Gene disruption of the DNA topoisomerase IB small subunit induces a non-viable phenotype in the hemoflagellate Leishmania major.
BACKGROUND
The unusual heterodimeric leishmanial DNA topoisomerase IB consists of a large subunit containing the phylogenetically conserved "core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelate Trypanosoma brucei. Unfortunately, this approach is not suitable in Leishmania where gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. In this work, we have successfully generated null mutants in the small subunit of the L. major DNA topoisomerase IB using two selection markers, each conferring resistance to hygromycin B and puromycin, respectively.


RESULTS
We have successfully replaced both topS loci with two selection markers. However, to achieve the second transfection round, we have had to rescue the null-homozygous with an episomal vector carrying the Leishmania major topS gene. Phenotypic characterization of the L. major rescued strain and a L. major strain, which co-overexpresses both subunits, shows few differences in DNA relaxation and camptothecin cytotoxicity when it was compared to the wild-type strain. Studies on phosphatidylserine externalization show a poor incidence of camptothecin-induced programmed cell death in L. major, but an effective cell-cycle arrest occurs within the first 24 h. S-Phase delay and G2/M reversible arrest was the main outcome at lower concentrations, but irreversible G2 arrest was detected at higher camptothecin pressure.


CONCLUSIONS
Results obtained in this work evidence the essentiality of the topS gene encoding the L. major DNA topoisomerase IB small subunit. Reversibility of the camptothecin effect points to the existence of effective checkpoint mechanisms in Leishmania parasites.
Balaña-Fouce
Rafael
R
Departamento de Farmacología y Toxicología (INTOXCAL), Universidad de León, Campus de Vegazana s/n; 24071 León, Spain. rbalf@unileon.es
García-Estrada
Carlos
C
Pérez-Pertejo
Yolanda
Y
Reguera
Rosa M
RM
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000254:growth & development; D002166:Camptothecin / Q000633:toxicity; D002455:Cell Division / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D016054:DNA, Protozoan / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000633:toxicity; D015971:Gene Expression Regulation, Enzymologic; D020043:Genes, Essential / Q000235:genetics*; D018320:Leishmania major / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D010641:Phenotype; D010718:Phosphatidylserines / Q000378:metabolism; D011156:Population Density
D016054:DNA, Protozoan; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin