Heterologous expression of a mammalian protein tyrosine phosphatase gene in <i>Leishmania</i>: effect on differentiation

Nascimento, Mirna; Abourjeily, Nay; Ghosh, Anirban; Zhang, Wen-Wei; Matlashewski, Greg

doi:10.1046/j.1365-2958.2003.03811.x

Cited by 17 publications

(11 citation statements)

References 31 publications

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“…It was therefore important to identify a prototype PTP-related gene in the Leishmania genome, determine its role in the parasite life cycle, and determine its potential as a drug target. We were particularly interested in identifying a Leishmania PTP1B-type gene because we had previously observed that overexpression of the human PTP1B increased the virulence of L. donovani and appeared to mediate certain aspects of differentiation toward the amastigote stage (7). The present study confirmed that Leishmania parasites do have a functional PTP1B-like gene, which we termed LPTP1.…”

Section: Discussionsupporting

confidence: 70%

“…Protein-tyrosine Phosphatase Activity and Inhibition AssaysThe 4 p-nitrophenylphosphate (pNPP) assay was used for detection of total phosphatase activity as described previously (7). Briefly, 30 l of His-bind resin containing purified PTP proteins was washed once in 1.0 ml of 150 mM NaCl, 1% Nonidet P-40, 20 mM Tris, pH 8.0, Roche Applied Science complete protease inhibitor buffer and placed in a 96-well plate.…”

Section: Methodsmentioning

confidence: 99%

“…and increases virulence in BALB/c mice (7). 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.…”

mentioning

confidence: 98%

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Identification and Characterization of a Protein-tyrosine Phosphatase in Leishmania

Nascimento

Zhang

Ghosh

et al. 2006

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 70%

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of a Protein-tyrosine Phosphatase in Leishmania

Nascimento

Zhang

Ghosh

et al. 2006

Journal of Biological Chemistry

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“…Supporting this idea, it was recently reported that the heterologous expression of the human PTP1B gene in Leishmania, together with the inhibition of tyrosine kinases, promoted partial differentiation from promastigote to amastigote forms (Nascimento et al, 2003). Here, we demonstrate that the activity of a T. brucei protein tyrosine phosphatase, TbPTP1, exhibits a pivotal function in parasite differentiation.…”

Section: Introductionsupporting

confidence: 52%

Protein tyrosine phosphatase TbPTP1: a molecular switch controlling life cycle differentiation in trypanosomes

Szöőr¹,

Wilson²,

McElhinney³

et al. 2006

The Journal of Experimental Medicine

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“…LPG is expressed on the surface of the promastigote form, but is minimally expressed by the amastigote form (Ilgoutz and McConville 2001) while that of the A2 gene is only express in amastigotes. A2 protein is expressed during protein tyrosine phosphorylation in amastigotes in determinate conditions of temperature and pH (Nascimento et al 2003). Historically, promastigotes have been readily cultured in cell-free media, whereas amastigotes began to be axenically cultivated only a few years ago (Bates et al 1992;Bates 1994;Zilberstein and Shapira 1994).…”

Section: Introductionmentioning

confidence: 99%

Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

et al. 2009

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Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, L: -glutamine, and HEPES at 37 masculineC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host-parasite relationship as well as the process of pathogenesis.

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Heterologous expression of a mammalian protein tyrosine phosphatase gene in Leishmania: effect on differentiation

Cited by 17 publications

References 31 publications

Identification and Characterization of a Protein-tyrosine Phosphatase in Leishmania

Identification and Characterization of a Protein-tyrosine Phosphatase in Leishmania

Protein tyrosine phosphatase TbPTP1: a molecular switch controlling life cycle differentiation in trypanosomes

Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

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