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Molecular analysis of FAS and PKS in Cryptosporidium parvum
Workshops / Parasitology International 47 ( SuppL ) (1998) 10,5-131
124
W-0054
MOLECULAR ANALYSISOF FAS AND PKS IN CRYPTOSPORIDIUM PARVUM
ZhuG. MarchewkaMJ, WoodK*, Upto~SJ*, KeithlyJS WadsworthCenter, New YorkState Depetmumtof Health, Albany,NY, USA and *Divisionof Biology, gmtt~t Sta~ Univenity, M~nhatm~KS, USA ltne we na~t the initial ~ of two fatty ~l/polyke~de synthases (FASIPKS) from Cryptosporidilmt parvum (cpfash~l and cp~). By soreeningtwo gDNAh'bimies,>22 kb of the cpfas/pksl gene has been isolated md ~ fi'~movnrlappingclones. However,it is still incomplete, twking the 3' natl. The idonafwd enzymatic dCanain~ form 3 cassettes, each contHdng lu~____,o~ acyl-ca~ wo~i~, Ic~mynd~meand (malonyl/acetyl)-lnmsferme. The size and o r ~ n t ~ a of the geae suggest it may encode a PKS. A 460 bp fa'asment of the cpf--,~tsl game has been extaessed as a GST-fusion protein, which is now being used to raise rabbit
polycional mtibody. Soothemblot mal~Asshowsthat this sizableepfas/pksl 8erie is a sialgle copy. A secondgene in the family (qKm/pk~) has also be~ idemified. Its IwtiMsequeace at the 3' md shows ~ homologyto the yeast FA.S 6-subenit. tlowcv~, nothing is yet lmown about the comlm'aneatatlouof these mzymesin C./mrvant Fatty acids me es~atial OOmlxmentsof all biomembranes,and me an storage ~ of all organisms.Their syntbr~s from acetyl..CoA~1 malonyl-CoA to p~lmitate ~.e-s about 50 ~ by either a single multifuncfimlal protein or a namber of ~ t i o n a l eazymes. PKS uses alu~tm~estar*e~andptocessesbefore condematiomis comply, resulting in the tetentiou of keto- and hydxoxyl gtmaps on carbons. Some polyketidm ame importantantibiotics, e.g., erylhromycin,monensln,avermectin. The discovery of FAS/PKS genes in C. par'ram SUgg~tsthe de novo fatty acid biosynthesis and/or polyl=tide productkm in this pumile, which providespotentially new parasi~spo:ific drag targets, In suppmt of this, our p~imln~,y drug tests showedthat the growthof C. parvwn in vitro could be intdbit~l 96% by an FAS inbibit~', cet~le~n. Since FAS inbibitors may also inhibit PKS, if C. parvum MmuldIm3ducepolyketide maibiotics (as well m FAS) to secure its colonization in the gut, inbibitors like these would not only block essential fattyacid biosynthesis, but would dea'eme its cempelitive advamtage over other
gut mic/oOrg:mi~ng.Thus, FAS [ &ug targets.
PKS could serveas new l~'asile-specific
W-0055 SECRETION AND TRAILING OF MICRONEME PROTEINS DURING MOTILITY AND INVASION OF EIMERIA TENELLA. Tomlev F, Bumstead J Institute for Animal Health, Compton, Newbury, Berkshire, UK Micronemes of Eimeria tenella sporozoites contain mound 11 proteins, several of which contain adhesive domains that are believed to promote the attachment of parasites to the host cell during invasion. Proteins are rapidly secreted from the micronemes when sporozoites are added to cultured epithelial cells (MDBK) and are readily detectable in culture supernatants by western blotting. Using indirect immunofluoresence and silverenhanced immuno-gold labeling, microneme proteins are seen to be released from the apical end of the parasite both before and during penetration and are also detected on the surface of parasites and host cells. As parasites glide over cells, microneme proteins are deposited onto the surface from the trailing posterior end. In cultures treated with cytochalasins or BDM (which interfere with actin and myosin respectively and inhibit parasite motility), secretion of microneme proteins from the apical end still occurs and the parasites attach to host cells. However, trails of microneme proteins are not seen and parasites fail to invade cells. Thus while microneme exocytosis is independent of actin/myosin, surface capping of micronemes and parasite invasion is dependent on these cytoskeletal elements.
W - 0 0 5 6 ENCYSTMEIqT IN GIARDIA: THE SYNTHESIS OF N-ACETYLGALACTOSAMINE AND THE CYST WALL Jarroll E*, Karr C*, Bulik D*, Steimle P**, van Keulen H***, Paget T'l', Gerwig GJ:I:, Kamerling JP'~, Vliegenthart JFG:I:, Erlandsen S:~ *Dept. of Biology, Northeastern Univ., Boston, MA, **Dept. of Biochemistry, Johns Hopkins University, Baltimore, MD, *** Dept. of Biology, Cleveland State Univ., Cleveland, OH, USA, "l'Dept. of Biological Sciences, Univ. of Hull, Hull, UK and :gBijvoet Center for Biomolecular Research, Utrecht Univ., Utrecht, NL, ~Dept. of Cell Biology and Neuroanatomy, Univ. of Minnesota Medical School, Minneapolis, MN, USA
Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events, related to the inducible synthesis of a novel N-acetyl-D-galactosamine (GalNAc) homopolymer, occur. Within the first 6 hrs of encystment, mRNA for glucosamine 6-P isomerase (Gpi), the first inducible enzyme, appears, oxygen uptake (OU) rates double from non-encysting levels and metronidazole (MTZ) inhibits OU. Within 12 hrs, Gpi and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and M'FZ's inhibition of OU cease. In contrast, aspartate uptake remained constant throughout the 40 hrs monitored. Gpi is encoded for by two genes (gpi 1 and 2) only gpi I is expressed during encystment. Glucosamine 6-P, the synthetic product of Gpi, activates UDP-N-acetylglucosamine (UDP-GIcNAc) pyrophosphorylase, a downstream enzyme, 3-5 fold in the synthetic direction. UDP-GIcNAc is epimerized to UDP-GalNAc and UDPGalNAc is polymerized by "cyst wall synthase" into a highly insoluble [3-1inked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia. This work was supported in part by NIHAID grants AI29591 and AI41230 to ELL
124
W-0054
MOLECULAR ANALYSISOF FAS AND PKS IN CRYPTOSPORIDIUM PARVUM
ZhuG. MarchewkaMJ, WoodK*, Upto~SJ*, KeithlyJS WadsworthCenter, New YorkState Depetmumtof Health, Albany,NY, USA and *Divisionof Biology, gmtt~t Sta~ Univenity, M~nhatm~KS, USA ltne we na~t the initial ~ of two fatty ~l/polyke~de synthases (FASIPKS) from Cryptosporidilmt parvum (cpfash~l and cp~). By soreeningtwo gDNAh'bimies,>22 kb of the cpfas/pksl gene has been isolated md ~ fi'~movnrlappingclones. However,it is still incomplete, twking the 3' natl. The idonafwd enzymatic dCanain~ form 3 cassettes, each contHdng lu~____,o~ acyl-ca~ wo~i~, Ic~mynd~meand (malonyl/acetyl)-lnmsferme. The size and o r ~ n t ~ a of the geae suggest it may encode a PKS. A 460 bp fa'asment of the cpf--,~tsl game has been extaessed as a GST-fusion protein, which is now being used to raise rabbit
polycional mtibody. Soothemblot mal~Asshowsthat this sizableepfas/pksl 8erie is a sialgle copy. A secondgene in the family (qKm/pk~) has also be~ idemified. Its IwtiMsequeace at the 3' md shows ~ homologyto the yeast FA.S 6-subenit. tlowcv~, nothing is yet lmown about the comlm'aneatatlouof these mzymesin C./mrvant Fatty acids me es~atial OOmlxmentsof all biomembranes,and me an storage ~ of all organisms.Their syntbr~s from acetyl..CoA~1 malonyl-CoA to p~lmitate ~.e-s about 50 ~ by either a single multifuncfimlal protein or a namber of ~ t i o n a l eazymes. PKS uses alu~tm~estar*e~andptocessesbefore condematiomis comply, resulting in the tetentiou of keto- and hydxoxyl gtmaps on carbons. Some polyketidm ame importantantibiotics, e.g., erylhromycin,monensln,avermectin. The discovery of FAS/PKS genes in C. par'ram SUgg~tsthe de novo fatty acid biosynthesis and/or polyl=tide productkm in this pumile, which providespotentially new parasi~spo:ific drag targets, In suppmt of this, our p~imln~,y drug tests showedthat the growthof C. parvwn in vitro could be intdbit~l 96% by an FAS inbibit~', cet~le~n. Since FAS inbibitors may also inhibit PKS, if C. parvum MmuldIm3ducepolyketide maibiotics (as well m FAS) to secure its colonization in the gut, inbibitors like these would not only block essential fattyacid biosynthesis, but would dea'eme its cempelitive advamtage over other
gut mic/oOrg:mi~ng.Thus, FAS [ &ug targets.
PKS could serveas new l~'asile-specific
W-0055 SECRETION AND TRAILING OF MICRONEME PROTEINS DURING MOTILITY AND INVASION OF EIMERIA TENELLA. Tomlev F, Bumstead J Institute for Animal Health, Compton, Newbury, Berkshire, UK Micronemes of Eimeria tenella sporozoites contain mound 11 proteins, several of which contain adhesive domains that are believed to promote the attachment of parasites to the host cell during invasion. Proteins are rapidly secreted from the micronemes when sporozoites are added to cultured epithelial cells (MDBK) and are readily detectable in culture supernatants by western blotting. Using indirect immunofluoresence and silverenhanced immuno-gold labeling, microneme proteins are seen to be released from the apical end of the parasite both before and during penetration and are also detected on the surface of parasites and host cells. As parasites glide over cells, microneme proteins are deposited onto the surface from the trailing posterior end. In cultures treated with cytochalasins or BDM (which interfere with actin and myosin respectively and inhibit parasite motility), secretion of microneme proteins from the apical end still occurs and the parasites attach to host cells. However, trails of microneme proteins are not seen and parasites fail to invade cells. Thus while microneme exocytosis is independent of actin/myosin, surface capping of micronemes and parasite invasion is dependent on these cytoskeletal elements.
W - 0 0 5 6 ENCYSTMEIqT IN GIARDIA: THE SYNTHESIS OF N-ACETYLGALACTOSAMINE AND THE CYST WALL Jarroll E*, Karr C*, Bulik D*, Steimle P**, van Keulen H***, Paget T'l', Gerwig GJ:I:, Kamerling JP'~, Vliegenthart JFG:I:, Erlandsen S:~ *Dept. of Biology, Northeastern Univ., Boston, MA, **Dept. of Biochemistry, Johns Hopkins University, Baltimore, MD, *** Dept. of Biology, Cleveland State Univ., Cleveland, OH, USA, "l'Dept. of Biological Sciences, Univ. of Hull, Hull, UK and :gBijvoet Center for Biomolecular Research, Utrecht Univ., Utrecht, NL, ~Dept. of Cell Biology and Neuroanatomy, Univ. of Minnesota Medical School, Minneapolis, MN, USA
Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events, related to the inducible synthesis of a novel N-acetyl-D-galactosamine (GalNAc) homopolymer, occur. Within the first 6 hrs of encystment, mRNA for glucosamine 6-P isomerase (Gpi), the first inducible enzyme, appears, oxygen uptake (OU) rates double from non-encysting levels and metronidazole (MTZ) inhibits OU. Within 12 hrs, Gpi and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and M'FZ's inhibition of OU cease. In contrast, aspartate uptake remained constant throughout the 40 hrs monitored. Gpi is encoded for by two genes (gpi 1 and 2) only gpi I is expressed during encystment. Glucosamine 6-P, the synthetic product of Gpi, activates UDP-N-acetylglucosamine (UDP-GIcNAc) pyrophosphorylase, a downstream enzyme, 3-5 fold in the synthetic direction. UDP-GIcNAc is epimerized to UDP-GalNAc and UDPGalNAc is polymerized by "cyst wall synthase" into a highly insoluble [3-1inked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia. This work was supported in part by NIHAID grants AI29591 and AI41230 to ELL