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. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.

Novel role of sphingolipid synthesis genes in regulating giardial encystation.

Although encystation (cyst formation) is important for the survival of Giardia lamblia outside its human host, the molecular events that prompt encystation have not been fully elucidated. Here, we demonstrate that sphingolipids (SLs), which are important for the growth and differentiation of many eukaryotes, play key roles in giardial encystation. Transcriptional analyses showed that only three genes in the SL biosynthesis pathways are expressed and transcribed differentially in nonencysting and encysting Giardia trophozoites. While the putative homologues of giardial serine palmitoyltransferase (gSPT) subunit genes (gspt-1 and -2) are differentially expressed in nonencysting and encysting trophozoites, the giardial ceramide glucosyltransferase 1 gene (gglct-1) is transcribed only in encysting cells. l-Cycloserine, an inhibitor of gSPT, inhibited the endocytosis and endoplasmic reticulum/perinuclear targeting of bodipy-ceramide in trophozoites, and this could be reversed by 3-ketosphinganine. On the other hand, D-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthesis, blocked karyokinesis and reduced cyst production in culture. PPMP also altered the expression of cyst wall protein transcripts in encysting cells. Phylogenetic analyses revealed that the gspt genes are paralogs derived from an ancestral spt sequence that underwent gene duplication early in eukaryotic history. This ancestral sequence, in turn, was probably derived from prokaryotic aminoacyl transferases. In contrast, gglct-1 is found in both prokaryotes and eukaryotes without any evidence of gene duplication. These studies indicate that SL synthesis genes are involved in key events in giardial biology and could serve as potential targets for developing new therapies against giardiasis.

Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia.

Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.

Transcriptional analysis of three major putative phosphatidylinositol kinase genes in a parasitic protozoan, Giardia lamblia.

The current investigation evaluates the expression of phosphatidylinositol kinase (PIK) genes in the parasitic protozoan, Giardia lamblia. The G. lamblia Genome Database revealed the presence of two putative phosphatidylinositol-3-kinase (gPI3K) and one phosphatidylinositol-4-kinase (gPI4K) genes resembling the catalytic subunit of eukaryotic PIKs. Primers, designed to amplify mRNA of these three genes, were used to measure transcription by quantitative reverse-transcriptase polymerase chain reactions. Results suggest that all three PIK genes are expressed in non-encysting and encysting trophozoites. The relative levels of the mRNA were highest in parasites cultured in pre-encysting medium that contained no bile. Two inhibitors of PI3K, LY 294002 and wortmannin were found to inhibit the growth of the trophozoite in culture. However, wortmannin was more effective than LY294002. Altogether, the present study indicates that Giardia is capable of expressing PIKs that are necessary for the growth and differentiation of this pathogen.