The Immunologically Protective P-4 Antigen ofLeishmania Amastigotes

Kar, Sudeshna; Soong, Lynn; Colmenares, Marı́a; Goldsmith-Pestana, Karen; McMahon-Pratt, Diane

doi:10.1074/jbc.m002149200

Cited by 35 publications

(24 citation statements)

References 59 publications

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“…It has shown that expression of this nuclease upregulated significantly in response to purine starvation [13, 17] which is consistent with the opinion that class I nuclease enzyme plays a vital role in providing purines for growth and development of Leishmania parasites. Since there is no a homologue enzyme with similar properties in mammalian tissues, this enzyme can serve as a chemotherapeutic target for selective targeting of Leishmania during infection [11].…”

Section: Resultssupporting

confidence: 80%

“…P-4 is a single-stranded specific class I nuclease that was identified initially in L. amazonensis [16] and then characterized in L. pifanoi by Kar et al [17]. Recently, we isolated a class I nuclease gene from amastigotes of L. major [18].…”

Section: Resultsmentioning

confidence: 99%

“…It has shown that Leishmania promastigotes possess a unique class I nuclease, [10, 11] that involves the salvage of preformed purines through the hydrolysis of either 3′-nucleotides or nucleic acids [12–16]. An intracellular amastigote-specific protein, P-4, with class I nuclease activity has also been identified in L. pifanoi by Kar et al [17] and by our group in L. major [18]. However, there was no data about the presence and characteristics of this nuclease in L. infantum , the causative agent of infantile human leishmaniasis.…”

Section: Introductionmentioning

confidence: 99%

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Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

et al. 2011

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Parasite of the genus Leishmania is reliant on the salvage pathway for recycling of ribonucleotides. A class I nuclease enzyme also known as P4 nuclease is involved in salvage of purines in cutaneous Leishmania species but the relevant enzymes have not been characterized in Leishmania infantum (L. infantum). The aim of this study was to clone and characterize the gene encoding class I nuclease in L. infantum. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4) by PCR. The product was cloned, sequenced, and expressed in E. coli for further characterization. Analysis of the sequence of Li-P4 revealed that the gene consists of an ORF of 951 bp. Sequence similarity analysis indicated that Li-P4 has a high homology to relevant enzymes of other kintoplastids with the highest homology (88%) to p1/s1 class I nuclease from L. donovani. Western blotting of antirecombinant Li-P4 with promastigote and amastigote stages of L. infantum showed that this nuclease is present in both stages of parasite with higher expression in amastigote stage. The highly conserved nature of this essential enzyme in Leishmania parasites suggests it as a promising drug target for leishmaniasis.

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

et al. 2011

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“…The latter include the following: 1) a Ն40-kDa bi-functional, 3Ј-nucleotidase/nuclease anchored in the cell surface membranes of several pathogenic species of Leishmania (i.e. L. donovani and Leishmania mexicana (5,7,9)) as well as Crithidia luciliae (8), and 2) a P-4 antigen/nuclease specifically localized to the endoplasmic reticulum of Leishmania pifanoi amastigote forms (39). In addition, a related P1/S1 gene homolog has been reported from an Iranian isolate of L. major, but no functional enzyme activity or subcellular localization was demonstrated for the product of that gene (22).…”

Section: Discussionmentioning

confidence: 99%

Molecular and Functional Analyses of a Novel Class I Secretory Nuclease from the Human Pathogen, Leishmania donovani

Joshi¹,

Dwyer

2007

Journal of Biological Chemistry

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The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc s , a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc s chimeric construct was used in conjunction with an anti-LdNuc s peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/ secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single-and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.Leishmania are a group of primitive pathogenic trypanosomatid protozoan parasites that cause over 2 million new cases of human cutaneous, mucocutaneous, and fatal visceral disease (i.e. leishmaniasis) per year worldwide (1). All Leishmania parasites undergo a digenetic life cycle, which includes differentiation, development, and transmission between both a sand fly vector and a mammalian host. Within their hosts, these parasites reside and multiply in highly restricted micro-environments (i.e. as extracellular, flagellated promastigote forms in the alimentary tract of their sandfly vectors and as obligate intracellular amastigote forms in the phagolysosomal compartments of infected mammalian macrophages) (2). It is important to point out that Leishmania, like other trypanosomatid parasites, are incapable of the de novo biosynthesis of purines (3, 4). Thus, it seems obvious that in order to survive, these purine auxotrophs must be capable of salvaging these essential nutrients from their host environments. One such purported purine salvage mechanism involves a unique cell surface membraneanchored, bi-functional 3Ј-nucleotidase/nuclease that...

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“…This analysis, shown in Figure 1C, indicated that LamAT-Y is expressed both in promastigotes and axenic amastigotes at relatively equivalent levels based on the amount of input RNA in each reaction. As a control, we examined the expression of P-4 nuclease [35], whose expression is upregulated in amastigotes (Figure 1C). …”

Section: Resultsmentioning

confidence: 99%

Molecular and Cellular Characterization of an AT-Hook Protein from Leishmania

2011

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AT-rich DNA, and the proteins that bind it (AT-hook proteins), modulate chromosome structure and function in most eukaryotes. Unlike other trypanosomatids, the genome of Leishmania species is unusually GC-rich, and the regulation of Leishmania chromosome structure, replication, partitioning is not fully understood. Because AT-hook proteins modulate these functions in other eukaryotes, we examined whether AT-hook proteins are encoded in the Leishmania genome, to test their potential functions. Several Leishmania ORFs predicted to be AT-hook proteins were identified using in silico approaches based on sequences shared between eukaryotic AT-hook proteins. We have used biochemical, molecular and cellular techniques to characterize the L. amazonensis ortholog of the L. major protein LmjF06.0720, a potential AT-hook protein that is highly conserved in Leishmania species. Using a novel fusion between the AT-hook domain encoded by LmjF06.0720 and a herpesviral protein, we have demonstrated that LmjF06.0720 functions as an AT-hook protein in mammalian cells. Further, as observed for mammalian and viral AT-hook proteins, the AT-hook domains of LmjF06.0720 bind specific regions of condensed mammalian metaphase chromosomes, and support the licensed replication of DNA in mammalian cells. LmjF06.0720 is nuclear in Leishmania, and this localization is disrupted upon exposure to drugs that displace AT-hook proteins from AT-rich DNA. Coincidentally, these drugs dramatically alter the cellular physiology of Leishmania promastigotes. Finally, we have devised a novel peptido-mimetic agent derived from the sequence of LmjF06.0720 that blocks the proliferation of Leishmania promastigotes, and lowers amastigote parasitic burden in infected macrophages. Our results indicate that AT-hook proteins are critical for the normal biology of Leishmania. In addition, we have described a simple technique to examine the function of Leishmania chromatin-binding proteins in a eukaryotic context amenable to studying chromosome structure and function. Lastly, we demonstrate the therapeutic potential of compounds directed against AT-hook proteins in Leishmania.

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The Immunologically Protective P-4 Antigen ofLeishmania Amastigotes

Cited by 35 publications

References 59 publications

Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

Molecular and Functional Analyses of a Novel Class I Secretory Nuclease from the Human Pathogen, Leishmania donovani

Molecular and Cellular Characterization of an AT-Hook Protein from Leishmania

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