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Expression and function of Zn-protease (GP63) in Leishmania amazonensis
Oral Sessions I Parasitology International 47 (Suppl.) (1998) 133-281
t bO37’)
O-0381
EXPRESSION AND FUNCTION OF ZN-PROTEASE (GP63) IN LEkS%/MANlA AMAZC’NEAWS
w, McGwire BS, Lu H-G, Chen D-Q, Yadava N Dept Microbiology/Immunology, Chicago Medical School, IL, USA The surface zinc protease is a glycoprotein of -63 kDa, especially abundant in the promastigote stage and encoded by clusters of tandemly repeated genes in Leishmnia spp. Recently, functional residues of gp63 essential in catalysis and its posttranslational modifications have been examined by site-specific mutagenesis. The availability of these mutants makes it possible to further examine these functional residues, as our ongoing work, to elucidate the expression and functions of the specific copy under study. We have also started to examine the multiple heterogeneous gp63 by inhibiting their expression using anti-sense transcription. The work began by cloning a 1.9 kb gp63 gene from 1. amazonensis in correct (C) orientation and in reverse (R) orientation separately into a Leishmania-specific vector, P6.5. Virulent promastigotes of the same species were transfected with both constructs plus the controls with irrelevant genes in P6.5. Transfectants were selected for tunicamycin (TM)-resistance. With the exposure to increasing TM concentrations. gp63 protems and their proteolytic activities increase and decrease In the transfectants with gp63-C and those with gp63-R. respectively. These and control transfectants were further compared for their infection of macrophages in vitro. Prelimmary results showed that the association of the gp63-R transfectants with macrophages and their intracellular survival are significantly reduced, while those with gp63-C is similar to the controls in these phenotypes of infectivity. Work is under to verify that gp63 anti-sense transcripts are produced in the former, accounting for the decrease seen in the expression of their gp63 and the loss of their infectivity.
O-0380
Molecular Cloning of Leishmanla homoiogue of Receptors for Activated C Kinase cDNA Leishmania donovani
219
REPIACEMENT OF P36 WlTH FOREIGN GENES FOR THEtRcHRoMosoMALAN)MTRAcHRoMosoMAL MPRESSlON IN LEk%Wh’~ AhW?UNEN.SlS
Lu HG, Kawazu S-l, Ito H, Chen D-Q, Fakruddin M, Akman L, Yadava N, w Dept Microbiology/Immunology. Chicago Medical School, IL, USA All species of Leishmania examined so far constitutively express a 36 kDa cytosolic protein (~36) homologous to c-crystallin/NADPH -oxidoreductase. The gene encoding p36 (P36) is single-copy per haploid genome. Both copies of P36 are dispensable, as they are replaceable with the marker genes. The latter remain structurally and functionally stable in situ even in the absence of selective pressures. Both alleles of P36 were replaced successively by homologous recombination first with NE0 and then with HYG. Replacement of P36 in 4 different clones is site-specific at both alleles. As expected, p36 is absent in double knockouts of P36 (DKOs) and reduced to half of the wildtype in the single knockouts (SKOs). DKOs emerged after exposure of SKOs to increasing concentrations of the relevant drugs. suggestive of inter-allelic recombination or unequal sister chromatid segregation during mitosis. The same conditions also increase the levels of immunologically detectable NE0 products, independent of gene amplification. All P36 null mutants grow as promastigotes or as axenic amastigotes equally well as their parental wildtype. Their infectivity to macrophages in vitro and to hamsters in viva are also comparable. DKOs recovered from infected macrophages or from cell-free cultures in the absence of drugs retain their drugresistance phenotypes, indicative of NE0 and HYG integrii and functionality. The chromosomal sites of P36 are thus expected to provide suitable locations lor stable expression of foreign genes in Leishmania. These sites have already proved useful to accommodate foreign genes for their extrachromosomal expression in Leisbmania. One vector developed is P6.5 where the gene, encoding N-acetylglucosamine-l-phosphate transferase, 2.3 kb downstream of P36 is used as the seletive marker for tunicamycin-resistance. Foreign genes cloned in P6.5 for expression in Leishmania include those from other protozoa, helminths and mammals. Transfectants with P6,5/porphobilinogen deaminase cDNA produced highly enzymatically active products.
O-0382 of
Takeuchi fl, Mataumoto Y’, Nakamura Y”, Matsumoto Y”, Otsuka H’ ‘LaboratoIy of Global Animmal Resource Science and “Department of Molecular Immunology. The University of Tokyo, Tokyo, Japan Leishmaniasis, which is classified into three main categories of cutaneous, mucocutaneous and visceral Leishmaniasis, is caused by intracellular protozoan parasite that belongs to the genus Leishmania Resistance and susceptibility to Leishmanfa is associated with the appearence of parasite-specific CD4+T helper 1 (Th 1) cells and T helper 2 (Th 2) cells, respectively. Leishmania homologue of receptors for activated C kinase (LACK) has been ldentifled as the target antigen for V o 8 V 8 4 T cell receptor expressing CD4’ Th 1 cells of BALB/c mice infected with Lmajor. Macrophages infected with Leishmania promastlgotes are able to present LACK antigen at initial phase of infection. inferring that LACK has an important role in protective immune response at initial phase of Leishmania infection, vaccination of L4CK antigen may have protective effect. In order to construct a recombinant vaccine which expresses LACK protein, we cloned LACK cDNA from Ldonovani, which causes a visceral Leishmaniasis, and determlned the nucleotides sequence. Total RNA was exuacted from Ldonovani (MHOM/CN/86/Sc-6) promastigotes. RT-PCR was carried out with the total RNA, using primers designed from known Lmajor LACK nucieotide sequence. A single band of the expected molecular size was amplified and the amplified cDNA was cloned into EcoRV site of pBiuescript KS plasmid vector. The nucieotide sequence of the clone was detemined and compared with those of LACK cDNA from Lmajor causing cutaneous Leishmaniasis and Lchagasi causing visceral Leishmaniasis. The nucleotlde sequence of Ldonovani LACK cDNA in the protein coding r&on showed 98.1% homology with Lmajor LACK and 99.9% with Lchagasi LACK. These findings demonstrated that LACK sequence 1s highly conserved among Leishmania species.
GENETIC VARIABILITY WITHIN L(V) BRAZILIENSIS (VENEZUELAN STRAINS) ISOLATED FROM PATIENTS WITH CUTANEOUS LEISHMANIASIS. &idauez. N: Barrios, M.A; De Lima, H; De CiugJielmo. 2; Roddguez, A and Barker, D.C. InstiM de Biomedicina - Universidad Central de Venezuela C-6 - Venezuela. 2.- University of Cambridge - Dep of Pathology Moltew Laboratories U.K Parasites of Leishmania brazilienas complex are the main causative agent of the New World cuuaeous and muco-cutaneous leisbnuiasis. Di5erem studies based on biological and molecular data have suggested that Leishmrmia (t? brazrhensis conbtitute a relatively bomogemus group of parasites, compared with other species, especially those of L. nexicona complex. Taxonomic studies in New World Leishmania, have revealed endiversity at both organismal and molecular levels, especially for the subgenus I’iannia (GrimaIdi Jr and Tesh 1993; Lainson et al. 1994). A number of methods have been developed and applied to the question of molecular diversity and relationship within Leii. In fhis work, we use a L (V) braziiiensis specific sequence probe (LbJ38), (Rcdrigucz et al, 1997). and pulse field gel electrophoresis 10 demonstrate genetic variation among Leuhmania fi7 brazilienss isolated from cutaneous leisbmaniasis c&eats from different geographicendemic areasin Venezuela L&&mania parasites were isolated and culturing in blood agar media. Collected by centrifuging at the iog&hmic grow?h phase Nuclear DNA and Gcnomic DNA plugs were prepared according to standard protocols. Digested DNAs were southern blotted and hybridised to the npetitive sequence gobe. Hybridisation results showed three different groups of parasite according to the hybridisation pattent The genetic distance between groups varied from 0.20 10 0.43, it was great between isolates from different geographic area, than between isolates from different villages in the same area Pulse field gel electmpboresis, also showed remarkable di&rencea between isolates from the different geographic areas. An small chromosome of 250 kh was obsened over all isolates. however differences were m& at the intermcdia~es ones. Polymorphism was observed at the Gp63 locus after hybridising the separated chromosomes 10 a ($63 probe. The genetic variation observed within the human isolates will be related witb the diversity of vectors and reservoirs for Lp3 bmzibensis previously described and the differences in the ccc-epidenuologlcal conditions. This poject is funded by The World Bank contract Ven/%/OO2/016
t bO37’)
O-0381
EXPRESSION AND FUNCTION OF ZN-PROTEASE (GP63) IN LEkS%/MANlA AMAZC’NEAWS
w, McGwire BS, Lu H-G, Chen D-Q, Yadava N Dept Microbiology/Immunology, Chicago Medical School, IL, USA The surface zinc protease is a glycoprotein of -63 kDa, especially abundant in the promastigote stage and encoded by clusters of tandemly repeated genes in Leishmnia spp. Recently, functional residues of gp63 essential in catalysis and its posttranslational modifications have been examined by site-specific mutagenesis. The availability of these mutants makes it possible to further examine these functional residues, as our ongoing work, to elucidate the expression and functions of the specific copy under study. We have also started to examine the multiple heterogeneous gp63 by inhibiting their expression using anti-sense transcription. The work began by cloning a 1.9 kb gp63 gene from 1. amazonensis in correct (C) orientation and in reverse (R) orientation separately into a Leishmania-specific vector, P6.5. Virulent promastigotes of the same species were transfected with both constructs plus the controls with irrelevant genes in P6.5. Transfectants were selected for tunicamycin (TM)-resistance. With the exposure to increasing TM concentrations. gp63 protems and their proteolytic activities increase and decrease In the transfectants with gp63-C and those with gp63-R. respectively. These and control transfectants were further compared for their infection of macrophages in vitro. Prelimmary results showed that the association of the gp63-R transfectants with macrophages and their intracellular survival are significantly reduced, while those with gp63-C is similar to the controls in these phenotypes of infectivity. Work is under to verify that gp63 anti-sense transcripts are produced in the former, accounting for the decrease seen in the expression of their gp63 and the loss of their infectivity.
O-0380
Molecular Cloning of Leishmanla homoiogue of Receptors for Activated C Kinase cDNA Leishmania donovani
219
REPIACEMENT OF P36 WlTH FOREIGN GENES FOR THEtRcHRoMosoMALAN)MTRAcHRoMosoMAL MPRESSlON IN LEk%Wh’~ AhW?UNEN.SlS
Lu HG, Kawazu S-l, Ito H, Chen D-Q, Fakruddin M, Akman L, Yadava N, w Dept Microbiology/Immunology. Chicago Medical School, IL, USA All species of Leishmania examined so far constitutively express a 36 kDa cytosolic protein (~36) homologous to c-crystallin/NADPH -oxidoreductase. The gene encoding p36 (P36) is single-copy per haploid genome. Both copies of P36 are dispensable, as they are replaceable with the marker genes. The latter remain structurally and functionally stable in situ even in the absence of selective pressures. Both alleles of P36 were replaced successively by homologous recombination first with NE0 and then with HYG. Replacement of P36 in 4 different clones is site-specific at both alleles. As expected, p36 is absent in double knockouts of P36 (DKOs) and reduced to half of the wildtype in the single knockouts (SKOs). DKOs emerged after exposure of SKOs to increasing concentrations of the relevant drugs. suggestive of inter-allelic recombination or unequal sister chromatid segregation during mitosis. The same conditions also increase the levels of immunologically detectable NE0 products, independent of gene amplification. All P36 null mutants grow as promastigotes or as axenic amastigotes equally well as their parental wildtype. Their infectivity to macrophages in vitro and to hamsters in viva are also comparable. DKOs recovered from infected macrophages or from cell-free cultures in the absence of drugs retain their drugresistance phenotypes, indicative of NE0 and HYG integrii and functionality. The chromosomal sites of P36 are thus expected to provide suitable locations lor stable expression of foreign genes in Leishmania. These sites have already proved useful to accommodate foreign genes for their extrachromosomal expression in Leisbmania. One vector developed is P6.5 where the gene, encoding N-acetylglucosamine-l-phosphate transferase, 2.3 kb downstream of P36 is used as the seletive marker for tunicamycin-resistance. Foreign genes cloned in P6.5 for expression in Leishmania include those from other protozoa, helminths and mammals. Transfectants with P6,5/porphobilinogen deaminase cDNA produced highly enzymatically active products.
O-0382 of
Takeuchi fl, Mataumoto Y’, Nakamura Y”, Matsumoto Y”, Otsuka H’ ‘LaboratoIy of Global Animmal Resource Science and “Department of Molecular Immunology. The University of Tokyo, Tokyo, Japan Leishmaniasis, which is classified into three main categories of cutaneous, mucocutaneous and visceral Leishmaniasis, is caused by intracellular protozoan parasite that belongs to the genus Leishmania Resistance and susceptibility to Leishmanfa is associated with the appearence of parasite-specific CD4+T helper 1 (Th 1) cells and T helper 2 (Th 2) cells, respectively. Leishmania homologue of receptors for activated C kinase (LACK) has been ldentifled as the target antigen for V o 8 V 8 4 T cell receptor expressing CD4’ Th 1 cells of BALB/c mice infected with Lmajor. Macrophages infected with Leishmania promastlgotes are able to present LACK antigen at initial phase of infection. inferring that LACK has an important role in protective immune response at initial phase of Leishmania infection, vaccination of L4CK antigen may have protective effect. In order to construct a recombinant vaccine which expresses LACK protein, we cloned LACK cDNA from Ldonovani, which causes a visceral Leishmaniasis, and determlned the nucleotides sequence. Total RNA was exuacted from Ldonovani (MHOM/CN/86/Sc-6) promastigotes. RT-PCR was carried out with the total RNA, using primers designed from known Lmajor LACK nucieotide sequence. A single band of the expected molecular size was amplified and the amplified cDNA was cloned into EcoRV site of pBiuescript KS plasmid vector. The nucieotide sequence of the clone was detemined and compared with those of LACK cDNA from Lmajor causing cutaneous Leishmaniasis and Lchagasi causing visceral Leishmaniasis. The nucleotlde sequence of Ldonovani LACK cDNA in the protein coding r&on showed 98.1% homology with Lmajor LACK and 99.9% with Lchagasi LACK. These findings demonstrated that LACK sequence 1s highly conserved among Leishmania species.
GENETIC VARIABILITY WITHIN L(V) BRAZILIENSIS (VENEZUELAN STRAINS) ISOLATED FROM PATIENTS WITH CUTANEOUS LEISHMANIASIS. &idauez. N: Barrios, M.A; De Lima, H; De CiugJielmo. 2; Roddguez, A and Barker, D.C. InstiM de Biomedicina - Universidad Central de Venezuela C-6 - Venezuela. 2.- University of Cambridge - Dep of Pathology Moltew Laboratories U.K Parasites of Leishmania brazilienas complex are the main causative agent of the New World cuuaeous and muco-cutaneous leisbnuiasis. Di5erem studies based on biological and molecular data have suggested that Leishmrmia (t? brazrhensis conbtitute a relatively bomogemus group of parasites, compared with other species, especially those of L. nexicona complex. Taxonomic studies in New World Leishmania, have revealed endiversity at both organismal and molecular levels, especially for the subgenus I’iannia (GrimaIdi Jr and Tesh 1993; Lainson et al. 1994). A number of methods have been developed and applied to the question of molecular diversity and relationship within Leii. In fhis work, we use a L (V) braziiiensis specific sequence probe (LbJ38), (Rcdrigucz et al, 1997). and pulse field gel electrophoresis 10 demonstrate genetic variation among Leuhmania fi7 brazilienss isolated from cutaneous leisbmaniasis c&eats from different geographicendemic areasin Venezuela L&&mania parasites were isolated and culturing in blood agar media. Collected by centrifuging at the iog&hmic grow?h phase Nuclear DNA and Gcnomic DNA plugs were prepared according to standard protocols. Digested DNAs were southern blotted and hybridised to the npetitive sequence gobe. Hybridisation results showed three different groups of parasite according to the hybridisation pattent The genetic distance between groups varied from 0.20 10 0.43, it was great between isolates from different geographic area, than between isolates from different villages in the same area Pulse field gel electmpboresis, also showed remarkable di&rencea between isolates from the different geographic areas. An small chromosome of 250 kh was obsened over all isolates. however differences were m& at the intermcdia~es ones. Polymorphism was observed at the Gp63 locus after hybridising the separated chromosomes 10 a ($63 probe. The genetic variation observed within the human isolates will be related witb the diversity of vectors and reservoirs for Lp3 bmzibensis previously described and the differences in the ccc-epidenuologlcal conditions. This poject is funded by The World Bank contract Ven/%/OO2/016