Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays.
In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.
Guimond
Chantal
C
Centre de Recherche en Infectiologie du Centre de Recherche du CHUL and Division de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada.
Trudel
Nathalie
N
Brochu
Christian
C
Marquis
Nathalie
N
El Fadili
Amal
A
Peytavi
Régis
R
Briand
Guylaine
G
Richard
Dave
D
Messier
Nadine
N
Papadopoulou
Barbara
B
Corbeil
Jacques
J
Bergeron
Michel G
MG
Légaré
Danielle
D
Ouellette
Marc
M
D000818:Animals; D000965:Antimony / Q000494:pharmacology; D018053:Arsenites / Q000494:pharmacology; D015152:Blotting, Northern; D018432:Drug Resistance, Multiple / Q000235:genetics; D005493:Folic Acid Antagonists / Q000494:pharmacology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D017125:Genes, Protozoan / Q000235:genetics; D007891:Leishmania / Q000187:drug effects / Q000235:genetics*; D018320:Leishmania major / Q000187:drug effects / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
D018053:Arsenites; D005493:Folic Acid Antagonists; D016053:RNA, Protozoan; D000965:Antimony; C015001:arsenite; D008727:Methotrexate