The pivotal role of guanine phosphoribosyltransferase in purine salvage by Giardia lamblia.
Giardia lamblia is an anaerobic binucleate flagellated protozoan known to lack de novo synthesis of purine nucleotides. Our previous metabolic studies indicated two major parallel pathways mediated by adenine phosphoribosyltransferase (APRT) and guanine phosphoribosyltransferase (GPRT) that constitute the primary route of purine salvage in this organism. To verify further that these enzymes play a pivotal role in replenishing the purine ribonucleotide pool and are required for replicative growth of Giardia, a knock-out of GPRT gene expression in this organism was attempted. A hammerhead ribozyme flanked by two arms of GPRT antisense RNA (GPRZ) was designed, synthesized and found capable of cleaving a GPRT mRNA fragment in vitro at the designated site. GPRZ cDNA was then cloned into a viral vector pC631pac, derived from the genome of giardiavirus (GLV), and its transcript was introduced into GLV-infected Giardia trophozoites by electroporation. Stable transformants selected under increasing concentrations of puromycin displayed parallel increases in ribozyme levels and associated decreases in GPRT mRNA levels, GPRT enzyme activity and replicative cell growth to less than 10-20% of wild-type levels. Metabolite analyses showed specific depletion of the guanine ribonucleotide pools in parallel with slower cell growth. We conclude that GPRT plays an essential role in supplying guanine nucleotides required for growth and multiplication of Giardia, emphasizing its potential as a bona fide target for antigiardiasis chemotherapy.
Munagala
Narsimha
N
Department of Pharmaceutical Chemistry, University of California, 513 Parnassus, San Francisco, CA 94143-0446, USA.
Wang
Ching C
CC
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D011687:Purines / Q000378:metabolism*; D011691:Puromycin / Q000494:pharmacology; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D006150:Guanine Nucleotides; D011687:Purines; D016337:RNA, Catalytic; D012333:RNA, Messenger; D016053:RNA, Protozoan; C427204:hammerhead ribozyme; D011691:Puromycin; D007041:Hypoxanthine Phosphoribosyltransferase