Cloning, functional analysis and post-transcriptional regulation of a type II DNA topoisomerase from Leishmania infantum. A new potential target for anti-parasite drugs.
We identified a type II topoisomerase enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li topo II, which displays a variable C-terminal end, is located in the kinetoplast. The cloned gene encoding Li-TOP2 compensates for the slow growth of topo II-deficient mutants of Saccharomyces cerevisiae, resulting in a catalytically active DNA topoisomerase in yeast. Analysis of the specific mRNA levels of the Li-TOP2 gene showed variations throughout the parasite cell cycle in synchronized cells as well as between the distinct forms of the parasite. Thus, the enzyme had higher levels of mRNA expression in the highly infective intracellular form of the parasite, the amastigote, than in the extracellular promastigote form, suggesting a relation with the distinct developmental and infectious phases of the protozoon. In addition, western blot analysis showed differences in protein expression between the proliferative and non-proliferative forms of L.infantum promastigotes, which displayed similar levels of mRNA. This indicated possible post-transcriptional regulation mechanisms. The data suggest that Li topo II has a part in DNA decatenation and probably at the initial stages of proliferation in the intracellular form of L.infantum, a parasite that has to proliferate into the host macrophage to survive its hostile environment in its first moments of intracellular infection.
Hanke
Tobias
T
Centro de Investigaciones Biológicas C.S.I.C., Velázquez 144, Madrid 28006, Spain.
Ramiro
María J
MJ
Trigueros
Sonia
S
Roca
Joaquim
J
Larraga
Vicente
V
D000595:Amino Acid Sequence; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D003001:Cloning, Molecular; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D018314:Leishmania infantum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D012323:RNA Processing, Post-Transcriptional; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D059005:Topoisomerase II Inhibitors
D000981:Antiprotozoal Agents; D018076:DNA, Complementary; D004278:DNA, Superhelical; D011994:Recombinant Proteins; D059005:Topoisomerase II Inhibitors; D004250:DNA Topoisomerases, Type II