The Role of Plasmodium falciparum Food Vacuole Plasmepsins

Abstract

Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions. Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions. Malaria kills 1–2 million people every year, most of whom are children under the age of 5. The economic burden caused by malaria in endemic countries is large, estimated to be approximately 1–2% of the gross domestic product in these countries (1World Health Organization (2004) WHO Fact Sheet No. 94—Malaria, GenevaGoogle all malarial are caused by Plasmodium a parasite in in P. falciparum is an and antimalarial are In the to for the study of hemoglobin degradation in the food vacuole of the parasite has generated substantial interest because is a of the parasite and is to be for the of the parasite of and of P. falciparum aspartic plasmepsins has been to be important for the of hemoglobin degradation in and in and is in plasmepsin inhibitors II for the be and the is has been the of drug design the in of generated has been The malaria has are plasmepsins in the P. falciparum Four of PM I and II, protease as PM and PM reside in the food vacuole and hemoglobin The four plasmepsins are homologous to each than amino HAP is in has a in of the aspartic is an protease may function by an aspartic protease P. of the of the of the food vacuole plasmepsins in has been and of these genes is to the parasite has been and to a plasmepsin be as the drug all four plasmepsin results suggest the four plasmepsins have overlapping in the hemoglobin degradation and is a for P. falciparum to the food vacuole plasmepsin genes in and P. falciparum clone 3D7 cultured in under and as in rich with and by for under drug an amino acid-limited by all amino and rich Parasite by by with of maintained in for by the of to the medium. in a of to The by and by and of parental The and to the are in All 3D7 by with the and the of The of all for to as for as for as for as for as for in a clones each knock-out each of the for the PM I clones and for the PM II clones and for the HAP clones and and for the PM IV/I clones and 3D7 as a for each The an for and clones and an for and a generated an and with the and All a by the of with with the with for PM for and for In the of an with PM and and and in and and generated as the by with in for by a Parasite by in The by and to a for for for and and for All to The with and In parasite in in and The with in for and with The in and an in for each maintained and by approximately every a of and by The of and to the growth is the parasite doubling and is the parasite the growth of with the of the parasite to have a parasite for each and is in and of knock-out clones in amino acid-limited medium. in amino acid-limited and by and for clones and clones and clones and and clones and and are as in of each clone with and by and in in a the and by the of of The of and of each by The to and the of in time. The to an function is the of in the is the is the of the function in is to by the and are are to the of the In all the of the than of for and as of with of in rich for of to the and the for in and in a The of the of the drug and the the in employed to the of the doubling times and cell cycle times of the knockouts as with The each are in of knock-out with is for all in the are cycle is for all in the are the of acid-limited is for all in the are The is for all in the The are the of cycle in a as of I, II, and HAP are in the plasmepsin a employed of homologous the and of each gene 3D7 and the to the The of each plasmepsin gene for the of the be double crossover with and with to double crossover recombination gene disruption of plasmepsin I, II, and is of plasmepsin I, II, and HAP gene The four genes in in the gene is approximately and the genes are is to of gene disruptions by is are to to the and for each of clones of and 3D7 as and are under each the of the to with individual disruptions of PM I and II and This by and by clones each corresponding to the a in of the with parental 3D7 as the gene as the in the 3D7 and of the for recombination for the clones to PM with an a double knock-out with homologous to the PM and the PM gene is for the PM IV/I and of with disruption of PM and PM I The PM the in the 3D7 and the in the to a of the in the the disruption of each gene by and of in each for each a and for an In with the in the clone of All the corresponding plasmepsin in the 3D7 the of plasmepsins of for of the as by with plasmepsin and of knock-out parasite by I II and I and with as a The in with knock-out clones morphologically normal. parental and knock-out growth parasite by clones each knock-out line and maintained 3D7 in rich and approximately every for The and with the and by the of clones approximately of 3D7 and approximately of 3D7 for the clones The growth of the and clones to 3D7 and these to an growth and doubling times Compared with 3D7, the doubling and doubling longer the the slow growth of and caused by an cell the growth of for with of by and the of the the and an function of the cell cycle times 3D7 and of the knockouts results by the of of cycle of knock-out in rich for and every to The parasite by and the of the time. maintained 3D7 and and the clones for each and and and and and and and and and the of The to an and the are to and to in amino acid-limited to parasite This the amino are in hemoglobin and the parasite to hemoglobin degradation to amino to growth growth to in rich All in amino acid-limited medium, with of the growth in rich The slow growth phenotype of the clones the most approximately of the 3D7 level in and the growth of and clones of the 3D7 The doubling times and are in times for and clones longer than for 3D7, for clones all of to and aspartic and cysteine protease in hemoglobin degradation in the food to the of knockouts to aspartic and cysteine protease inhibitors The of and for each clone and are as the of to 3D7 All knock-out clones to to the clones The of the protease inhibitors the aspartic and cysteine to the of the a substantial of by overlapping of of and The of each knock-out clone to 3D7 for for the of all Plasmodium and P. falciparum has been to have food vacuole plasmepsins gene disruption study the is a for P. falciparum in all four food vacuole plasmepsin genes The growth of PM II and IV/I knock-out is than of 3D7 in rich medium, and the growth of all four knockouts is in amino acid-limited RPMI approximately amino than the may have a phenotype is important for in a growth has a substantial parasite This a for why P. falciparum may have a growth plasmepsin gene and why has maintained four overlapping genes in plasmepsins a growth The of each plasmepsin is P. P. P. and of plasmepsins have been to be in hemoglobin in is the plasmepsins have in hemoglobin degradation doubling of 3D7 in amino acid-limited is approximately longer than in rich medium. the amino for are to most amino hemoglobin as an amino to 3D7 in than in rich This degradation of hemoglobin for as an amino is important for parasite growth and plasmepsins are important for hemoglobin the plasmepsin knockouts slower the is is productive progeny cycle for with cycle for 3D7 the of decreased in the cell be the slow growth all knock-out clones to be in and morphologically in growth This is a substantial of The be caused by an by overlapping of genes The food vacuole cysteine protease has been by and is a in the food vacuole of the knock-out is growth the of cysteine protease inhibitors as in the the of the aspartic protease decreased appears the of be by an is the for and changed in the plasmepsin for plasmepsin the overlapping of the plasmepsin of plasmepsins in the and the of plasmepsins in the P. falciparum may lack the for a II has been the of most plasmepsin antimalarial drug study with the of of the four food vacuole plasmepsins are the study under in to plasmepsin the to P. falciparum the is the substantial redundancy in the food vacuole plasmepsin the of a plasmepsin is to in an with is to a with and of plasmepsins be to plasmepsin be to inhibitors to the to appears a a the the of all the of the PM IV/I double disruption in study is the of a double in Plasmodium and is the disruption of genes in Plasmodium with a is why to a PM PM I is PM PM as a for PM I, and in PM be to the to the of the knock-out knock-out to a the gene is a for the PM IV/I is a the of the are for Plasmodium and the gene in in the Plasmodium the double knock-out employed in study be to study Plasmodium gene function in the Malaria kills 1–2 million people every year, most of whom are children under the age of 5. The economic burden caused by malaria in endemic countries is large, estimated to be approximately 1–2% of the gross domestic product in these countries (1World Health Organization (2004) WHO Fact Sheet No. 94—Malaria, GenevaGoogle all malarial are caused by Plasmodium a parasite in in P. falciparum is an and antimalarial are In the to for the study of hemoglobin degradation in the food vacuole of the parasite has generated substantial interest because is a of the parasite and is to be for the of the parasite of and of P. falciparum aspartic plasmepsins has been to be important for the of hemoglobin degradation in and in and is in plasmepsin inhibitors II for the be and the is has been the of drug design the in of generated has been The malaria has are plasmepsins in the P. falciparum Four of PM I and II, protease as PM and PM reside in the food vacuole and hemoglobin The four plasmepsins are homologous to each than amino HAP is in has a in of the aspartic is an protease may function by an aspartic protease P. of the of the of the food vacuole plasmepsins in has been and of these genes is to the parasite has been and to a plasmepsin be as the drug all four plasmepsin results suggest the four plasmepsins have overlapping in the hemoglobin degradation and is a for P. falciparum to the food vacuole plasmepsin genes in and P. falciparum clone 3D7 cultured in under and as in rich with and by for under drug an amino acid-limited by all amino and rich Parasite by by with of maintained in for by the of to the medium. in a of to The by and by and of parental The and to the are in All 3D7 by with the and the of The of all for to as for as for as for as for as for in a clones each knock-out each of the for the PM I clones and for the PM II clones and for the HAP clones and and for the PM IV/I clones and 3D7 as a for each The an for and clones and an for and a generated an and with the and All a by the of with with the with for PM for and for In the of an with PM and and and in and and generated as the by with in for by a Parasite by in The by and to a for for for and and for All to The with and In parasite in in and The with in for and with The in and an in for each maintained and by approximately every a of and by The of and to the growth is the parasite doubling and is the parasite the growth of with the of the parasite to have a parasite for each and is in and of each clone with and by and in in a the and by the of of The of and of each by The to and the of in time. The to an function is the of in the is the is the of the function in is to by the and are are to the of the In all the of the than of for and as of with of in rich for of to the and the for in and in a The of the of the drug and the the in employed to the of the doubling times and cell cycle times of the knockouts as with The each are in of knock-out with is for all in the are cycle is for all in the are the of acid-limited is for all in the are The is for all in the The are the of cycle in a as Parasite and P. falciparum clone 3D7 cultured in under and as in rich with and by for under drug an amino acid-limited by all amino and rich Parasite by by with of maintained in for by the of to the medium. in a of to The by and by and of parental The and to the are in All 3D7 by with the and the of The of all clones each knock-out each of the for the PM I clones and for the PM II clones and for the HAP clones and and for the PM IV/I clones and 3D7 as a for each The an for and clones and an for and a generated an and with the and All a by the of with with the with for PM for and for In the of an with PM and and and in and and generated as the by with in for by a Parasite by in The by and to a for for for and and for All to The with and In parasite in in and The with in for and with The in and an in for each Parasite maintained and by approximately every a of and by The of and to the growth is the parasite doubling and is the parasite the growth of with the of the parasite to have a parasite for each and is in and 5. Parasite of each clone with and by and in in a the and by the of of The of and of each by The to and the of in time. The to an function is the of in the is the is the of the function in is to by the and are are to the of the In all the of the than of for and as of with of in rich for of to the and the for in and in a The of the of the drug and the the in employed to the of the doubling times and cell cycle times of the knockouts as with The each are in as of I, II, and HAP are in the plasmepsin a employed of homologous the and of each gene 3D7 and the to the The of each plasmepsin gene for the of the be double crossover with and with to double crossover recombination to with individual disruptions of PM I and II and This by and by clones each corresponding to the a in of the with parental 3D7 as the gene as the in the 3D7 and of the for recombination for the clones to PM with an a double knock-out with homologous to the PM and the PM gene is for the PM IV/I and of with disruption of PM and PM I The PM the in the 3D7 and the in the to a of the in the the disruption of each gene by and of in each for each a and for an In with the in the clone of All the corresponding plasmepsin in the 3D7 the of plasmepsins of for of the as by with plasmepsin and of knock-out parasite by I II and I and with as a The in with knock-out clones morphologically normal. parental and knock-out growth parasite by clones each knock-out line and maintained 3D7 in rich and approximately every for The and with the and by the of clones approximately of 3D7 and approximately of 3D7 for the clones The growth of the and clones to 3D7 and these to an growth and doubling times Compared with 3D7, the doubling and doubling longer the the slow growth of and caused by an cell the growth of for with of by and the of the the and an function of the cell cycle times 3D7 and of the knockouts results by the of of cycle of knock-out in rich for and every to The parasite by and the of the time. maintained 3D7 and and the clones for each and and and and and and and and and the of The to an and the are to and to in amino acid-limited to parasite This the amino are in hemoglobin and the parasite to hemoglobin degradation to amino to growth growth to in rich All in amino acid-limited medium, with of the growth in rich The slow growth phenotype of the clones the most approximately of the 3D7 level in and the growth of and clones of the 3D7 The doubling times and are in times for and clones longer than for 3D7, for clones all of to and aspartic and cysteine protease in hemoglobin degradation in the food to the of knockouts to aspartic and cysteine protease inhibitors The of and for each clone and are as the of to 3D7 All knock-out clones to to the clones The of the protease inhibitors the aspartic and cysteine to the of the a substantial of by overlapping of of and The of each knock-out clone to 3D7 for for the of of I, II, and HAP are in the plasmepsin a employed of homologous the and of each gene 3D7 and the to the The of each plasmepsin gene for the of the be double crossover with and with to double crossover recombination to with individual disruptions of PM I and II and This by and by clones each corresponding to the a in of the with parental 3D7 as the gene as the in the 3D7 and of the for recombination for the clones to PM with an a double knock-out with homologous to the PM and the PM gene is for the PM IV/I and of with disruption of PM and PM I The PM the in the 3D7 and the in the to a of the in the the disruption of each gene by and of in each for each a and for an In with the in the clone of All the corresponding plasmepsin in the 3D7 the of plasmepsins of for of the as by with plasmepsin and Parasite knock-out clones morphologically normal. parental and knock-out growth parasite by clones each knock-out line and maintained 3D7 in rich and approximately every for The and with the and by the of clones approximately of 3D7 and approximately of 3D7 for the clones The growth of the and clones to 3D7 and these to an growth and doubling times Compared with 3D7, the doubling and doubling longer the the slow growth of and caused by an cell the growth of for with of by and the of the the and an function of the cell cycle times 3D7 and of the knockouts results by the of of Parasite in amino acid-limited to parasite This the amino are in hemoglobin and the parasite to hemoglobin degradation to amino to growth growth to in rich All in amino acid-limited medium, with of the growth in rich The slow growth phenotype of the clones the most approximately of the 3D7 level in and the growth of and clones of the 3D7 The doubling times and are in times for and clones longer than for 3D7, for clones all of to and aspartic and cysteine protease in hemoglobin degradation in the food to the of knockouts to aspartic and cysteine protease inhibitors The of and for each clone and are as the of to 3D7 All knock-out clones to to the clones The of the protease inhibitors the aspartic and cysteine to the of the a substantial of by overlapping of all Plasmodium and P. falciparum has been to have food vacuole plasmepsins gene disruption study the is a for P. falciparum in all four food vacuole plasmepsin genes The growth of PM II and IV/I knock-out is than of 3D7 in rich medium, and the growth of all four knockouts is in amino acid-limited RPMI approximately amino than the may have a phenotype is important for in a growth has a substantial parasite This a for why P. falciparum may have a growth plasmepsin gene and why has maintained four overlapping genes in plasmepsins a growth The of each plasmepsin is P. P. P. and of plasmepsins have been to be in hemoglobin in is the plasmepsins have in hemoglobin degradation doubling of 3D7 in amino acid-limited is approximately longer than in rich medium. the amino for are to most amino hemoglobin as an amino to 3D7 in than in rich This degradation of hemoglobin for as an amino is important for parasite growth and plasmepsins are important for hemoglobin the plasmepsin knockouts slower the is is productive progeny cycle for with cycle for 3D7 the of decreased in the cell be the slow growth all knock-out clones to be in and morphologically in growth This is a substantial of The be caused by an by overlapping of genes The food vacuole cysteine protease has been by and is a in the food vacuole of the knock-out is growth the of cysteine protease inhibitors as in the the of the aspartic protease decreased appears the of be by an is the for and changed in the plasmepsin for plasmepsin the overlapping of the plasmepsin of plasmepsins in the and the of plasmepsins in the P. falciparum may lack the for a II has been the of most plasmepsin antimalarial drug study with the of of the four food vacuole plasmepsins are the study under in to plasmepsin the to P. falciparum the is the substantial redundancy in the food vacuole plasmepsin the of a plasmepsin is to in an with is to a with and of plasmepsins be to plasmepsin be to inhibitors to the to appears a a the the of all the of the PM IV/I double disruption in study is the of a double in Plasmodium and is the disruption of genes in Plasmodium with a is why to a PM PM I is PM PM as a for PM I, and in PM be to the to the of the knock-out knock-out to a the gene is a for the PM IV/I is a the of the are for Plasmodium and the gene in in the Plasmodium the double knock-out employed in study be to study Plasmodium gene function in the all Plasmodium and P. falciparum has been to have food vacuole plasmepsins gene disruption study the is a for P. falciparum in all four food vacuole plasmepsin genes The growth of PM II and IV/I knock-out is than of 3D7 in rich medium, and the growth of all four knockouts is in amino acid-limited RPMI approximately amino than the may have a phenotype is important for in a growth has a substantial parasite This a for why P. falciparum may have a growth plasmepsin gene and why has maintained four overlapping genes in plasmepsins a growth The of each plasmepsin is P. P. P. and of plasmepsins have been to be in hemoglobin in is the plasmepsins have in hemoglobin degradation The doubling of 3D7 in amino acid-limited is approximately longer than in rich medium. the amino for are to most amino hemoglobin as an amino to 3D7 in than in rich This degradation of hemoglobin for as an amino is important for parasite growth and plasmepsins are important for hemoglobin the plasmepsin knockouts slower the is is productive progeny cycle for with cycle for 3D7 the of decreased in the cell be the slow growth all knock-out clones to be in and morphologically in growth This is a substantial of The be caused by an by overlapping of genes The food vacuole cysteine protease has been by and is a in the food vacuole of the knock-out is growth the of cysteine protease inhibitors as in the the of the aspartic protease decreased appears the of be by an is In the for and changed in the plasmepsin for plasmepsin the overlapping of the plasmepsin of plasmepsins in the and the of plasmepsins in the P. falciparum may lack the for a PM II has been the of most plasmepsin antimalarial drug study with the of of the four food vacuole plasmepsins are the study under in to plasmepsin the to P. falciparum the is the substantial redundancy in the food vacuole plasmepsin the of a plasmepsin is to in an with is to a with and of plasmepsins be to plasmepsin be to inhibitors to the to appears a a the the of all the of the PM IV/I double disruption in study is the of a double in Plasmodium and is the disruption of genes in Plasmodium with a is why to a PM PM I is PM PM as a for PM I, and in PM be to the to the of the knock-out knock-out to a the gene is a for the PM IV/I is a the of the are for Plasmodium and the gene in in the Plasmodium the double knock-out employed in study be to study Plasmodium gene function in the for the for and for and for for and for