Attenuation of Leishmania infantum chagasi metacyclic promastigotes by sterol depletion.

The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro.

Mapping of VSG similarities in Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei switches its variant surface glycoprotein (VSG) to subvert its mammalian hosts' immune responses. The T. brucei genome contains as many as 1600 VSG genes (VSGs), but most are silent noncoding pseudogenes.

Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples.

The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established.

The effects of macrophage source on the mechanism of phagocytosis and intracellular survival of Leishmania.

Leishmania spp. protozoa are obligate intracellular parasites that replicate in macrophages during mammalian infection. Efficient phagocytosis and survival in macrophages are important determinants of parasite virulence.

Differential protein expression throughout the life cycle of Trypanosoma congolense, a major parasite of cattle in Africa.

Trypanosoma congolense is an important pathogen of livestock in Africa. To study protein expression throughout the T. congolense life cycle, we used culture-derived parasites of each of the three main insect stages and bloodstream stage parasites isolated from infected mice, to perform differential protein expression analysis.

Proteomic examination of Leishmania chagasi plasma membrane proteins: Contrast between avirulent and virulent (metacyclic) parasite forms.

Purpose: About two million new cases of leishmaniasis with 50 000 associated deaths occur worldwide each year. Promastigotes of the causative Leishmania spp. develop from the procyclic stage to the highly virulent metacyclic stage within the sand fly vector.

Leishmania infantum chagasi: a genome-based approach to identification of excreted/secreted proteins.

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite's interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L.

Analysis of expressed sequence tags from the four main developmental stages of Trypanosoma congolense.

Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.

Differential expression of a protease gene family in African trypanosomes.

During their life cycle African trypanosomes must quickly adapt to the different environments of the tsetse fly midgut and the mammalian bloodstream by modulating expression of many of their genes. One group of these differentially expressed genes encodes different forms of a major surface protease. Using a luciferase reporter gene transiently or permanently transfected into trypanosomes, we show here that the 3'-UTRs of these protease genes are responsible for their differential expression.

Different trans RNA splicing events in bloodstream and procyclic Trypanosoma brucei.

Most trypanosomatid genes are transcribed into polycistronic precursor RNAs that are processed into monocistronic mRNAs possessing a 39-nucleotide spliced leader (SL) at their 5'-ends and polyadenylation at their 3'-ends. We show here that precursor RNA derived from a luciferase gene integrated in reverse orientation at the rDNA locus of Trypanosoma brucei is processed into three major SL-containing RNAs in bloodstream cells and a single SL-containing RNA in procyclic RNAs. This difference in trans RNA splicing between bloodstream and procyclic cells is independent of the 5'- and 3'-UTRs flanking the luciferase coding region.

The major surface protease (MSP or GP63) in the intracellular amastigote stage of Leishmania chagasi.

The Leishmania spp. protozoa have an abundant surface metalloprotease called MSP (major surface protease), which in Leishmania chagasi is encoded by three distinct gene classes (MSPS, MSPL, MSPC). Although MSP has been characterized primarily in extracellular promastigotes, it also facilitates survival of intracellular amastigotes.

A function for a specific zinc metalloprotease of African trypanosomes.

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes.

Leishmania chagasi: homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model.

Homogenous metacyclic promastigotes of Leishmania chagasi were isolated by buoyant density from in vitro heterogeneous cultures and used for biochemical characterization of isoforms of the major surface protease (MSP). Compared to stationary phase promastigotes, metacyclic cells had three times more MSP, produced 3-fold higher parasite loads in a mouse model in vivo, and were more resistant to complement-mediated lysis in vitro. These metacyclic L.

Internal and surface-localized major surface proteases of Leishmania spp. and their differential release from promastigotes.

Major surface protease (MSP), also called GP63, is a virulence factor of Leishmania spp. protozoa. There are three pools of MSP, located either internally within the parasite, anchored to the surface membrane, or released into the extracellular environment.

Leishmania chagasi: a tetracycline-inducible cell line driven by T7 RNA polymerase.

Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P(T7)) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P(T7)-driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches.

Leishmania chagasi T-cell antigens identified through a double library screen.

Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp.

Procyclic Trypanosoma brucei expresses separate sialidase and trans-sialidase enzymes on its surface membrane.

The procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T.

The genome of the African trypanosome Trypanosoma brucei.

African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T.

Comparative genomics of trypanosomatid parasitic protozoa.

A comparison of gene content and genome architecture of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, revealed a conserved core proteome of about 6200 genes in large syntenic polycistronic gene clusters. Many species-specific genes, especially large surface antigen families, occur at nonsyntenic chromosome-internal and subtelomeric regions. Retroelements, structural RNAs, and gene family expansion are often associated with syntenic discontinuities that-along with gene divergence, acquisition and loss, and rearrangement within the syntenic regions-have shaped the genomes of each parasite.

Leishmania chagasi: the alpha-tubulin intercoding region results in constant levels of mRNA abundance despite protein synthesis inhibition and growth state.

The intercoding regions of many Leishmania sp. genes have been implicated in the regulation of mRNA processing, stability, and translation. Herein we show that the intercoding region of the Leishmania chagasi alpha-tubulin gene (alpha-TUB) confers stable beta-galactosidase (beta-GAL) reporter mRNA levels during promastigote growth and development in vitro and during protein synthesis inhibition.

Regulation of genes encoding the major surface protease of Leishmania chagasi via mRNA stability.

The intercoding regions between many Leishmania sp. genes regulate their mRNA expression. The MSPL mRNA, encoding a subclass of the major surface protease (MSP) of Leishmania chagasi, increases in abundance, when protein synthesis is arrested, while alpha-tubulin (alpha-TUB) mRNA and most other mRNAs do not.

Internal and surface subpopulations of the major surface protease (MSP) of Leishmania chagasi.

Major surface protease (MSP) facilitates Leishmania promastigote evasion of complement-mediated lysis in the mammalian host and enhances host macrophage phagocytosis of the promastigotes. We previously showed that the steady-state abundance of MSP protein increases 14-fold during in vitro cultivation of L. chagasi promastigotes from logarithmic to stationary phase, despite the fact that the total amount of MSP mRNA does not increase.

Multiple products of the Leishmania chagasi major surface protease (MSP or GP63) gene family.

The major surface protease (MSP or GP63) of the Leishmania spp. protozoa facilitates parasite evasion of complement-mediated killing, phagocytosis by macrophages, and intracellular survival in macrophage phagolysosomes. Immunoblots of several Leishmania species have shown there are distinct MSP isoforms, but the biochemical bases for these differences are unknown.

Trypanosoma brucei: a first-generation CRE-loxP site-specific recombination system.

The bacteriophage CRE-loxP system of DNA recombination is widely used to manipulate segments of the genomes of mice and other eukaryotes for the purpose of studying the regulation and functions of their genes. Since this recombination system could have similar applications in analyzing the genomes of trypanosomatids, we assessed the action of CRE recombinase on its loxP DNA recognition sites in Trypanosoma brucei after inserting tetracycline-regulated CRE and two 34-bp loxP sites into the T. brucei genome.