A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania.

Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population.

Diverse viscerotropic isolates of Leishmania all express a highly conserved secretory nuclease during human infections.

Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L.

Identification, characterization, and expression of a unique secretory lipase from the human pathogen Leishmania donovani.

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani.

Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice.

Chitinases of trypanosomatid parasites have been proposed to fulfil various roles in their blood-feeding arthropod vectors but so far none have been directly tested using a molecular approach. We characterized the ability of Leishmania mexicana episomally transfected with LmexCht1 (the L. mexicana chitinase gene) to survive and grow within the permissive sand fly vector, Lutzomyia longipalpis.

Proteomic analysis of the secretome of Leishmania donovani.

Background: Leishmania and other intracellular pathogens have evolved strategies that support invasion and persistence within host target cells. In some cases the underlying mechanisms involve the export of virulence factors into the host cell cytosol. Previous work from our laboratory identified one such candidate leishmania effector, namely elongation factor-1alpha, to be present in conditioned medium of infectious leishmania as well as within macrophage cytosol after infection.

Molecular characterization and expression of a novel kinesin which localizes with the kinetoplast in the human pathogen, Leishmania donovani.

Using a variety of molecular and cell biological approaches, we identified, characterized and expressed a novel kinesin, LdK39B in the protozoan pathogen Leishmania donovani. Results of RT-PCR revealed two distinct LdK39B products with different splice leader mini-exon sites, indicative of two potentially mature mRNA transcripts. Analyses indicated that LdK39B had a calculated molecular mass of >261, 327 Da and contained multiple amino acid repeat units.

Episomally driven antisense mRNA abrogates the hyperinducible expression and function of a unique cell surface class I nuclease in the primitive trypanosomatid parasite, Crithidia luciliae.

Here, we show that Crithidia luciliae, a primitive trypanosomatid, purine auxotroph, up-expressed its unique, bi-functional, surface membrane 3'-nucleotidase/nuclease (Cl 3'NT/NU) activity by approximately 1000-fold in response to purine starvation. A second surface membrane phospho-monoesterase, i.e.

Identification and biochemical characterization of unique secretory nucleases of the human enteric pathogen, Entamoeba histolytica.

The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients.

Down-regulation of the trypanosomatid signal recognition particle affects the biogenesis of polytopic membrane proteins but not of signal peptide-containing proteins.

Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma. The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins.

Molecular and functional analyses of a novel class I secretory nuclease from the human pathogen, Leishmania donovani.

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).

Molecular dissection and expression of the LdK39 kinesin in the human pathogen, Leishmania donovani.

In this study we show for the first time the intracellular distribution of a K39 kinesin homologue in Leishmania donovani, a medically important parasite of humans. Further, we demonstrated that this motor protein is expressed in both the insect and mammalian developmental forms (i.e.

Rab5b localization to early endosomes in the protozoan human pathogen Leishmania donovani.

Leishmania donovani is a primitive trypanosomatid pathogen of humans. This protozoan is apically polarized such that the flagellar reservoir, the exclusive site of endocytosis and exocytosis, is situated at the anterior end. Recent evidence for the existence of an endocytic pathway in Leishmania has prompted us to investigate candidate temporal markers for endocytosis.

Molecular characterization, expression, and in vivo analysis of LmexCht1: the chitinase of the human pathogen, Leishmania mexicana.

Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an approximately 50 kDa protein, with well conserved substrate binding and catalytic domains characteristic of members of the chitinase-18 protein family.

LdARF1 in trafficking and structural maintenance of the trans-Golgi cisternal network in the protozoan pathogen Leishmania donovani.

Adenosine diphosphate ribosylation factors (ARFs) are small guanosine-5'-triphosphatases that are essential in vesicular trafficking and in the maintenance of the Golgi network. In this report, we identified a homolog of the mammalian ARF1 in the human pathogenic protozoan parasite, Leishmania donovani (Ld). Ld ARF1 is a 549 bp gene encoding a 183-amino acid deduced protein of approximately 20 kDa.

The human pathogen Leishmania donovani secretes a histidine acid phosphatase activity that is resistant to proteolytic degradation.

Promastigotes of all pathogenic Leishmania species secrete acid phosphatase (SAcP) activity during their growth in vitro. It has been suggested that this enzyme may play a role in the survival of the parasite within its sandfly-vector host. To carry out such functions, SAcP would have to be relatively resistant to endogenous sandfly gut-proteases.

Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics.

In this report, we describe an in vitro culture system for the generation and propagation of axenic amastigotes from the well characterised 1S-CL2D line of Leishmania donovani. Fine structure analyses of these in vitro-grown amastigotes demonstrated that they possessed morphological features characteristic of L. donovani tissue-derived amastigotes.

Extracellular release of the surface metalloprotease, gp63, from Leishmania and insect trypanosomatids.

Protease activity was found in spent culture medium collected from Leishmania donovani, L. mexicana, L. major, as well as the insect trypanosomatids, Crithidia luciliae and Leptomonas seymouri.

Members of a unique histidine acid phosphatase family are conserved amongst a group of primitive eukaryotic human pathogens.

Recently, we identified and characterized the genes encoding several distinct members of the histidine-acid phosphatase enzyme family from Leishmania donovani, a primitive protozoan pathogen of humans. These included genes encoding the heavily phosphorylated/glycosylated, tartrate-sensitive, secretory acid phosphatases (Ld SAcP-1 and Ld SAcP-2) and the unique, tartrate-resistant, externally-oriented, surface membrane-bound acid phosphatase (Ld MAcP) of this parasite. It had been previously suggested that these enzymes may play essential roles in the growth, development and survival of this organism.

Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages.

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins.

Molecular dissection of the functional domains of a unique, tartrate-resistant, surface membrane acid phosphatase in the primitive human pathogen Leishmania donovani.

The primitive trypanosomatid pathogen of humans, Leishmania donovani, constitutively expresses a unique externally oriented, tartrate-resistant, acid phosphatase on its surface membrane. This is of interest because these organisms are obligate intracellular protozoan parasites that reside and multiply within the hydrolytic milieu of mammalian macrophage phago-lysosomes. Here we report the identification of the gene encoding this novel L.

Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana.

Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail.