Inactivation of the Leishmania tarentolae Pterin Transporter (BT1) and Reductase (PTR1) Genes Leads to Viable Parasites with Changes in Folate Metabolism and Hypersensitivity to the Antifolate Methotrexate

The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The null mutant is to in a we to in cells. to a folate and cells with biopterin folate are of The cells have the gene the we a in polyglutamylation in cells. These the of Leishmania cells to the of The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The null mutant is to in a we to in cells. to a folate and cells with biopterin folate are of The cells have the gene the we a in polyglutamylation in cells. These the of Leishmania cells to the of The protozoan parasite Leishmania is a of with a of is the to the the of the is the The of is Leishmania from in from and from in the of the and leading to in pterin and folate of Leishmania in a pterin (BT1) and a of folate folate folylpolyglutamate folate folylpolyglutamate the of folate and pterin These be the parasite synthetase and a pterin reductase PTR1 Leishmania have folylpolyglutamate synthetase Leishmania to are are in the of are growth of Leishmania and a of the Leishmania to and to in the of the been to pterin with parasite cells the methotrexate the of the be gene of of the folate transporter genes Leishmania is a folate this gene to be and we have in Leishmania tarentolae in the main folate transporter is deleted, the biopterin transporter is The BT1 gene been in a of and cells in are in pterin in parasite The PTR1 gene been in a of and PTR1 null to a to and in to be of the of are folate and pterin and Leishmania in are been have a a in the parasite the this is is In to of pterin in we in generating in both the main pterin transporter (BT1) and the pterin reductase (PTR1) of folate was BT1 to in the transporter in tarentolae we of in a null mutant folate of the cells to be tarentolae and the null in with and The null mutant was in a to to and to cells in with was and following The PTR1, and tarentolae with and inactivation was a The PTR1 was in the BT1 null mutant in the BT1 the gene was with one PTR1 of the PTR1 was of the to and of the null characterized the PTR1, and in activity of Leishmania cells was and of to the of of from the the was a of and and from and cells of and in the of of was at and the cells in and in and The of was with Leishmania and of in cells from and of from The in Leishmania cells and and the of polyglutamylation was the same with of a BT1 and PTR1 of tarentolae cells to the biopterin transporter BT1 gene BT1 is a biopterin transporter we to from a BT1 null in we of PTR1 to a null mutant in the a be folate cells in PTR1 in BT1 have been null The tarentolae BT1 null mutant is in The BT1 gene was the and a BT1 null mutant was of the of BT1 is of a this in the BT1 null mutant and is of and with the of the in the BT1 gene and In this BT1 null we a to the PTR1 gene of the BT1 null mutant with this to the at the level of one PTR1 the of the and of the we to of a PTR1 null mutant in a BT1 null mutant the PTR1 is of this to in the null mutant and with a to the of in Leishmania we we a tarentolae null the mutant growth in the and a growth in the folate tarentolae cells in PTR1 BT1 to be to cells with PTR1 and BT1 to be to the BT1 null mutant was to the PTR1 null mutant was a The null mutant was to be to with cells in The of PTR1 and BT1 in gene PTR1 to BT1 when in The of Leishmania cells is the folate of the and the of cells to is in with in and the null mutant was to with of in PTR1 to BT1 cells to with cells in the this in and be to a in folate when BT1 in with the of to in the properties of Leishmania cells. The growth of tarentolae of the null of the null mutant level methotrexate and in in of cells in of and at at was to The of is of the tarentolae null Leishmania cells in and and growth was at the of the and cells with genes in PTR1 was the PTR1 in BT1 was the BT1 cells in both BT1 and PTR1 the cells from the and the of cells in PTR1 BT1 of was The of a of is of the BT1 and PTR1 of the of in be Leishmania cells in the of PTR1 and in to the The mutant growth and cells have been a is are in this mutant to the of BT1 and from a BT1 null is biopterin in the null mutant In tarentolae cells in folate is we have increased of biopterin and are folate and pterin we folate was in the null we both folate and in the null mutant and of folate was in Leishmania cells folate in are is in the of in tarentolae the both of folate and of increased in the null mutant with cells and The of the null mutant to the of with of in Leishmania cells. The in the tarentolae null mutant and null mutant cells methotrexate and and Leishmania cells in and was The was with Leishmania and in cells was The of one in is been with in tarentolae and of are The and of are in a of methotrexate in tarentolae and of are The and of are in a The in folate and polyglutamylation in the null mutant with cells to the of genes was genes are in folate and we a of this gene of folate transporter genes was in the null mutant a in cells was in the mutant and The of was in the and null mutant and activity was in the null mutant with cells of genes in Leishmania cells. with the and and and with to and with the the and from the synthetase activity in Leishmania cells. The activity was from of Leishmania cells in and and The of are of in the the null mutant cells to we in generating in the of succeeded in generating and These a growth with mutant was in a in folate and and with folate Leishmania cells a biopterin transporter and with a folate the and and Leishmania cells gene and this be a of be in the and the gene the of a in tarentolae The of the mutant was in mutant to a and gene in the folate transporter gene The gene in the mutant the of The of was in the with the with a in and and in and The of in the was with a from and in the to the of in and a of with in in polyglutamylation is with a in activity are growth Leishmania and have been to be in parasite cells main biopterin transporter and main pterin reductase are viable the The pterin of the null mutant be the of to a pterin the a in of to Leishmania the of Leishmania to have of the and in the of one of to biopterin to the cells to In we have of the and this to a folate in the null mutant The transporter gene in a null mutant is a be one of the gene and the of to the of a to the of The to the main folate be to to this in we to of the in a and is in is biopterin the biopterin of the is a been to in Leishmania PTR1 is in the same the of is Leishmania a of and this activity been in a parasite to reductase have been characterized In to the null mutant increased activity to in the level of increased polyglutamylation to increased of and the of folate from gene of this is gene in a protozoan parasite to in the of in gene is and this be when at a phenotype following a gene to of of increased are in the same the gene study, is the of gene be at the level the are following gene the null mutant growth a phenotype of to to of the of the of PTR1 and PTR1 is BT1 was of BT1 been in a of Leishmania and In to and we BT1 to in BT1 was in in BT1 folate in cells in the folate folate and we the in BT1 is to a in folate the to be the in folate at folate should folate should with null to to activity of the transporter in tarentolae is cells BT1 the activity of the folate was the a of both folate and was in and These have a growth one have when both biopterin and folate are Leishmania is to folate and this at least in the of cells is to in of the gene of the gene been in in Leishmania tarentolae when the PTR1 gene is both the of the gene and the of the the with a be in Leishmania Leishmania are In the of is with in and in tarentolae The the is to a in activity be to is in in tarentolae the inactivation of BT1 and PTR1, cells have increased activity was folate was in the In the null we in the polyglutamylation of is a in cells in and inactivation of a of in tarentolae to The of in is to a in activity and is to increased activity of the of be to to is The level of polyglutamylation in cells is the of and a in a mutant of be from cells. is from the Leishmania the of a and the level of been in Leishmania. the level of cells is the the to the However, in we in this the gene was be to Leishmania to the of the of this study, we have succeeded in generating a tarentolae null These the are of folate is The the parasite to gene inactivation of when one the phenotype of a the null mutant to we to the cells with folate and pterin and to the of a of and pterin a of with cells to The protozoan parasite Leishmania is a of with a of is the to the the of the is the The of is Leishmania from in from and from in the of the and leading to in pterin and folate of Leishmania in a pterin (BT1) and a of folate folate folylpolyglutamate folate folylpolyglutamate the of folate and pterin These be the parasite synthetase and a pterin reductase PTR1 Leishmania have folylpolyglutamate synthetase Leishmania to are are in the of are growth of Leishmania and a of the Leishmania to and to in the of the been to pterin with parasite Leishmania cells the methotrexate the of the be gene of of the folate transporter genes Leishmania is a folate this gene to be and we have in Leishmania tarentolae in the main folate transporter is deleted, the biopterin transporter is The BT1 gene been in a of and cells in are in pterin in parasite The PTR1 gene been in a of and PTR1 null to a to and in to be of the of are folate and pterin and Leishmania in are been have a a in the parasite the this is is In to of pterin in we in generating in both the main pterin transporter (BT1) and the pterin reductase (PTR1) of folate was BT1 to in the transporter in tarentolae we of in a null mutant folate of the cells to be tarentolae and the null in with and The null mutant was in a to to and to cells in with was and following The PTR1, and tarentolae with and inactivation was a The PTR1 was in the BT1 null mutant in the BT1 the gene was with one PTR1 of the PTR1 was of the to and of the null characterized the PTR1, and in activity of Leishmania cells was and of to the of of from the the was a of and and from and cells of and in the of of was at and the cells in and in and The of was with Leishmania and of in cells from and of from The in Leishmania cells and and the of polyglutamylation was the same with tarentolae and the null in with and The null mutant was in a to to and to cells in with was and following The PTR1, and tarentolae with and inactivation was a The PTR1 was in the BT1 null mutant in the BT1 the gene was with one PTR1 of the PTR1 was of the to and of the null characterized the PTR1, and in activity of Leishmania cells was and of to the of of from the the was a of and and from and cells of and in the of of was at and the cells in and in and The of was with Leishmania and of in cells from and of from The in Leishmania cells and and the of polyglutamylation was the same with of a BT1 and PTR1 of tarentolae cells to the biopterin transporter BT1 gene BT1 is a biopterin transporter we to from a BT1 null in we of PTR1 to a null mutant in the a be folate cells in PTR1 in BT1 have been null The tarentolae BT1 null mutant is in The BT1 gene was the and a BT1 null mutant was of the of BT1 is of a this in the BT1 null mutant and is of and with the of the in the BT1 gene and In this BT1 null we a to the PTR1 gene of the BT1 null mutant with this to the at the level of one PTR1 the of the and of the we to of a PTR1 null mutant in a BT1 null mutant the PTR1 is of this to in the null mutant and with a to the of in Leishmania we we a tarentolae null the mutant growth in the and a growth in the folate tarentolae cells in PTR1 BT1 to be to cells with PTR1 and BT1 to be to the BT1 null mutant was to the PTR1 null mutant was a The null mutant was to be to with cells in The of PTR1 and BT1 in gene PTR1 to BT1 when in The of Leishmania cells is the folate of the and the of cells to is in with in and the null mutant was to with of in PTR1 to BT1 cells to with cells in the this in and be to a in folate when BT1 in with the of to in the of the tarentolae null Leishmania cells in and and growth was at the of the and cells with genes in PTR1 was the PTR1 in BT1 was the BT1 cells in both BT1 and PTR1 the cells from the and the of cells in PTR1 BT1 of was The of a of is of the BT1 and PTR1 of the of in be Leishmania cells in the of PTR1 and in to the The mutant growth and cells have been a is are in this mutant to the of BT1 and from a BT1 null is biopterin in the null mutant In tarentolae cells in folate is we have increased of biopterin and are folate and pterin we folate was in the null we both folate and in the null mutant and of folate was in Leishmania cells folate in are is in the of in tarentolae the both of folate and of increased in the null mutant with cells and The of the null mutant to the of with of in Leishmania cells. The in the tarentolae null mutant and null mutant cells methotrexate and and Leishmania cells in and was The was with Leishmania and in cells was The of one in is been with in tarentolae and of are The and of are in a of methotrexate in tarentolae and of are The and of are in a The in folate and polyglutamylation in the null mutant with cells to the of genes was genes are in folate and we a of this gene of folate transporter genes was in the null mutant a in cells was in the mutant and The of was in the and null mutant and activity was in the null mutant with cells of genes in Leishmania cells. with the and and and with to and with the the and from the synthetase activity in Leishmania cells. The activity was from of Leishmania cells in and and The of are of in the the null mutant cells to we in generating in the of succeeded in generating and These a growth with mutant was in a in folate and and with folate Leishmania cells a biopterin transporter and with a folate the and and Leishmania cells gene and this be a of be in the and the gene the of a in tarentolae The of the mutant was in mutant to a and gene in the folate transporter gene The gene in the mutant the of The of was in the with the with a in and and in and The of in the was with a from and in the to the of in and a of with in in polyglutamylation is with a in activity of a BT1 and PTR1 of tarentolae cells to the biopterin transporter BT1 gene BT1 is a biopterin transporter we to from a BT1 null in we of PTR1 to a null mutant in the a be folate Leishmania cells in PTR1 in BT1 have been null The tarentolae BT1 null mutant is in The BT1 gene was the and a BT1 null mutant was of the of BT1 is of a this in the BT1 null mutant and is of and with the of the in the BT1 gene and In this BT1 null we a to the PTR1 gene of the BT1 null mutant with this to the at the level of one PTR1 the of the and of the we to of a PTR1 null mutant in a BT1 null mutant the PTR1 is of this to in the null mutant and with a to the of in Leishmania we we a tarentolae null the mutant growth in the and a growth in the folate tarentolae cells in PTR1 BT1 to be to cells with PTR1 and BT1 to be to the BT1 null mutant was to the PTR1 null mutant was a The null mutant was to be to with cells in The of PTR1 and BT1 in gene PTR1 to BT1 when in The of Leishmania cells is the folate of the and the of cells to is in with in and the null mutant was to with of in PTR1 to BT1 cells to with cells in the this in and be to a in folate when BT1 in with the of to in the of the BT1 and PTR1 of the of in be Leishmania cells in the of PTR1 and in to the The mutant growth and cells have been a is are in this mutant to the of BT1 and from a BT1 null is biopterin in the null mutant In tarentolae cells in folate is we have increased of biopterin and are folate and pterin we folate was in the null we both folate and in the null mutant and of folate was in Leishmania cells folate in are is in the of in tarentolae the both of folate and of increased in the null mutant with cells and The of the null mutant to the of with The in folate and polyglutamylation in the null mutant with cells to the of genes was genes are in folate and we a of this gene of folate transporter genes was in the null mutant a in cells was in the mutant and The of was in the and null mutant and activity was in the null mutant with cells of in the the null mutant cells to we in generating in the of succeeded in generating and These a growth with mutant was in a in folate and and with folate Leishmania cells a biopterin transporter and with a folate the and and Leishmania cells gene and this be a of be in the and the gene the of a in tarentolae The of the mutant was in mutant to a and gene in the folate transporter gene The gene in the mutant the of The of was in the with the with a in and and in and The of in the was with a from and in the to the of in and a of with in in polyglutamylation is with a in activity are growth Leishmania and have been to be in parasite cells main biopterin transporter and main pterin reductase are viable the The pterin of the null mutant be the of to a pterin the a in of to Leishmania the of Leishmania to have of the and in the of one of to biopterin to the cells to In we have of the and this to a folate in the null mutant The transporter gene in a null mutant is a be one of the gene and the of to the of a to the of The to the main folate be to to this in we to of the in a and is in is biopterin the biopterin of the is a been to in Leishmania PTR1 is in the same the of is Leishmania a of and this activity been in a parasite to reductase have been characterized In to the null mutant increased activity to in the level of increased polyglutamylation to increased of and the of folate from gene of this is gene in a protozoan parasite to in the of in gene is and this be when at a phenotype following a gene to of of increased are in the same the gene study, is the of gene be at the level the are following gene the null mutant growth a phenotype of to to of the of the of PTR1 and PTR1 is BT1 was of BT1 been in a of Leishmania and In to and we BT1 to in BT1 was in in BT1 folate in cells in the folate folate and we the in BT1 is to a in folate the to be the in folate at folate should folate should with null to to activity of the transporter in tarentolae is cells BT1 the activity of the folate was the a of both folate and was in and These have a growth one have when both biopterin and folate are Leishmania is to folate and this at least in the of cells is to in of the gene of the gene been in in Leishmania tarentolae when the PTR1 gene is both the of the gene and the of the the with a be in Leishmania Leishmania are In the of is with in and in tarentolae The the is to a in activity be to is in in tarentolae the inactivation of BT1 and PTR1, cells have increased activity was folate was in the In the null we in the polyglutamylation of is a in cells in and inactivation of a of in tarentolae to The of in is to a in activity and is to increased activity of the of be to to is The level of polyglutamylation in cells is the of and a in a mutant of be from cells. is from the Leishmania the of a and the level of been in Leishmania. the level of cells is the the to the However, in we in this the gene was be to Leishmania to the of the of this study, we have succeeded in generating a tarentolae null These the are of folate is The the parasite to gene inactivation of when one the phenotype of a the null mutant to we to the cells with folate and pterin and to the of a of and pterin a of with cells to are growth Leishmania and have been to be in parasite cells main biopterin transporter and main pterin reductase are viable the The pterin of the null mutant be the of to a pterin the a in of to Leishmania the of Leishmania to have of the and in the of one of to biopterin to the cells to In we have of the and this to a folate in the null mutant The transporter gene in a null mutant is a be one of the gene and the of to the of a to the of The to the main folate be to to this in we to of the in a and is in is biopterin the biopterin of the is a been to in Leishmania PTR1 is in the same the of is Leishmania a of and this activity been in a parasite to reductase have been characterized In to the null mutant increased activity to in the level of increased polyglutamylation to increased of and the of folate from gene of this is gene in a protozoan parasite to in the of in gene is and this be when at a phenotype following a gene to of of increased are in the same the gene study, is the of gene be at the level the are following gene the null mutant growth a phenotype of to to of the of the of PTR1 and PTR1 is BT1 was of BT1 been in a of Leishmania and In to and we BT1 to in BT1 was in in BT1 folate in cells in the folate folate and we the in BT1 is to a in folate the to be the in folate at folate should folate should with null to to activity of the transporter in tarentolae is cells BT1 the activity of the folate was the a of both folate and was in and These have a growth one have when both biopterin and folate are Leishmania is to folate and this at least in the of cells is to in of the gene of the gene been in in Leishmania tarentolae when the PTR1 gene is both the of the gene and the of the the with a be in Leishmania In Leishmania are In the of is with in and in tarentolae The the is to a in activity be to is in in tarentolae the inactivation of BT1 and PTR1, cells have increased activity was folate was in the In the null we in the polyglutamylation of is a in cells in and inactivation of a of in tarentolae to The of in is to a in activity and is to increased activity of the of be to to is The level of polyglutamylation in cells is the of and a in a mutant of be from cells. is from the Leishmania the of a and the level of been in Leishmania. the level of cells is the the to the However, in we in this the gene was be to Leishmania to the of the of In this study, we have succeeded in generating a tarentolae null These the are of folate is The the parasite to gene inactivation of when one the phenotype of a the null mutant to we to the cells with folate and pterin and to the of a of and pterin a of with cells to the of the of the

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