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Leishmania mexicana: investigation offlagellar attachment by promastigotesin vitro
Poster Sessions I Parasitology
330
International
47 (Suppl.) (1998) 283-389
D. Molecular Biology and Biochemistry ~~______ p-0774
dXkWM4NIA FLAGELLAR IN Ul??O
MEXICANA: INVESTIGATION OF ATTACHMENT BY PROMASTIGOTES
l&b&E&& Bates PA Liierpool School of Tropical Medicine, Liverpool, UK Flagellar attachment via the formation of hemidesmosome-like StNctures is a common feature of trypanosomatids in their insect vectors. We have developed an in vitro attachment system to investigate the molecular and biochemical basis of flagellar attachment in Leishmania mexicana In infected sandflies attachment occurs at the stomodeal valve and foregut in mature infections. Three types of p&tic film were used as potential substrates: plastic coverslips, Melinex film and Thermanox tissue culture coverslips. These were both used as supplied (smooth) and after roughening the sufice with Emery paper (scratched). In general the scratched coverslips gave better attachment; Meliex and Themumox were better than plastic. Melinex was chosen for further experiments. Promastigotes were grown in two media: Medium 199 supplemented with 10% f&al calf serum, and M199+, a se.rum free chemically defined medium. The presence of serum was found to promote attachment. Different species and strains were also compared Inter-strain and inter-species variations in attachment rate were found. Highest rates were obtained with L. bruziliensis, followed by L. mexicmta, then L. major. The correct type of Bagellar attachment in vifro was confirmed by scanning and transmission electron microscopy, which showed the expanded fIagellar tips and associated hemidesmosonie-like structures. Flagellar cytoskeletons were prepared from attached and free parasites. These were compared by SDS-PAGE and protein staining, which revealed the presence of at least one protein, of molecular mass ISOkDa, which was unique to the attached promastigotes. The identity and %nction of this protein is under investigation. P-0775
LEISHMANIA MAJOR: CHARACTERIZATION
OF
PROTEINS SECRETED BY PROMASTIGOTES IN KXQO Alkhahfe ISM, Bates PA Liverpool School of Tropical Medicine, Liverpool, UK Promastigotes of Leishmania major were adapted to grow in a proteinfree, chemicallyd&ed medium based on Medium 199 (M 199t). The cell-f%e culture supematant of stationary phase cultures was collected to study the biochemical natureandtimtion of secrctcdor releasedproteins. Thesewxc analysedwithrespectto totalproteinprofile,for the presence of nuckascandproteinase activities, by immunoblotting with mouse infection serum, and by &tin blotting to reveal glycoproteins. A range of secreted or released proteins was observed ranging from 6 kDa to 155 kDa in molecular mass. Amongst these there were at least 17 clearly distinct major bands, and a number of additional minor bands Substrate gels incorporatin8 polyadenylic acid revealedthe presence of three bands with nuclease activity of 3 1, 35 and 38 kDa. The possible role of these in purine salvage is under investigation. Substrate gels incorporating gelatin revealed the presence of one proteinase actwity of 66 kDa. The relationship of this protein to the major promastigote surface prot&ase of similar molecular mass, and potential function is being assessed. hnmunoblotting showed tbat many of the protein bands were recognised as antigens by mouse infection serum, and therefore share antigenic epitopes with the amastigote form of the parasite. Lectin blotting also revealed many of the protein bands to be glycosylated. The effect of external pH on secretory activity was investigated by culturing promastigotes in media at different pH values. There were no obvious morphological changes in the parasites, all retaining the promastigote form from pH 5 5 to pH 8.0. However, secretory activity was markedly affected by PH. At alkaline pH the till range of protein bands was observed. However, with increasing acidity secretory activity was inhibited, with little detected below pH 6.5. This suggests that the parasites may down-regulate secretion in acidic environments, such as that experienced by the amastigote form in macropbage lysosomes
P-0776 LEISHMdNL4 DONOVXNZ INFANTUM: CLONING AND CHARACTERIZATION OF A RAR PROTEIN HOMOLOGUE Cibrelus P., Walas F., Carret C., Carey B., Lemesre JL*, Pr&cigout E., Gore&lot A. Laboratoire de Biologic Cellulaire et MolCulaire, EA MESR 2413, UFR des Sciences Pharmaceutiques et Biologiques, 15 Avenue C. Flahault, 34060 Montpellier Cedex 2, France. *Unit& de Biologie Parasitaire, ORSTOM, BP 5045,34032 Montpellier Cedex 1, France Leishmania are dimorphic parasitic protozoa which have two morphologically distinct forms during their life cycle : flagellated promastigotes in the digestive tract of sat&lies vectors and nonmotile amastigotes within phagolysosomes of mammalian macrophage. A polyclonai rabbit antiserum raised against the antigens excreted/secreted by the amastigote form of Leishmaniu infintum allowed us to screen an amastigote cDNA library. The open-reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. Rab proteins are implicated as important components in vesicular traflicking in the eukaryotes and the conservation of sequence suggests a similar function in different organisms. As all the GTPases, rab protein of L. infanturn contains the four highly conserved sequence motifs which are required for guanine nucleotide binding and GTP hydrolysis. However, while all the rab proteins examined to date have subterminal cysteine residues, involved in posttranslationally modification essential for function and membrane association, the protein found in L. infatium does not contain this typical carboxyterminal end. This suggests a particular role for the rab protein of Leishmuniu infanturn. To raise a polyclonal rabbit antiserum agaisnt this protein, a portion of the gene was cloned in the pGEX vector and expressed in E. co/i as a fusion protein. P-0777
NOVEL ORGANIZATION OF THE GFNES FOR THE INITIAL THRBE ENZYMES IN DE NOVO PYRIMIDINB BIOSYNTHES IS IN ~pypAN0S0’M CRUD
&gQ, Gao G, Nakajima-Shimada I, Aoki T Department of Parasitology, Juntendo University School of Medicine, Tokyo, Japan De novo pyrimidine biosynthesis is essential in most organisms and the initial three enzymes in the synthetic pathway, carbamoyl-phosphate synthetase II (CPS II), aspartate carbamoyltransferase (ACT), and dihydroorotase (DHOase), are expressed as a multi-functional protein CAD in the order CPS IIDHOase-ACT in mammals. In bacteria, however, these enzyme Our activities reside on individual subunits or polypeptides. previous study demonstrated that CPS II and ACT genes are independent in T. cruzi. Since trypanosomatids are thought to be the earliest eukaryotic lineage, we hypothesize that the genome may have retained an ancient form, possibly a gene cluster that contains open reading frames for these enzymes. The present study was undertaken to examine the genomic organization of the CPS II, Aa and DHOase genes in T. cruzi. Pulsed field gel electrophoresis reveal&l the simultaneous localization of a pair of CPS II and ACT genes on two chromosomal DNAs of 1,000 and 800 kb. An extra copy of the ACT gene was found on the 800 kb chromosomal DNA. A T. cnrri genomic library was screened using CPS II- and Amspecific DNA probes, and the positive clones obtained were aligned so as to detect any overlapping region. The results show that the CPS II gene is located 14 kb up&ream of the ACT gene. The DHOase gene was identified and found to be located between the CPS II and ACT genes, in the order 5’- CPS ILDHOase-ACT -3’. This gene organization is conserved in both the 1,000 and 800 kb DNA.% This is the first report demonstrating the organization of the genes responsible for pyrimidine biosynthesis in protozoan parasites.
330
International
47 (Suppl.) (1998) 283-389
D. Molecular Biology and Biochemistry ~~______ p-0774
dXkWM4NIA FLAGELLAR IN Ul??O
MEXICANA: INVESTIGATION OF ATTACHMENT BY PROMASTIGOTES
l&b&E&& Bates PA Liierpool School of Tropical Medicine, Liverpool, UK Flagellar attachment via the formation of hemidesmosome-like StNctures is a common feature of trypanosomatids in their insect vectors. We have developed an in vitro attachment system to investigate the molecular and biochemical basis of flagellar attachment in Leishmania mexicana In infected sandflies attachment occurs at the stomodeal valve and foregut in mature infections. Three types of p&tic film were used as potential substrates: plastic coverslips, Melinex film and Thermanox tissue culture coverslips. These were both used as supplied (smooth) and after roughening the sufice with Emery paper (scratched). In general the scratched coverslips gave better attachment; Meliex and Themumox were better than plastic. Melinex was chosen for further experiments. Promastigotes were grown in two media: Medium 199 supplemented with 10% f&al calf serum, and M199+, a se.rum free chemically defined medium. The presence of serum was found to promote attachment. Different species and strains were also compared Inter-strain and inter-species variations in attachment rate were found. Highest rates were obtained with L. bruziliensis, followed by L. mexicmta, then L. major. The correct type of Bagellar attachment in vifro was confirmed by scanning and transmission electron microscopy, which showed the expanded fIagellar tips and associated hemidesmosonie-like structures. Flagellar cytoskeletons were prepared from attached and free parasites. These were compared by SDS-PAGE and protein staining, which revealed the presence of at least one protein, of molecular mass ISOkDa, which was unique to the attached promastigotes. The identity and %nction of this protein is under investigation. P-0775
LEISHMANIA MAJOR: CHARACTERIZATION
OF
PROTEINS SECRETED BY PROMASTIGOTES IN KXQO Alkhahfe ISM, Bates PA Liverpool School of Tropical Medicine, Liverpool, UK Promastigotes of Leishmania major were adapted to grow in a proteinfree, chemicallyd&ed medium based on Medium 199 (M 199t). The cell-f%e culture supematant of stationary phase cultures was collected to study the biochemical natureandtimtion of secrctcdor releasedproteins. Thesewxc analysedwithrespectto totalproteinprofile,for the presence of nuckascandproteinase activities, by immunoblotting with mouse infection serum, and by &tin blotting to reveal glycoproteins. A range of secreted or released proteins was observed ranging from 6 kDa to 155 kDa in molecular mass. Amongst these there were at least 17 clearly distinct major bands, and a number of additional minor bands Substrate gels incorporatin8 polyadenylic acid revealedthe presence of three bands with nuclease activity of 3 1, 35 and 38 kDa. The possible role of these in purine salvage is under investigation. Substrate gels incorporating gelatin revealed the presence of one proteinase actwity of 66 kDa. The relationship of this protein to the major promastigote surface prot&ase of similar molecular mass, and potential function is being assessed. hnmunoblotting showed tbat many of the protein bands were recognised as antigens by mouse infection serum, and therefore share antigenic epitopes with the amastigote form of the parasite. Lectin blotting also revealed many of the protein bands to be glycosylated. The effect of external pH on secretory activity was investigated by culturing promastigotes in media at different pH values. There were no obvious morphological changes in the parasites, all retaining the promastigote form from pH 5 5 to pH 8.0. However, secretory activity was markedly affected by PH. At alkaline pH the till range of protein bands was observed. However, with increasing acidity secretory activity was inhibited, with little detected below pH 6.5. This suggests that the parasites may down-regulate secretion in acidic environments, such as that experienced by the amastigote form in macropbage lysosomes
P-0776 LEISHMdNL4 DONOVXNZ INFANTUM: CLONING AND CHARACTERIZATION OF A RAR PROTEIN HOMOLOGUE Cibrelus P., Walas F., Carret C., Carey B., Lemesre JL*, Pr&cigout E., Gore&lot A. Laboratoire de Biologic Cellulaire et MolCulaire, EA MESR 2413, UFR des Sciences Pharmaceutiques et Biologiques, 15 Avenue C. Flahault, 34060 Montpellier Cedex 2, France. *Unit& de Biologie Parasitaire, ORSTOM, BP 5045,34032 Montpellier Cedex 1, France Leishmania are dimorphic parasitic protozoa which have two morphologically distinct forms during their life cycle : flagellated promastigotes in the digestive tract of sat&lies vectors and nonmotile amastigotes within phagolysosomes of mammalian macrophage. A polyclonai rabbit antiserum raised against the antigens excreted/secreted by the amastigote form of Leishmaniu infintum allowed us to screen an amastigote cDNA library. The open-reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. Rab proteins are implicated as important components in vesicular traflicking in the eukaryotes and the conservation of sequence suggests a similar function in different organisms. As all the GTPases, rab protein of L. infanturn contains the four highly conserved sequence motifs which are required for guanine nucleotide binding and GTP hydrolysis. However, while all the rab proteins examined to date have subterminal cysteine residues, involved in posttranslationally modification essential for function and membrane association, the protein found in L. infatium does not contain this typical carboxyterminal end. This suggests a particular role for the rab protein of Leishmuniu infanturn. To raise a polyclonal rabbit antiserum agaisnt this protein, a portion of the gene was cloned in the pGEX vector and expressed in E. co/i as a fusion protein. P-0777
NOVEL ORGANIZATION OF THE GFNES FOR THE INITIAL THRBE ENZYMES IN DE NOVO PYRIMIDINB BIOSYNTHES IS IN ~pypAN0S0’M CRUD
&gQ, Gao G, Nakajima-Shimada I, Aoki T Department of Parasitology, Juntendo University School of Medicine, Tokyo, Japan De novo pyrimidine biosynthesis is essential in most organisms and the initial three enzymes in the synthetic pathway, carbamoyl-phosphate synthetase II (CPS II), aspartate carbamoyltransferase (ACT), and dihydroorotase (DHOase), are expressed as a multi-functional protein CAD in the order CPS IIDHOase-ACT in mammals. In bacteria, however, these enzyme Our activities reside on individual subunits or polypeptides. previous study demonstrated that CPS II and ACT genes are independent in T. cruzi. Since trypanosomatids are thought to be the earliest eukaryotic lineage, we hypothesize that the genome may have retained an ancient form, possibly a gene cluster that contains open reading frames for these enzymes. The present study was undertaken to examine the genomic organization of the CPS II, Aa and DHOase genes in T. cruzi. Pulsed field gel electrophoresis reveal&l the simultaneous localization of a pair of CPS II and ACT genes on two chromosomal DNAs of 1,000 and 800 kb. An extra copy of the ACT gene was found on the 800 kb chromosomal DNA. A T. cnrri genomic library was screened using CPS II- and Amspecific DNA probes, and the positive clones obtained were aligned so as to detect any overlapping region. The results show that the CPS II gene is located 14 kb up&ream of the ACT gene. The DHOase gene was identified and found to be located between the CPS II and ACT genes, in the order 5’- CPS ILDHOase-ACT -3’. This gene organization is conserved in both the 1,000 and 800 kb DNA.% This is the first report demonstrating the organization of the genes responsible for pyrimidine biosynthesis in protozoan parasites.