In vitro culture of freshly isolated Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes

Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were taken from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains. MAK65 and MAK98, which have undergone only 3 rodent passages since isolation from Ugandan cattle. Adaptation of these strains to culture was accompanied by changes in gene copy numbers, some of which were also evident when other lab-adapted strains, field isolates of T. rhodesiense, and the genome strain TREU927 were compared. Reproducible increases were seen for genes encoding histones, enzymes of mRNA processing and degradation, the cytosolic chaperone HSP70, and two proteins required for the DNA damage response. These results indicate that similar work with other eukaryotic pathogens would be worthwhile. Meanwhile, the two new T. brucei strains should be useful to researchers interested in trypanosome differentiation and pathogenicity. They have differing pathogenicities in mice and may also differ in their propensity for stumpy-form differentiation, as judged by morphology and mRNA expression. MAK65 grows better than MAK98 in bloodstream-form culture, and is uniformly diploid, whereas MAK98 is triploid for chromosome 5. Genome sequence exceeding 100-fold coverage is available for both strains.

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Structure and transcription of the actin gene of Trypanosoma brucei

Molecular and Cellular Biology ◽

10.1128/mcb.8.5.2166-2176.1988 ◽

1988 ◽

Vol 8 (5) ◽

pp. 2166-2176 ◽

Cited By ~ 1

Author(s):

M F Ben Amar ◽

A Pays ◽

P Tebabi ◽

B Dero ◽

T Seebeck ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Splice Site ◽

Gene Copy Number ◽

Gene Copy ◽

Actin Gene ◽

S1 Nuclease ◽

Actin Genes ◽

Gene Copies ◽

Actin Mrna

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

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Structure and transcription of the actin gene of Trypanosoma brucei.

Molecular and Cellular Biology ◽

10.1128/mcb.8.5.2166 ◽

1988 ◽

Vol 8 (5) ◽

pp. 2166-2176 ◽

Cited By ~ 61

Author(s):

M F Ben Amar ◽

A Pays ◽

P Tebabi ◽

B Dero ◽

T Seebeck ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Splice Site ◽

Gene Copy Number ◽

Gene Copy ◽

Actin Gene ◽

S1 Nuclease ◽

Actin Genes ◽

Gene Copies ◽

Actin Mrna

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

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Comparison of the cytolytic effects in vitro on Trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein A-I from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection.

The Journal of Lipid Research ◽

10.1016/s0022-2275(20)41617-7 ◽

1992 ◽

Vol 33 (4) ◽

pp. 513-523

Author(s):

MP Gillett ◽

JS Owen

Keyword(s):

Trypanosoma Brucei ◽

High Density ◽

High Density Lipoproteins ◽

Trypanosoma Brucei Brucei ◽

Apolipoprotein A ◽

Plasma High Density ◽

Cattle And Sheep

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Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00332-10 ◽

2010 ◽

Vol 54 (7) ◽

pp. 2893-2900 ◽

Cited By ~ 69

Author(s):

Antoaneta Y. Sokolova ◽

Susan Wyllie ◽

Stephen Patterson ◽

Sandra L. Oza ◽

Kevin D. Read ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Human African Trypanosomiasis ◽

Parent Drug ◽

Cross Resistance ◽

Pharmacokinetic Studies ◽

African Trypanosomiasis ◽

Trypanosoma Brucei Brucei ◽

Resistant Lines

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

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

Molecular and Biochemical Parasitology ◽

10.1016/0166-6851(96)02665-5 ◽

1996 ◽

Vol 81 (2) ◽

pp. 127-136 ◽

Cited By ~ 41

Author(s):

Nobuko Minagawa ◽

Yoshisada Yabu ◽

Kiyoshi Kita ◽

Kazuo Nagai ◽

Nobuo Ohta ◽

...

Keyword(s):

Trypanosoma Brucei ◽

In Vitro Growth ◽

Trypanosoma Brucei Brucei

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In Vitro, In Vivo and Phytochemical Screening, of Extracts of Piper guineense for trypanocidal Activities Against Trypanosoma brucei brucei.

International Journal of Biology ◽

10.5539/ijb.v5n3p85 ◽

2013 ◽

Vol 5 (3) ◽

Cited By ~ 2

Author(s):

J. A. Abedo ◽

A. O. Jonah ◽

M. R. Mazadu ◽

R. S. Abdullahi ◽

H. Y. Idris ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Phytochemical Screening ◽

Trypanosoma Brucei Brucei ◽

Piper Guineense

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In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

Eukaryotic Cell ◽

10.1128/ec.00116-06 ◽

2006 ◽

Vol 5 (8) ◽

pp. 1276-1286 ◽

Cited By ~ 15

Author(s):

Sara D. Faulkner ◽

Monika W. Oli ◽

Rudo Kieft ◽

Laura Cotlin ◽

Justin Widener ◽

...

Keyword(s):

High Density Lipoprotein ◽

Trypanosoma Brucei ◽

Density Lipoprotein ◽

High Density ◽

Surface Glycoprotein ◽

Trypanosoma Brucei Brucei ◽

African Trypanosomes ◽

Expression Site ◽

Human High Density Lipoprotein

ABSTRACT The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

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Isothiochromenothiazoles—A Class of Fused Thiazolidinone Derivatives with Established Anticancer Activity That Inhibits Growth of Trypanosoma brucei brucei

Scientia Pharmaceutica ◽

10.3390/scipharm86040047 ◽

2018 ◽

Vol 86 (4) ◽

pp. 47 ◽

Cited By ~ 4

Author(s):

Anna Kryshchyshyn ◽

Danylo Kaminskyy ◽

Igor Nektegayev ◽

Philippe Grellier ◽

Roman Lesyk

Keyword(s):

Acute Toxicity ◽

Anticancer Activity ◽

Trypanosoma Brucei ◽

In Vitro Assay ◽

Parasite Growth ◽

Trypanosoma Brucei Brucei ◽

Vitro Assay ◽

Antiparasitic Agents ◽

Micromolar Range

Recently, thiazolidinone derivatives have been widely studied as antiparasitic agents. Previous investigations showed that fused 4-thiazolidinone derivatives (especially thiopyranothiazoles) retain pharmacological activity of their synthetic precursors—simple 5-ene-4-thiazolidinones. A series of isothiochromeno[4a,4-d][1,3] thiazoles was investigated in an in vitro assay towards bloodstream forms of Trypanosoma brucei brucei. All compounds inhibited parasite growth at concentrations in the micromolar range. The established low acute toxicity of this class of compounds along with a good trypanocidal profile indicates that isothiochromenothiazole derivatives may be promising for designing new antitrypanosomal drugs.

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