Proteome Mapping of the Protozoan Parasite Leishmania and Application to the Study of Drug Targets and Resistance Mechanisms

Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of α- and β-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be post-translationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms. Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of α- and β-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be post-translationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms. Leishmania is a protozoan parasite that can cause a species-dependent spectrum of disease, ranging from self-healing cutaneous lesions (Leishmania major) to visceral infections that are fatal if untreated (Leishmania donovani). With an estimated 12 million cases of leishmaniasis worldwide and 1.5–2 million new cases reported each year (1.Herwaldt B.L. Leishmaniasis..Lancet. 1999; 354: 1191-1199Google Scholar), this protozoan has a significant impact on human populations. Leishmania is transmitted by a sandfly vector as a motile, elongated promastigote. Upon transmission, the parasite is engulfed by a macrophage where it transforms into a round, non-motile amastigote able to spread within the host. The parasite is primarily found in South America, Asia, southern Europe, and Africa, but recent reports have confirmed the visceral form of the disease in dogs in 21 states in the United States and in Canada (2.Gaskin A.A. Schantz P. Jackson J. Birkenheuer A. Tomlinson L. Gramiccia M. Levy M. Steurer F. Kollmar E. Hegarty B.C. Ahn A. Breitschwerdt E.B. Visceral leishmaniasis in a New York foxhound kennel.J. Vet. Intern. Med. 2002; 16: 34-44Google Scholar). In some regions, up to 80% of cases exhibit clinical drug resistance (3.Sundar S. Drug resistance in Indian visceral leishmaniasis.Trop. Med. Int. Health. 2001; 6: 849-854Google Scholar) leading to high levels of treatment failure. While the number of global proteomic expression profiling studies involving infectious disease caused by bacteria and viruses are numerous, the application of these methodologies to protozoan parasites has lagged behind. This phenomenon can be attributed to a number of causes. Protozoan parasites tend to be challenging to culture and manipulate in vitro, making it difficult to obtain enough pure material for proteomic analysis. However, Leishmania promastigotes can be cultured relatively easily, and the amastigote form of several species can be propagated in cell-free culture (for a review, see Ref. 4.Pan A.A. Duboise S.M. Eperon S. Rivas L. Hodgkinson V. Traub-Cseko Y. McMahon-Pratt D. Developmental life cycle of Leishmania—cultivation and characterization of cultured extracellular amastigotes.J. Eukaryot. Microbiol. 1993; 40: 213-223Google Scholar). In addition, methods for gene transfection, overexpression, and disruption are available for Leishmania. A more substantial impediment is the lack of genome sequences for parasites in general. There are ongoing genome projects for only 20 species of protozoan parasites as opposed to several hundred bacterial and archaeal examples (see wit.integratedgenomics.com/GOLD). Among these are two species of Leishmania. An expressed sequence tag database of Leishmania chagasi is being created, and the 34-Mb genome of the human pathogen L. major Friedlin is being completely sequenced by a consortium of laboratories (www.sanger.ac.uk/Projects/L_major/). To date, the sequences of five of the 36 chromosomes have been completed, a further 21 are nearing completion, and the entire genome is projected to be finished in 2003. It is estimated that the Leishmania genome contains 8000 genes, and while numerous post-translational modifications are known to exist in this organism, the genes in general have no introns, and there is no alternative mRNA splicing. Thus, unlike the predicted case in higher eukaryotes, the proteome of Leishmania should not contain a drastically higher number of proteins than genes. Its significance as a human pathogen has made Leishmania the focus of numerous studies in areas such as host-parasite interactions, cell differentiation, and our area of specialization, mechanisms of drug resistance. The availability of molecular methods, cell-free culture, and genomic sequence makes the application of proteomic methods to the study of these and other aspects of Leishmania biology possible and practical. Preliminary efforts in this direction include the generation of a partial two-dimensional (2D) 1The abbreviations used are: 2D, two-dimensional; DIG, digoxigenin; MTX, methotrexate; PTR1, pteridine reductase 1; TRYR, trypanothione reductase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; MS/MS, tandem MS; IPG, immobilized pH gradient. gel map for Leishmania guyanensis (5.Acestor N. Masina S. Walker J. Saravia N.G. Fasel N. Quadroni M. Establishing two-dimensional gels for the analysis of Leishmania proteomes.Proteomics. 2002; 2: 877-879Google Scholar), and in another early analysis proteins differentially expressed in Leishmania donovani infantum amastigotes versus promastigotes were pinpointed (6.El Fakhry Y. Ouellette M. A proteomic to proteins in Leishmania 2002; 2: Scholar). A proteome map of the species being sequenced is and the a framework for and mass to quantitative in L. major and other We have also these methods to the study of resistance mechanisms and novel drug targets and show that are tools for L. major L. donovani and Leishmania donovani donovani were in with and Leishmania and L. major with the trypanothione reductase gene Ouellette M. J. S. M. of the trypanothione reductase gene of Leishmania to in J. 16: Scholar) were in M. and in vitro culture forms of in a Scholar) also with and was were at were to as by at were by at in and in CHAPS, was to for at and were to was by the the were into and at In the first were on and on an as by the with modifications as in Ref. S. D. J. two-dimensional gel at pH using immobilized pH 2002; 2: for on pH were in pH of for and in for and to gels using in was in a at and the the were by was by the of aspects of for proteomic 2001; Scholar). gels were for in in and in a of in the from to were with and using a laser were using a laser and and by at were to obtain of gels were for at in were from to and in were with the at resolution using a and To the proteome gels of pH were using of TRYR overexpression, gels were with the proteins of were by by the and for mass were in and with were on a to the using sequencing from the gel into were in a and in of The was with an of 20 in and a The was to at and was with of Mass were on a mass in the were using and by for mass in a protein database generated by of L. major Friedlin sequence available the (www.sanger.ac.uk/Projects/L_major/). The used were complete of partial and mass than A of than was significant We also at protein and the of the to and molecular tandem mass were by to an mass with a An of the protein was to with and a New were from the with a of in at a of Mass were using a in which each mass spectrum was by of the most The was and the was to were using the An to tandem mass of with sequences in a protein Mass Scholar) and proteins in the for protein of and of were in the A protein was a if at two were identification of a a of and for and identification was confirmed by of the were in and on were to by the of J. of proteins from gels to and some Scholar). The was for in in A an of in Leishmania was in and for with the The was in 20 and with in The was as with and to was from Leishmania using was with to the and the fragments were on a gel using as a A was generated by and with to the The gel was to with the with fragments and and to all to the were from L. major Friedlin promastigotes with a of and gel the of gels for each pH were using and into two The first generated from gels the pH and contains spots, while the is of a pH of and contains of the number of on the pH gel was challenging to significant a with in the S. J. high two-dimensional gel using Scholar). there to be if the of and the in to resolution in these we that we have protein was to a number of these spots, to as landmark proteins and to the of by MS, the of the genome sequencing project and the that from the Leishmania genome project have been to of were and with and the were subjected to analysis. the were There was a and spectrum some by In proteins were found the α- and β-tubulins were identified in four and spots, and with and most to the α- and and and were identified in two related proteins are present in the Leishmania and while there are no reports in the of post-translational of we have identified a a of from the L. major of 20 protein no protein protein protein in a new The number of α- and species identified in our and we were that this was are known to be post-translationally for a review, see Ref. S. The 2001; Scholar), and we that these modifications for the of found in the (see and in However, several identified as but than the were also in our gels The we contains as as which has been to L. D. of of in 2002; Scholar), but we were that the number of fragments identified was the of To this in in vitro, were in and with an the of the protein that numerous fragments of were present in of the on the in mass to fragments identified on our While amounts of the fragments are present in some to increase in amount with the age of the culture as by the number and of revealed using an early for gels are in which could the number and amount of fragments We were in be able to L. donovani proteins in the of a genome sequence for this species and the L. major protein map could be used as a for gel studies of other such as the related L. We in using proteins sequences were known in L. major and L. donovani and the number of and that in The of from a high of for to a of for the with an of mass This that it be to identification of L. donovani proteins gel were We gels of pH of L. major L. major L. donovani L. donovani and L. of the protein of these that the of in protein can be to the of the species in while there are in the of protein spots, the related the of a protein map for expression studies of Leishmania in general. In at the five species were and the results were However, the of the more expressed that if were the protein could be used as While we not to in all the we that a more of L. major and L. donovani was of pH of cell of L. major and L. donovani under were using a pH we were able to these gels to our proteome map and some we of L. major proteins spots, while of L. donovani only of gel and using as being in the two of of these to be the more expressed was in the pH gels protein were from the gels and for 12 protein of were identified as the protein We not obtain for of It is possible that of this protein in and identification by In case the L. donovani protein was identified as while the L. major protein was not However, two of the major were the mass of these In cases we only relatively protein of by to high levels of It is possible that the number of mass was at the for the four no were in L. major L. but a number of were the in and that are the the identification were in of cases where were of the proteins in our was TRYR and an responsible for an is found in trypanosomatid parasites P. P. A. a novel for glutathione reductase in Scholar). It is also in of the drug from the cell and as such is an of drug resistance in Leishmania S. N. D. J. Ouellette M. Trypanothione and resistance to and in S. A. D. A. S. Ouellette M. and increased trypanothione levels in Scholar). TRYR is the of glutathione reductase in other A. and of trypanothione in the Microbiol. Scholar), and has TRYR under considerable scrutiny as a specific parasite drug A. and of trypanothione in the Microbiol. M. studies on trypanothione a for 16: of parasite as drug 1999; Scholar). We the TRYR gene in a L. major strain to see we could the protein in our gels and also to see the if of TRYR on the expression of While we see a increase in the of the we identified as TRYR, we also an increase in the of four other in the general area of the gel identification confirmed four of the five as being TRYR most material for have molecular and a in of pH This of is with proteins but the from not possible modifications for other proteins in the pH to be TRYR The primary of our into is to study mechanisms of drug resistance in Leishmania of the drug we are in such as on resistance mechanisms have increased our of and in Leishmania and have pinpointed novel potential drug targets (for a review, see Ref. M. J. A. D. and in Leishmania and related trypanosomatid J. 2002; Scholar). To proteomic L. major were generated in vitro by in of analysis of gels of and revealed a number of differentially expressed The most is an present in L. major but in the identified this protein as the pteridine reductase and sequence of which is a known primary resistance mechanism Ouellette M. A novel resistance gene on the of J. S.M. A of the reductase resistance in Scholar). drug resistance in Leishmania is by gene S.M. in Microbiol. M. A. D. of gene and of other resistance genes in Leishmania and study by gene and gene Scholar), and we the of was to this The of was such that it was possible to a gene by gels of the of and of genomic of the L. major and strain with a revealed that this gene was present on the a mechanism for the observed increase in to have as several to the we present a high resolution gel map of L. This 3700 protein spots, the most complete gel representation of a parasite proteome to date. A of this map is available our protein using L. donovani infantum (6.El Fakhry Y. Ouellette M. A proteomic to proteins in Leishmania 2002; 2: Scholar), while a recent study using gel analysis of Leishmania guyanensis (5.Acestor N. Masina S. Walker J. Saravia N.G. Fasel N. Quadroni M. Establishing two-dimensional gels for the analysis of Leishmania proteomes.Proteomics. 2002; 2: 877-879Google Scholar). In another protein from were using gels and and with gels that it should be possible to and of global protein expression by two-dimensional and mass of J. 2002; Scholar). on our this is the in genome and Leishmania and two comparative proteome of the life of were using a mass approach. In proteins were of the predicted from the genome sequence E. Y. A. N. M. of the proteome by mass 2002; Scholar), while in the proteins were L. M. N. A.A. D. Y. A.A. A proteomic of the life 2002; Scholar). In a case our proteome representation of the predicted 8000 genes in the Leishmania the of post-translational and this of the gel generated using species and of Leishmania the of these using and of the Leishmania revealed by of and gene for a of Scholar) show that L. major and L. human are while the pathogen L. is in a The of the two L. major are as is the case with the two L. donovani the L. major and the L. donovani species there is more and the relatively L. species to the human The on these be the but these results also the that some protein not found in the on gels could be as We to a expressed of such a protein and but no identification was from the mass spectrum A more is that the L. major genome be valuable in the proteomic analysis of other species of Leishmania. The of our at L. donovani protein identification the made our in analysis of L. donovani is at of the currently a at for it be that in more cases methods to where this is a more from these studies is the of being able to to using P. E. Y. A. N. M. of the proteome by mass 2002; L. M. N. A.A. D. Y. A.A. A proteomic of the life 2002; Scholar) comparative in with species of Leishmania. A recent Two-dimensional gel in but it up the 2002; 2: Scholar) the and of the two proteomic methods have numerous and are more and but gel analysis is a relatively and with in and the analysis of protein The two are and each is to novel aspects of parasite TRYR which is the major responsible for an in and replaces glutathione in these to of the TRYR gene in Leishmania have been Ouellette M. J. S. M. of the trypanothione reductase gene of Leishmania to in J. 16: J. S. that trypanothione reductase is an essential in Leishmania by of the gene Microbiol. Scholar), that the protein is essential for parasite and levels of TRYR to in Ouellette M. J. S. M. of the trypanothione reductase gene of Leishmania to in J. 16: Scholar). Trypanothione is also in the resistance mechanism to the drug of for the treatment of The of this to the and of trypanosomatid parasites and in the has to significant in the of using TRYR as a drug target. TRYR the related glutathione reductase is known to be in it is that numerous of glutathione reductase are present in In the were able to of the in by of in the of in gel Scholar). While some of these could be the of genes and these could also be with the of modifications on the The Leishmania and genome sequencing projects have identified only a TRYR gene in each organism, and in our case the in were all by the of a An analysis of the of and glutathione levels on glutathione reductase in while glutathione reductase could be increased by there was no in the amount of glutathione reductase protein of gene S. by glutathione of glutathione reductase and gene expression in L. Scholar). for by of glutathione reductase in a recent study M. J. the increased generation of species and the of in 2002; Scholar) where it was that in a of in several the increased of glutathione While more was no in protein amount was that the of the protein was being modified. a post-translational of as opposed to by for a of from is essential for the of the parasite in of TRYR amounts of the to be present in the cell in an levels to be in the within the macrophage is the could be the cell to It is also possible that is The of the glutathione reductase is in by a number of of the of glutathione reductase by Scholar), F. of glutathione reductase by 1999; Scholar), and glutathione J. Scholar). have revealed a number of possible potential of and N. S. S. and of protein 1999; F. E. A. E. The in protein 2002; Scholar). J. and S. in is and which have been to the of Y. M. Mass of from two-dimensional gels as a major in 2002; Scholar) and the of P. of protein in two-dimensional of and 2002; Scholar) and could cause to observed for the TRYR We are currently to the and the of the revealed by our using and The primary of our proteomic is to the proteins in resistance. studies could to more by that the of the parasite to of a global analysis of this is that proteins be identified that not have been the estimated of Leishmania proteins of with no S.M. S. A. E. S.M. E. S. The Leishmania genome new into gene and Microbiol. 2001; Scholar) (www.sanger.ac.uk/Projects/L_major/). of resistance new drug targets this of proteins has the that these are to the making to with proteins and than a of that has in the treatment of as as some (for a review, see Ref. M. E. resistance mechanisms from bacteria to with on Med. Scholar). The is a and the of reductase is responsible for to the form in numerous essential In this study of a mutant a protein was identified as PTR1, a primary resistance Ouellette M. A novel resistance gene on the of J. S.M. A of the reductase resistance in Scholar). is able to to at a D. L. S.M. a reductase of and resistance in the protozoan parasite Leishmania S. A. J. E. Ouellette M. and by the Leishmania Scholar), and of this can the of The gene is in this mutant and this constitutes a that gene can to protein analysis has confirmed that this mutant has increased mRNA and M. no to is in the we gene is to protein proteomic studies of bacteria have reported the expression of in P. E. L. L. A proteomic analysis of resistance in 1999; Scholar) and of reductase in of differentially proteins in by proteome 2001; Scholar), but the of these proteins in resistance are A number of protein expression studies of have also been (for a review, see Ref. P. for and the of 2001; Scholar), but the of most of the proteins identified by these studies in resistance In the protein was found to be in The of this protein was to resistance of the to several Y. Y. Y. D. is with of 2002; Scholar), but the mechanism of this is is the clearest of a primary resistance mechanism identified using global proteomic methods to date, and this is in the first study of drug resistance in parasites using this approach. to is and further analysis has identified a number of other proteins differentially and We are currently the of these proteins in resistance. resistance mechanisms are on in such as D. Ouellette M. A new of high in the protozoan parasite Leishmania and of gene in methotrexate-resistant 2002; Scholar). proteins are not to analysis by but we are other for the proteomic analysis of this of Here we present a high resolution gel map of L. major a number of landmark proteins identified by mass We have also the of these to study the as species L. analysis has to the of in We have that trypanothione a protein with potential as a drug be post-translationally a phenomenon not can a significant in and of an and this if could be to the of this target. using comparative expression we have identified a primary drug resistance mechanism in a methotrexate-resistant strain of L. the of this to proteins in resistance. this study a framework for the of proteomic in Leishmania and other parasites and the of these to study and aspects of parasite We and for the Leishmania TRYR and for and and from the for on the L. major protein

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