Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis.
We have isolated and characterized two superoxide dismutase (SOD) cDNAs from a Leishmania chagasi promastigote cDNA library using degenerate primers derived from conserved amino acid residues of previously isolated manganese and iron SODs. Comparison of these two L. chagasi SOD deduced amino acid sequences with previously isolated MnSOD and FeSOD amino acid sequences revealed that they have higher homology to, and complete conservation of, invariant residues found in iron-containing SODs. Southern blot analysis showed that one gene, L.c.FeSODA, is a single copy gene, whereas the other gene, L.c.FeSODB, belongs to a multi-gene family. Transcript levels and enzyme activities of L.c.FeSODA and L.c.FeSODB show differential stage expression, with higher levels present in the amastigote stage of the parasite compared to the promastigote stage. Expression of the L.c.FeSODs in an E. coli SOD null strain protected the bacteria against free radical generating agents. Overexpression of these FeSODs in L. chagasi parasites also showed enhanced protection against the free radical generating agents, paraquat and nitroprusside. The cloning, characterization and overexpression of the L.c.FeSODA and L.c.FeSODB genes and proteins demonstrates the possible role of SODs in Leishmania pathogenesis.
Paramchuk
W J
WJ
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Ismail
S O
SO
Bhatia
A
A
Gedamu
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test; D018314:Leishmania infantum / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity*; D008969:Molecular Sequence Data; D009599:Nitroprusside / Q000494:pharmacology; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D009599:Nitroprusside; D013482:Superoxide Dismutase; D010269:Paraquat