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. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.

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. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes.

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. Northern blots show that transcripts of the three tandem gene classes encoding Leishmania chagasi MSP (MSPS, MSPL, MSPC) are differentially expressed during parasite growth in vitro. Cell-associated MSPs increase in abundance during growth, correlating directly with parasite virulence. We examined whether distinct products of these >18 MSP genes are either differentially expressed or differentially processed during parasite growth. Two-dimensional gel electrophoresis and immunoblots delineated more than 10 MSP isoforms in stationary phase L. chagasi, distributed between pIs of 5.2-6.1 and masses of 58-63 kDa. Post-translational modifications including N-glycosylation, GPI anchor addition and phosphorylation did not account for all differences among the isoforms. MALDI-TOF mass spectrometry demonstrated that at least some L. chagasi MSPs were the products of different MSP genes. One isoform was not available for surface biotinylation, suggesting it could be located internally. Parasites in logarithmic growth expressed only four MSP isoforms, and an attenuated strain of L. chagasi (L5) did not express one of the MSP classes (MSPS). These data demonstrate that the products of individual MSP genes are differentially expressed during Leishmania development. We hypothesize they may play different roles during parasite migration through its two hosts.

Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi.

The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi beta-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.

Characterization of cDNA clones encoding ribonucleoprotein antigens expressed in Trypanosoma cruzi amastigotes.

Sera from patients with Chagas' disease were used to screen a Trypanosoma cruzi amastigote cDNA library. Characterization of 50 positive clones showed that 21 (42%) encode previously identified T. cruzi ribosomal and flagellar proteins, heat-shock proteins or proteins with repetitive motifs. Twenty-nine clones (58%) correspond to nine genes not previously described in T. cruzi. Three cDNAs, encoding novel repetitive antigens with homology to ribosomal proteins and to other RNA binding proteins, were further characterized. Patient humoral responses against the recombinant proteins encoded by these cDNAs were evaluated in anticipation that they may constitute potential new targets for serodiagnostic assays.

Trypanosoma cruzi: RNA structure and post-transcriptional control of tubulin gene expression.

Changes in tubulin expression are among the biochemical and morphological adaptations that occur during the life cycle of Trypanosomatids. To investigate the mechanism responsible for the differential accumulation of tubulin mRNAs in Trypanosoma cruzi, we determine the sequences of alpha- and beta-tubulin transcripts and analyzed their expression during the life cycle of the parasite. Two beta-tubulin mRNAs of 1.9 and 2.3 kb were found to differ mainly by an additional 369 nucleotides at the end of the 3' untranslated region (UTR). Although their transcription rates are similar in epimastigotes and amastigotes, alpha- and beta-tubulin transcripts are 3- to 6-fold more abundant in epimastigotes than in trypomastigotes and amastigotes. Accordingly, the half-lives of alpha- and beta-tubulin mRNAs are significantly higher in epimastigotes than in amastigotes. Transient transfection experiments indicated that positive regulatory elements occur in the 3' UTR plus downstream intergenic region of the alpha-tubulin gene and that both positive and negative elements occur in the equivalent regions of the beta-tubulin gene.