Identification and Characterization of a Protein-tyrosine Phosphatase in Leishmania

Leishmania parasites are eukaryotic protozoans responsible for a variety of human diseases known as leishmaniasis, which ranges from skin lesions to fatal visceral infections. Leishmania is transmitted by the bite of an infected sandfly where it exists as promastigotes and, upon entry into a mammalian host, differentiates into amastigotes, which replicate exclusively in macro-phages. The biochemical pathways enabling Leishmania to differentiate and survive in the mammalian host are poorly defined. We have therefore examined the role of protein-tyrosine phosphorylation, which is essential in regulating cell function in higher eukaryotes. Using the recently completed Leishmania genome, we have identified and cloned a Leishmania protein-tyrosine phosphatase (PTP) gene (LPTP1) by virtue of its homology with the human protein-tyrosine phosphatase 1B gene (hPTP1B). The enzyme activity of recombinant LPTP1 was confirmed using a combination of PTP-specific substrates and inhibitors. We further demonstrate, by creating LPTP1 null mutants through gene targeting, that LPTP1 is necessary for survival as amastigotes in mice, but it is dispensable for survival as promastigotes in culture. Human PTPs, including the PTP1B enzyme, are actively pursued drug targets for a variety of diseases. The observations with the LPTP1 mutants in mice suggest that it may also represent a drug target against the mammalian amastigote stage. However, in silico structure analysis of LPTP1 revealed a striking similarity with hPTP1B in the active site suggesting that, although this is an attractive drug target, it may be difficult to develop an inhibitor specific for the Leishmania LPTP1. Leishmania parasites are eukaryotic protozoans responsible for a variety of human diseases known as leishmaniasis, which ranges from skin lesions to fatal visceral infections. Leishmania is transmitted by the bite of an infected sandfly where it exists as promastigotes and, upon entry into a mammalian host, differentiates into amastigotes, which replicate exclusively in macro-phages. The biochemical pathways enabling Leishmania to differentiate and survive in the mammalian host are poorly defined. We have therefore examined the role of protein-tyrosine phosphorylation, which is essential in regulating cell function in higher eukaryotes. Using the recently completed Leishmania genome, we have identified and cloned a Leishmania protein-tyrosine phosphatase (PTP) gene (LPTP1) by virtue of its homology with the human protein-tyrosine phosphatase 1B gene (hPTP1B). The enzyme activity of recombinant LPTP1 was confirmed using a combination of PTP-specific substrates and inhibitors. We further demonstrate, by creating LPTP1 null mutants through gene targeting, that LPTP1 is necessary for survival as amastigotes in mice, but it is dispensable for survival as promastigotes in culture. Human PTPs, including the PTP1B enzyme, are actively pursued drug targets for a variety of diseases. The observations with the LPTP1 mutants in mice suggest that it may also represent a drug target against the mammalian amastigote stage. However, in silico structure analysis of LPTP1 revealed a striking similarity with hPTP1B in the active site suggesting that, although this is an attractive drug target, it may be difficult to develop an inhibitor specific for the Leishmania LPTP1. Leishmaniasis is a disease caused by infection with Leishmania protozoan parasites that results in a spectrum of clinical manifestations ranging from self-healing cutaneous lesions to fatal visceral disease (reviewed in Ref. 1Murray H.W. Berman J.D. Davies C.R. Saravia N.G. Lancet. 2005; 366: 1561-1577Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar). There are over two million new cases of leishmaniasis each year and over 12 million people currently suffering from this infection in 88 tropic and subtropic countries (2Desjeux P. Nat. Rev. Microbiol. 2004; 2: 692Crossref PubMed Scopus (141) Google Scholar, 3Desjeux P. Trans. R Soc. Trop. Med. Hyg. 2001; 95: 239-243Abstract Full Text PDF PubMed Scopus (782) Google Scholar). During its life cycle, Leishmania alternates between promastigotes in the sandfly vector and amastigotes in the mammalian host. Once transmitted to the mammalian host through the bite of an infected sandfly, the promastigotes differentiate into nonflagellated intracellular amastigotes, whereupon they multiply exclusively in the phagolysosome organelle of infected macrophages. Amastigotes are responsible for the diverse pathologies associated with leishmaniasis, which depends to a large extent on the Leishmania species (reviewed in Ref. 1Murray H.W. Berman J.D. Davies C.R. Saravia N.G. Lancet. 2005; 366: 1561-1577Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar). The biochemical changes associated with differentiation from promastigotes to amastigotes and with the long term survival of amastigotes in the mammalian host are poorly understood, and consequently the biological role of protein phosphorylation remains largely unknown in Leishmania. Protein phosphorylation is among the most important regulatory biochemical changes in higher eukaryotic cells. Phosphorylation of tyrosine residues is controlled by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). 5The abbreviations used are: PTP, protein-tyrosine phosphatase; pNPP, 4-p-nitrophenylphosphate; IR, insulin receptor; bpV (phen), potassium bisperoxo(1,10-phenanthroline)oxovanadate (V).5The abbreviations used are: PTP, protein-tyrosine phosphatase; pNPP, 4-p-nitrophenylphosphate; IR, insulin receptor; bpV (phen), potassium bisperoxo(1,10-phenanthroline)oxovanadate (V). Particularly, protein-tyrosine phosphorylation and dephosphorylation regulate multiple central processes including cellular phenotypic functions, differentiation, proliferation, and cell death (reviewed in Ref. 4Tonks N.K. FEBS Lett. 2003; 546: 40-148Crossref Scopus (316) Google Scholar). The overall protein phosphorylation pattern in Leishmania parasites and related trypanosomatids has been shown to change during differentiation associated with different life cycle stages, suggesting that protein kinases and phosphatases play a role in these processes (5Bengs F. Scholz A. Kuhn D. Wiese M. Mol. Microbiol. 2005; 55: 1606-1615Crossref PubMed Scopus (100) Google Scholar). More recently, it has been established that L. major promastigote extracts contain protein-tyrosine phosphatase activity, although the corresponding gene(s) has not yet been identified (6Aguirre-Garcia M.M. Escalona-Montano A.R. Bakalara N. Perez-Torres A. Gutierrez-Kobeh L. Becker I. Parasitology. 2006; 132: 641-649Crossref PubMed Scopus (17) Google Scholar). Because of the central role of tyrosine phosphorylation in higher eukaryotic cell function, we have begun to investigate the potential contribution of protein-tyrosine phosphorylation to the Leishmania life cycle and virulence. We had previously observed that heterologous expression of a prototype human protein-tyrosine phosphatase 1B (hPTP1B) in Leishmania donovani mediates partial differentiation toward the amastigote stage, induces expression of amastigote-specific proteins, and increases virulence in BALB/c mice (7Nascimento M. Abourjeily N. Ghosh A. Zhang W.-W. Matlashewski G. Mol. Microbiol. 2003; 50: 1517-1526Crossref PubMed Scopus (15) Google Scholar). These observations argue that PTPs play a role in amastigote survival in the mammalian host and provide strong justification for the characterization of endogenous Leishmania PTP genes as detailed within. In the present study, we describe the identification of a PTP gene from Leishmania major, Leishmania infantum, and L. donovani that has extensive sequence and corresponding structural homology with the human PTP1B gene product. These Leishmania genes have been designated LmPTP1, LiPTP1, and LdPTP1, respectively. The enzyme activity of LmPTP1 has been confirmed with relevant substrates and inhibitors. We have also developed LdPTP1 heterozygous and homozygous null mutant knock-out clones that proliferated in a similar fashion to wild type promastigotes in culture but were severely impaired with respect to survival as amastigotes in BALB/c mice. Through in silico structural analysis, we also show that the L. infantum PTP1 (LiPTP1) and human PTP1B (hPTP1B) shared remarkable structural conservation in the active site; however, notable differences outside this region are also present. This observation suggests that from a phenotypic perspective, the LPTP1 represents an attractive drug target. However, from a structural perspective, it may be difficult to develop a small molecule specific to the active site of LPTP1. Parasite Cultures—The L. donovani 1S/Cl2D and L. major Friedlin V9 promastigotes were routinely cultured at pH 7.2 and 27 °C in Medium 199 medium (Invitrogen) supplemented with 10% fetal bovine serum. L. donovani differentiation into amastigotes was performed by shifting to amastigote culture medium (37 °C, pH 5.5 in RPMI 1640 plus 10% fetal bovine serum) overnight, which mimics the temperature and pH of the host macrophage phagolysosome. Cloning, Sequencing, and Tagging of L. major PTP1—The 1.5-kb L. major DNA fragment homologous to the human PTP1B was identified in the L. major data base by BLAST search (Entry GeneDB LmjF36.5370; www.genedb.org). Based on the sequence obtained from the data base, two primers were designed to PCR amplify the LmPTP1 gene and to incorporate a sequence encoding a His tag at the N terminus, and the amplified product was then ligated into the mammalian expression vector pcDNA3. The primers used were HisF 5′-aagcttATGGGCCATCATCATCATCATCATCATATGTGTGAAAAGCAACTCAAGGAG-3′, which contained a HindIII site, and reverse R 5′-ggatccTTACACAAACGAAGGCGAGAAGCGC-3′, which contained a The LmPTP1 was used as in a where a new a tag from the L. donovani specific protein that is by an Matlashewski G. Mol. PubMed Scopus Google with a HindIII site and the R with a site was The amplified product was then cloned into the vector and as shown in a for the human PTP1B sequence was also with the using the primers with an site and the with a The L. donovani LdPTP1 gene was from L. donovani 1S/Cl2D DNA with the primers used for L. major and In the amplified fragment was cloned into the vector (Invitrogen) and and reverse and primers were used in with the of were performed by a with DNA and was by extensive of and of LmPTP1 and cell was using to the a of was with a of of DNA of and of a expression in of The the were with medium and with pH with on for The cell were and were performed to the of in each and to the of protein used in each was used for of the LmPTP1 and hPTP1B to the was in pH with of was to of cell and with for at was in of pH with and of the with the LmPTP1 hPTP1B was used for activity and the used for analysis with tag and was used for of phosphatase activity as previously (7Nascimento M. Abourjeily N. Ghosh A. Zhang W.-W. Matlashewski G. Mol. Microbiol. 2003; 50: 1517-1526Crossref PubMed Scopus (15) Google Scholar). of PTP was in of pH inhibitor and in a of pH was to each and the was at °C The were at The phosphatase activity with the insulin is a specific for the is a major of this enzyme A. N.K. D. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. P. D. A. PubMed Scopus Google Scholar). The was performed to the phosphatase of and of potassium bisperoxo(1,10-phenanthroline)oxovanadate were used to further the activity of LmPTP1 and the hPTP1B as the bpV is a of a of and specific PTP Full Text Full Text PDF PubMed Scopus Google Scholar, D. G. A. Full Text PDF PubMed Google Scholar). the LmPTP1 and hPTP1B was with of pH inhibitor and then with for at the PTPs was with of pH inhibitor and then for phosphatase activity using as as detailed of the LdPTP1 from L. L. donovani PTP1 gene were by homologous gene as in and fragment the L. donovani PTP1 gene from the was into a The was with to the region of the LdPTP1 The fragment the gene was from the vector G. M. Mol. PubMed Scopus Google with and and into the site the LdPTP1 the Hyg. The fragment the gene and the was then into L. were in the targeting, with to the heterozygous LdPTP1 knock-out The knock-out homozygous null mutant for the LdPTP1 gene was by the to in culture of the knock-out null mutant with a the LdPTP1 gene was as The primers with a HindIII site and the reverse with a site were used to amplify a fragment from L. donovani DNA that contained the LdPTP1 gene including and The fragment was then into HindIII and of G. M. Mol. PubMed Scopus Google to the as shown in analysis, of Leishmania DNA was with and and in a and were performed as previously Matlashewski G. Mol. PubMed Scopus Google Scholar). The LdPTP1 active encoding DNA fragment from was used as a for DNA from the and knock-out clones to the of the LdPTP1 gene using the gene specific into the LdPTP1 gene The L. major 1.5-kb LmPTP1 gene fragment was used in the to the of the LdPTP1 to the knock-out null of BALB/c and of BALB/c mice were with promastigotes in of as previously Matlashewski G. PubMed Scopus Google Scholar). of the mice were examined for L. donovani by the of amastigotes in the as was by the of cell were by in as previously detailed Matlashewski G. Mol. Microbiol. 2001; PubMed Scopus Google Scholar). in was by of the at of from in from between the L. infantum PTP1 sequence and human PTP1B obtained with in with the PTP1B structure were used in A. A. Protein PubMed Scopus Google to the L. infantum PTP1 homology of present in the Leishmania PTP1 sequence that was outside of the enzyme active site was not present in the human enzyme sequence and, as a was not In the used to the human enzyme structure not contain residues these two were from the Leishmania homology We this by a BLAST search of the L. major data base L. G. M. A. P. N. 2005; PubMed Scopus Google for that represent PTP genes by virtue of homology with the human PTP1B In potential PTP genes were identified in the Leishmania The with the with human PTP1B was on which we have designated The L. infantum PTP1 (LiPTP1) sequence was also identified in this from the L. infantum data base www.genedb.org). Based on these we designed PCR primers to and sequence the L. donovani LdPTP1 as detailed of the Leishmania PTP1 and hPTP1B revealed they sequence including a of important residues the hPTP1B which the essential and residues for enzyme activity region in (reviewed in Ref. 4Tonks N.K. FEBS Lett. 2003; 546: 40-148Crossref Scopus (316) Google Scholar). In to the relevant with hPTP1B including the and the residues region with type in which play a role in the of the active The and hPTP1B also a region from that are responsible for type and cellular in hPTP1B N.K. FEBS Lett. 2003; 546: 40-148Crossref Scopus (316) Google Scholar). analysis of DNA from L. major that LmPTP1 was a gene in the W.-W. and G. in is the PTP sequence from which is from hPTP1B is the the Leishmania PTP1 shown in suggest that they for a hPTP1B it was necessary to this using relevant enzyme substrates and inhibitors. We designed primers to and the LmPTP1 gene into a eukaryotic expression vector for expression in cells. and the LmPTP1 from a tag from the L. donovani by a tag encoding were at the of the LmPTP1 gene In this the cell LmPTP1 be with Matlashewski G. Mol. PubMed Scopus Google and from cell by with We also the using the human hPTP1B gene as a for of the LmPTP1 and hPTP1B genes in was by analysis with shown in the hPTP1B and LmPTP1 were at the of and and The vector not the of the for The were from the cell using and by analysis with shown in and the His contained of hPTP1B and These data confirmed that it was to and similar of hPTP1B and LmPTP1 from cells. We we PTP enzyme activity in the the hPTP1B and LmPTP1 from the cells. protein phosphatase substrates were used for these including and a specific tyrosine phosphatase and this the hPTP1B and LmPTP1 expression were into on for activity, and further to analysis to similar of hPTP1B and in each shown in and LmPTP1 and hPTP1B enzyme were on the with the and substrates with the from that the activity in the shown was to PTP activity, we it was to this activity using the protein phosphatase inhibitor and a specific protein-tyrosine phosphatase inhibitor bpV D. G. A. Full Text PDF PubMed Google Scholar, M. Full Text Full Text PDF PubMed Scopus Google Scholar). shown in and the LmPTP1 and hPTP1B were with and bpV confirmed were similar of LmPTP1 and hPTP1B in the and of these inhibitors. the not the activity observed on His from the vector these data the that the LmPTP1 gene for a protein-tyrosine phosphatase enzyme that, these had a of activity similar to that of Once the Leishmania PTP1 gene had been identified and as detailed it was necessary to its role in the life cycle of the by null PTP1 We performed and of the of the PTP1 gene from L. donovani and the mutant L. donovani was for this analysis it be cultured in as promastigotes and amastigotes, L. major be cultured as it has been previously shown that of a hPTP1B gene in L. donovani in virulence in the amastigote (7Nascimento M. Abourjeily N. Ghosh A. Zhang W.-W. Matlashewski G. Mol. Microbiol. 2003; 50: 1517-1526Crossref PubMed Scopus (15) Google Scholar). Because Leishmania are the two of the of the LdPTP1 gene were for as in The enzyme fragment the LdPTP1 was with a fragment the gene The was and into the LdPTP1 site of the L. donovani by and were for The of gene was using of and the was using of In this LdPTP1 be with were then to to LdPTP1 knock-out were performed to the heterozygous and homozygous of the of the LdPTP1 gene in the cloned L. donovani mutant this analysis, the enzyme which was by gene was used as the shown in the LdPTP1 gene fragment encoding the was from the L. donovani clones as by the of the this sequence in the null mutant and further by a in the as with wild type L. donovani culture. PCR analysis of the LdPTP1 gene in these mutant clones confirmed the data of the not We further confirmed the of the gene into the LdPTP1 on the clones by analysis with a specific for the shown in the gene was present in the specific site for the LdPTP1 these that the active site region of the LdPTP1 gene was and in the null mutant for phenotypic the of the LdPTP1 null mutants was with that of the wild type L. donovani using established in culture for promastigotes and observed in the two homozygous LdPTP1 null mutant clones cultured promastigote °C, pH proliferated at a with wild type L. donovani The heterozygous knock-out proliferated at a similar to the null mutant amastigote culture (37 °C, pH the homozygous null mutants and also a in with the knock-out and the wild type culture respect to promastigote not to be between the null mutant clones and the wild type as shown in The most for L. donovani virulence is its to survive in the visceral in a mammalian host. We therefore the of the LdPTP1 null mutant clones and the wild type L. donovani to survive in the in the of BALB/c mice. shown in the two LdPTP1 null mutant clones and virulence with the wild type parasites as by the of amastigotes in and by the by the of infection by LdPTP1 null mutant clones the with an in virulence as by to survive in the The knock-out a in with expression of These results argue that the LdPTP1 gene a role in survival in the mammalian host. We it was to the virulence of the null mutant by with the wild type LdPTP1 This was performed by the LdPTP1 gene including its regulatory into a The LdPTP1 was into the null mutant and were in and cloned by in mice, the wild type L. donovani promastigotes that had been in culture for and used to the null mutants in were the heterozygous knock-out also a relevant it had wild type PTP1 was the null mutant clones and had the as the null mutant analysis with the LdPTP1 gene confirmed that the had been into the null mutant and We the virulence of the mutant the to the null mutant and the heterozygous mutant which endogenous LdPTP1 In a established L. donovani culture was used for shown in and the LdPTP1 gene to the null mutant virulence in the in with the null mutant The parasites the LdPTP1 was similar in virulence to the heterozygous and which endogenous for the LdPTP1 is also that the infection shown in and for the heterozygous and null mutant were to the shown in the culture of wild type L. donovani and was the L. donovani culture that was used to the LdPTP1 null mutants and which had been in culture for and further the of the LdPTP1 gene to virulence to the null we infection in the shown in the LdPTP1 gene to the null mutant in with the null mutant and was similar in virulence to the heterozygous knock-out these infection results in the and that the LdPTP1 gene on a the in the null mutant the for LdPTP1 in amastigote survival in the mammalian host. The observations provide an that of Leishmania PTP1 in amastigotes may have potential and therefore may represent a drug target. this in in silico homology was performed to the of the L. infantum PTP with the hPTP1B This was performed to were differences in the active between these that be to develop specific small molecule inhibitors. The structure of the between a and human PTP Protein was used to the of the into the L. infantum homology residues of the were used to the The pattern in the between the and the of the protein to be in the L. infantum PTP1 homology and a of structural conservation between the human and Leishmania is in and the active site and it that the active of the two are although are differences in residues further from the active site, including residues and differences to the active site are although the L. infantum enzyme has a at the human enzyme has an in the L. infantum sequence is a in human tyrosine phosphatases is a at this These differences the of the and the This may provide for an inhibitor that to the L. infantum enzyme to the human the most important in eukaryotic are that protein-tyrosine phosphorylation and dephosphorylation N.K. FEBS Lett. 2003; 546: 40-148Crossref Scopus (316) Google Scholar). was therefore important to a prototype gene in the Leishmania genome, its role in the life cycle, and its potential as a drug target. We were in a Leishmania gene we had previously observed that of the human PTP1B the virulence of L. donovani and to of differentiation toward the amastigote (7Nascimento M. Abourjeily N. Ghosh A. Zhang W.-W. Matlashewski G. Mol. Microbiol. 2003; 50: 1517-1526Crossref PubMed Scopus (15) Google Scholar). The present confirmed that Leishmania parasites have a which we LPTP1. of the L. donovani PTP1 gene in amastigotes in BALB/c mice but not promastigote survival in culture. These results argue that Leishmania PTP1 a role in biochemical pathways associated with amastigote survival in the mammalian host. the we not that Leishmania PTP1 was in differentiation into amastigotes as previously (7Nascimento M. Abourjeily N. Ghosh A. Zhang W.-W. Matlashewski G. Mol. Microbiol. 2003; 50: 1517-1526Crossref PubMed Scopus (15) Google the knock-out mutant promastigotes were of into and as amastigotes in culture. In we not changes in the expression of the amastigote-specific protein in the null mutant clones not This further the that L. major has PTP enzyme activity (6Aguirre-Garcia M.M. Escalona-Montano A.R. Bakalara N. Perez-Torres A. Gutierrez-Kobeh L. Becker I. Parasitology. 2006; 132: 641-649Crossref PubMed Scopus (17) Google and into the and structural of the Leishmania PTP1 of the structure of the human PTP1B and the Leishmania PTP1 revealed striking the active site the central is with a in this revealed in and human PTP1B and the Leishmania PTP1 important including the in the and the in the which are also in the of the N.K. FEBS Lett. 2003; 546: 40-148Crossref Scopus (316) Google Scholar). The in silico structural analysis in the of a further the between the Leishmania and human This is further by the observations in which that in the of the substrates and the Leishmania PTP1 and human PTP1B had similar of of the active site, the region at the of human PTP1B is in its with substrates N.K. FEBS Lett. 2003; 546: 40-148Crossref Scopus (316) Google Scholar, PubMed Scopus Google this of the region was also present in the Leishmania PTP1 of the human PTP1B enzyme is largely through the which this enzyme to the D. N.K. A. 95: Scopus Google Scholar, M. A. Mol. Med. PubMed Scopus Google Scholar, N. 2004; PubMed Scopus Google Scholar). the of Leishmania PTP1 are also suggesting a potential with cell similar to the human PTP1B This is with the that PTP activity is associated with L. major (6Aguirre-Garcia M.M. Escalona-Montano A.R. Bakalara N. Perez-Torres A. Gutierrez-Kobeh L. Becker I. Parasitology. 2006; 132: 641-649Crossref PubMed Scopus (17) Google Scholar). the role of the Leishmania PTP1 gene in the life cycle and the active site of the enzyme was by gene is that although LdPTP1 is a it was to develop heterozygous and homozygous PTP1 gene in that this enzyme not play a major role in the survival of promastigotes in culture. the LdPTP1 mutant promastigotes were similar to wild type However, the LdPTP1 mutants were severely in with the wild type L. donovani with respect to survival in the and of BALB/c mice. be necessary to the endogenous targets of Leishmania PTP1 to the biochemical pathways in survival in the mammalian host. is sequence on the Leishmania PTP1 enzyme, its strong homology with human PTP1B it to that it also target host cell This be similar to macrophage including and of which PTP into the host cell that play in the virulence of these (reviewed in P. G. I. Microbiol. 2005; PubMed Scopus Google and Microbiol. Full Text Full Text PDF PubMed Scopus Google Scholar). This represents an of including the Leishmania and in silico protein structure analysis be with biological including gene and virulence analysis in to potential drug We have on PTPs of the central role of tyrosine phosphorylation in the of higher eukaryotic this among the most of cell and drug This revealed a conservation between the Leishmania and human Because the active of these are it is difficult to that Leishmania PTP1 represents a target for drug may be differences in the active site, suggesting that a of PTP for Leishmania may be The combination of the used in this however, in drug targets against this important human We of the for during the of this and and for with

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