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The Killing of African Trypanosomes by Ethidium Bromide


Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.


Vyšlo v časopise: The Killing of African Trypanosomes by Ethidium Bromide. PLoS Pathog 6(12): e32767. doi:10.1371/journal.ppat.1001226
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001226

Souhrn

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.


Zdroje

1. WatkinsTI

WoolfeG

1952 Effect of changing the quaternizing group on the trypanocidal activity of dimidium bromide. Nature 189 506

2. WaringMJ

1965 Complex formation between ethidium bromide and nucleic acids. J Mol Biol 13 269 282

3. HolmesPH

EislerMC

GeertsS

2004 Current chemotherapy of animal trypanosomiasis.

MaudlinI

HolmesPH

MilesMA

The trypanosomiases Wallingford, Oxfordshire, U.K. CABI Publishing 411 444

4. LiuB

LiuY

MotykaSA

AgboEEC

EnglundPT

2005 Fellowship of the rings: The replication of kinetoplast DNA. Trends Parasitol 21 363 369

5. ShlomaiJ

2004 The structure and replication of kinetoplast DNA. Curr Mol Med 4 623 647

6. StuartKD

SchnauferA

ErnstNL

PanigrahiAK

2005 Complex management: RNA editing in trypanosomes. Trends Biochem Sci 30 97 105

7. RiouG

BaltzT

GabillotM

PautrizelR

1980 Absence of kinetoplast DNA in a late antigenic variant of Trypanosoma equiperdum. Mol Biochem Parasitol 1 97 105

8. StuartKD

1971 Evidence for the retention of kinetoplast DNA in an acriflavine-induced dyskinetoplastic strain of Trypanosoma brucei which replicates the altered central element of the kinetoplast. J Cell Biol 49 189 195

9. ZweygarthE

KaminskyR

WesbsterP

1990 Trypanosoma brucei evansi: dyskinetoplasia and loss of infectivity after long-term in vitro cultivation. Acta Trop 48 95 99

10. SimpsonL

1968 Effect of acriflavin on the kinetoplast of Leishmania tarentolae. Mode of action and physiological correlates of the loss of kinetoplast DNA. J Cell Biol 37 660 682

11. SchnauferA

PanigrahiAK

PanicucciB

IgoRPJr

SalavatiR

2001 An RNA ligase essential for RNA editing and survival of the bloodstream form of Trypanosoma brucei. Science 291 2159 2162

12. SchnauferA

Clark-WalkerGD

SteinbergAG

StuartK

2005 The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function. EMBO J 24 4029 4040

13. BrownSV

HoskingP

LiJ

WilliamsN

2006 ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei. Eukaryot Cell 5 45 53

14. JensenRE

SimpsonL

EnglundPT

2008 What happens when Trypanosoma brucei leaves Africa. Trends Parasitol 24 428 431

15. LaiDH

HashimiH

LunZR

AyalaFJ

LukesJ

2008 Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei. Proc Natl Acad Sci U S A 105 1999 2004

16. CarpenterLR

EnglundPT

1995 Kinetoplast maxicircle DNA replication in Crithidia fasciculata and Trypanosoma brucei. Mol Cell Biol 15 6794 6803

17. HajdukSL

KleinVA

EnglundPT

1984 Replication of kinetoplast DNA maxicircles. Cell 36 483 492

18. LiuB

WangJ

YaffeN

LindsayME

ZhaoZ

2009 Trypanosomes have six mitochondrial DNA helicases with one controlling kinetoplast maxicircle replication. Mol Cell 35 490 501

19. ShapiroTA

EnglundPT

1990 Selective cleavage of kinetoplast DNA minicircles promoted by antitrypanosomal drugs. Proc Natl Acad Sci USA 87 950 954

20. LindsayME

GluenzE

GullK

EnglundPT

2008 A new function of Trypanosoma brucei mitochondrial topoisomerase II is to maintain kinetoplast DNA network topology. Mol Microbiol 70 1465 1476

21. ZhaoZ

LindsayME

Roy ChowdhuryA

RobinsonDR

EnglundPT

2008 p166, a link between the trypanosome mitochondrial DNA and flagellum, mediates genome segregation. EMBO J 27 143 154

22. RauchCA

Perez-MorgaD

CozzarelliNR

EnglundPT

1993 The absence of supercoiling in kinetoplast DNA minicircles. EMBO J 12 403 411

23. KitchinPA

KleinVA

EnglundPT

1985 Intermediates in the replication of kinetoplast DNA minicircles. J Biol Chem 260 3844 3851

24. CozzarelliNR

BolesTC

WhiteJH

1990 Primer on the topology and geometry of DNA supercoiling.

CozzarelliNR

WangJC

DNA topology and its biological effects Cold Spring Harbor, NY Cold Spring Harbor Labaoratory Press 139 184

25. LiuB

MolinaH

KalumeD

PandeyA

GriffithJD

2006 Role of p38 in replication of Trypanosoma brucei kinetoplast DNA. Mol Cell Biol 26 5382 5393

26. RyanKA

EnglundPT

1989 Synthesis and processing of kinetoplast DNA minicircles in Trypanosoma equiperdum. Mol Cell Biol 9 3212 3217

27. WangJC

1971 Interaction between DNA and an Escherichia coli protein omega. J Mol Biol 55 523 533

28. RichA

NordheimA

WangAH

1984 The chemistry and biology of left-handed Z-DNA. Annu Rev Biochem 53 791 846

29. StraussPR

WangJC

1990 The TOP2 gene of Trypanosoma brucei: a single-copy gene that shares extensive homology with other TOP2 genes encoding eukaryotic DNA topoisomerase II. Mol Biochem Parasitol 38 141 150

30. ScoccaJR

ShapiroTA

2008 A mitochondrial topoisomerase IA essential for late theta structure resolution in African trypanosomes. Mol Microbiol 67 820 829

31. BakshiRP

ShapiroTA

2004 RNA interference of Trypanosoma brucei topoisomerase IB: both subunits are essential. Mol Biochem Parasitol 136 249 255

32. XuC

RayDS

1993 Isolation of proteins associated with kinetoplast DNA networks in vivo. Proc Natl Acad Sci USA 90 1786 1789

33. LukesJ

HinesJC

EvansCJ

AvliyakulovNK

PrabhuVP

2001 Disruption of the Crithidia fasciculata KAP1 gene results in structural rearrangement of the kinetoplast disc. Mol Biochem Parasitol 117 179 186

34. LiuY

EnglundPT

2007 The rotational dynamics of kinetoplast DNA replication. Mol Microbiol 64 676 690

35. GuilbrideDL

EnglundPT

1998 The replication mechanism of kinetoplast DNA networks in several trypanosomatid species. J Cell Sci 111 675 679

36. HoeijmakersJHJ

WeijersPJ

1980 The segregation of kinetoplast DNA networks in Trypanosoma brucei. Plasmid 4 97 116

37. DomingoGJ

PalazzoSS

WangB

PannicucciB

SalavatiR

2003 Dyskinetoplastic Trypanosoma brucei contains functional editing complexes. Eukaryot Cell 2 569 577

38. BodleyAL

McGarryMW

ShapiroTA

1995 Drug cytotoxicity assay for African trypanosomes and Leishmania species. J Infect Dis 172 1157 1159

39. DelcrosJG

SturkenboomMC

BasuHS

ShaferRH

SzollosiJ

1993 Differential effects of spermine and its analogues on the structures of polynucleotides complexed with ethidium bromide. Biochem J 291 Pt 1 269 274

40. ShapiroTA

1993 Kinetoplast DNA maxicircles: Networks within networks. Proc Natl Acad Sci USA 90 7809 7813

41. ShapiroTA

KleinVA

EnglundPT

1989 Drug promoted cleavage of kinetoplast DNA minicircles: evidence for type II topoisomerase activity in trypanosome mitochondria. J Biol Chem 264 4173 4178

42. DrusanoGL

2004 Antimicrobial pharmacodynamics: critical interactions of ‘bug and drug’. Nat Rev Microbiol 2 289 300

43. RottenbergH

1984 Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81 127 138

44. KaminskyR

SchmidC

LunZR

1997 Susceptibility of dyskinetoplastic Trypanosoma evansi and T. equiperdum to isometamidium chloride. Parasitol Res 83 816 818

45. HirumiH

HirumiK

1989 Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J Parasitol 75 985 989

46. LeeSH

StephensJL

PaulKS

EnglundPT

2006 Fatty acid synthesis by elongases in trypanosomes. Cell 126 691 699

47. WirtzE

LealS

OchattC

CrossGA

1999 A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol 99 89 101

48. BrunR

ShonenbergerM

1979 Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Tropica 36 289 292

49. WangZ

EnglundPT

2001 RNA interference of a trypanosome topoisomerase II causes progressive loss of mitochondrial DNA. EMBO J 20 4674 4683

50. Pérez-MorgaD

EnglundPT

1993 The attachment of minicircles to kinetoplast DNA networks during replication. Cell 74 703 711

51. ThresherR

GriffithJ

1992 Electron microscopic visualization of DNA and DNA-protein complexes as adjunct to biochemical studies. Methods Enzymol 211 481 490

52. Pérez-MorgaDL

EnglundPT

1993 Microtechnique for electron microscopy of DNA. Nucleic Acids Res 21 1328 1329

53. RobinsonDR

GullK

1994 The configuration of DNA replication sites within the Trypanosoma brucei kinetoplast. J Cell Biol 126 641 648

54. ShapiroTA

KleinVA

EnglundPT

1999 Isolation of kinetoplast DNA. Methods Mol Biol 94 61 67

55. HolfordNH

SheinerLB

1981 Understanding the dose-effect relationship: clinical application of pharmacokinetic-pharmacodynamic models. Clin Pharmacokinet 6 429 453

56. WilliamsonJ

1970 Review of chemotherapeutic and chemoprophylactic agents.

MulliganHW

The African Trypanosomiases Great Britain Wiley-Interscience 125 221

57. BrownDG

SandersonMR

GarmanE

NeidleS

1992 Crystal structure of a berenil-d(CGCAAATTTGCG) complex. An example of drug-DNA recognition based on sequence-dependent structural features. J Mol Biol 226 481 490

58. WakelinLP

ChetcutiP

DennyWA

1990 Kinetic and equilibrium binding studies of amsacrine-4-carboxamides: a class of asymmetrical DNA-intercalating agents which bind by threading through the DNA helix. J Med Chem 33 2039 2044

59. DannO

BergenG

DemantE

VolzG

1971 Trypanocide diamidine des 2-phenyl-benzofurans, 2-Phenyl-indens, and 2-phenyl- indols. Liebigs Ann Chem 749 68 89

60. WilliamsonJ

McLarenDJ

1978 Effects of DAPI a new diamidine trypanocide, on the ultrastructure of Trypanosoma rhodesiense. Trans R Soc Trop Med Hyg 72 660 661

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Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

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