpessoai1

Crithidia fasciculata (Anopheles, Culex, and Nöller strains), C. hutneri, C. luciliae thermophila, and Herpetomonas samuelpessoai were grown in a defined medium with different values of osmolarity at different temperatures. C. fasciculata (all strains) grew best between 300 to 500 mOsm; H. samuelpessoai, 400–500 mOsm; and C. hutneri and C. luciliae thermophila, 500–800 mOsm. At higher temperatures better growth was obtained at the upper osmolarities.     

In Vitro Cultivation of Bloodstream Forms of Trypanosoma brucei,T. rhodesiense,and T. gambiense1

ABSTRACT. A series of new in vitro systems for the cultivation of bloodstream forms of Trypanosoma (Trypanozoon) brucei brucei, T. (T.) b. rhodesiense, and T. (T.) b. gambiense was developed. The standard system consists of a feeder layer of fibroblast-like cells derived from embryos of New Zealand White rabbits (REF) or a mountain vole, Microtus montanus (MEF), with HEPES-buffered Minimum Essential Medium (MEM), with Earle's salts, supplemented with 15% inactivated rabbit serum. These two and other feeder layers were cross-checked with different sera to test for growth support of bloodstream forms of the three trypanosome subspecies studied. Cultures could be initiated with bloodstream forms from mammalian hosts or from cryopreserved stabilates. Metacyclic forms from infected Glossina m. morsitans could also be used as inoculum; they transformed within 6 h to bloodstream forms. Maintenance of cultures and growth properties are described in detail. Experiments were undertaken to confirm that the cultivated bloodstream forms still possess some of the characteristic features of pleomorphic bloodstream populations. Cultivated bloodstream forms were always infective for mice, and a surface coat could be demonstrated by electron microscopy. They could also be cyclically transmitted through tsetse flies, and the metacyclic forms from these flies could be brought back into culture. In vitro cloning with single bloodstream forms and metacyclic forms could be achieved with high cloning efficiency. The consumption of glucose and the production of pyruvate and lactate were determined.     

Trypanosoma gambiense and T. equiperdum: characterization of variant specific antigens

It has been shown that by a simple procedure the variant specific protective antigen can be isolated from both T. gambiense and T. equiperdum. The protection afforded mice by this antigen is relatively long term and a high percentage of mice has complete protection. It has been suggested that the antigens are highly antigenic and although antigenically unique for each relapse strain, they have one or more common physical characteristic. It is therefore hypothesized that a multivalent vaccine might be prepared by similar procedures for protection against trypanosomiasis.     

Cultural and physiological observations on Trypanosoma rhodesiense and Trypanosoma gambiense. 1949

1. A diphasic medium of simple preparation is described for the indefinite cultivation of T. rhodesiense and T. gambiense. 2. The chief advantage of the medium is that it contains rabbit blood and thus obviates the necessity of using human blood. 3. The flagellates develop only to the proventricular stage; hence the cultures are noninfective. 4. The proventricular forms of both T. rhodesiense and T. gambiense consume sugar with the concomitant formation of acid. They are aerobic fermenters. 5. Very little, if any, ammonia is produced by the living parasites.     

Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.     

Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense.

A semi-defined medium for the cultivation of bloodstream forms of the African trypanosome brucei subgroup was developed. Out of 14 different strains tested, 10 could be cultured including Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. The presence of a reducing agent (2-mercaptoethanol or thioglycerol) was found to be essential for growth. The standard medium consisted of Hepes buffered minimum essential medium with Earle's salts supplemented with 0.2 mM 2-mercaptoethanol, 2 mM pyruvate and 10% inactivated serum either from rabbit (T. brucei, T. equiperdum, T. evansi and T. rhodesiense) or human (T. gambiense). Although a general medium could be defined for the long-term maintenance of trypanosome cultures, the initiation to culture nevertheless required particular conditions for the different strains. The cultured trypanosomes had all the characteristics of the in vivo bloodstream forms including: morphology, infectivity, antigenic variation and glucose metabolism.     

Effect of sugars,Na+-, and K+-ions on biopterin transport in Crithidia fasciculata

Biopterin uptake by Crithidia fasciculata is pH dependent with optimum at pH 6 and is strongly inhibited by 0.5 mM NAA and DNP,respectively. Both inhibitors also reduce respiration by 40% (NAA) and 97% (DNP). K⁺-ions (1.1%) and K⁺/Na⁺ (0.5% each) stimulate biopterin uptake to the same high extent, but ouabain has no effect, thereby ruling out involvement of Na⁺/K⁺ pump. In absence of these ions, even in 5% glucose solution biopterin uptake is reduced to minimum. Proton excretion seems to be linked to sugar uptake. Both these sugars seem to have the same site of entry, demonstrated by competitive uptake, though D-glucose is taken up much faster by Crithidia than D-galactose. DNP (0.5 mM) causes greater proton excretion in glucose than in galactose medium. With NAA (0.5 mM) proton excretion is inhibited in both glucose and galactose media. D-glucose promotes greater biopterin uptake than D-galactose.     

Nitric oxide-mediated cytostatic activity on Trypanosoma brucei gambiense and Trypanosoma brucei brucei.

Macrophages collected from BCG-infected mice or exposed in vitro to interferon-gamma plus lipopolysaccharide developed a cytostatic activity on Trypanosoma brucei gambiense and Trypanosoma brucei brucei. This trypanostatic activity of activated macrophages was inhibited by addition of N-monomethyl-L-arginine, an inhibitor of the L-arginine-nitric oxide (NO) metabolic pathway, indicating a role for NO as the effector molecule. Contrary to trypanosomes treated with N2gas, trypanosomes treated with NO gas did not proliferate in vitro on normal macrophages. Compared to mice infected with control parasites, mice infected with NO-treated parasites had decreased parasitemias in the first days postinfection and had a prolonged survival. Addition of excess iron reversed the trypanostatic effect of both activated macrophages and NO gas. These data show that activated macrophages exert an antimicrobial effect on T.b. gambiense and T.b. brucei through the L-arginine-NO metabolic pathway. In trypanosomes, NO could trigger iron loss from critical targets involved in parasite division. The participation of this effector mechanism among the other immune elements involved in the control of African trypanosomes (antibodies, complement, phagocytic events) remains to be defined.     

Effect of neocarzinostatin on the nucleus, kinetoplast and microtubules of Trypanosoma gambiense and Trypanosoma evansi.

The antibiotic neocarzinostatin (NCS) induces the anucleate form, not the dyskinetoplastic form, of Trypanosoma gambiense and Trypanosoma evansi. Light and electron microscopic studies indicated that production of the anucleate form is due to delay or inhibition of nuclear division. Excess pellicular microtubules are formed after treatment of trypanosomes with NCS, suggesting that in trypanosomes the microtubules replicate by induction, not by division. NCS also causes deformation of the axonemal and spindle microtubules. The K clone of T. evansi is more sensitive than the AK clone to the effects of NCS in inhibiting nuclear division and inducing the anucleate form.     

Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata.

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.     

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.     

Life-Cycle of Trypanosoma conorhini. Influence of Temperature and Other Factors on Growth and Morphogenesis

SYNOPSIS. In continued observations on the in vitro growth and multiplication of the bloodstream trypanosome stage of Trypanosoma conorhini , a better medium was found for cultivating these forms at 37°C, but no subcultures could be obtained. The infectivity for mice of the blood type trypanosomes grown in vitro was comparable to that of the metacyclic trypanosomes. The only reproducing forms of T. conorhini found in the vertebrate were in the trypanosome stage.
It was also found that the in vitro reversion of the bloodstream trypanosome into crithidia, such as occurs in the invertebrate host and in the usual diphasic culture medium, is dependent on at least two factors: if incubated at 25–28° reversion did not occur in any of the liquid media tried (all containing blood serum and hematin or hemoglobin), unless total blood was part of the inoculum or washed red blood cells were added to the media; on the other hand, no reversion was seen, even in the presence of red blood cells if the cultures were incubated at 37°.     

Therapeutic effect of bleomycin on experimental infection of mice with Trypanosoma gambiense and Trypanosoma evansi

Intraperitoneal injection of 10 mg/Kg bleomycin into mice 24 h after inoculation with Trypanosoma gambiense or Trypanosoma evansi, reduced the incidence of infection 62.1%, and 95.2%, respectively. No parasitemia was not found in these mice. Treatment of mice with parasitemia with 30 mg/Kg of bleomycin decreased the number of parasites within about 5 h and caused complete cure without relapse in 45% and 75% of mice infected with T. gambiense and T. evansi, respectively. Treatment of mice infected with T. gambiense with bleomycin in combination with ethidium bromide was highly effective and resulted in a high incidence of complete cure, even in heavily infected mice. The mode of action of bleomycin and ethidium bromide on trypanosomes in relation to p-rosaniline resistance is discussed.