Identification and Characterization of a Protein-tyrosine Phosphatase in Leishmania

Abstract

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 macrophages. 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. 1). 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 (2, 3). 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. 1). 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 co...

Down-regulation of dendritic cell signaling pathways by Leishmania amazonensis amastigotes

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Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein

“…The 10 amino acid tags were hemagglutinin epitope (HE) and A2 (monoclonal antibody against A2 was a kind gift of Prof. Greg Matlashewski, McGill University, Quebec, Canada) (62). To clone HAS1-FL and HAS1-Vs, firststrand cDNAs were synthesized from KMS-12BM cells using Superscript-III from Invitrogen following the manufacturer's protocol with minor modifications as follows.…”

Leishmania mexicana promastigotes secrete a protein tyrosine phosphatase

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