Transient and stable transfection in the protozoan parasite Entamoeba invadens

Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development.     

RNA interference (RNAi) inhibits growth of Plasmodium falciparum

RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. Erythrocyte-infecting stages of the malaria parasite Plasmodium falciparum were treated with double-stranded RNA (dsRNA) encoding a segment of the gene encoding dihydroorotate dehydrogenase (DHODH). DHODH is an enzyme in pyrimidine biosynthesis, essential for parasite growth. A decrease in parasite growth (P<0.0005) correlated with a decrease in levels of DHODH mRNA. Control treatments with single-stranded RNA, dsRNA encoding the circumsporozoite protein (a stage-specific protein not expressed in the asexual blood stage) and dsRNA encoding a gene from the related organism Toxoplasma gondii did not inhibit growth. As a test for the RNAi assay, parasites were treated with dsRNA encoding chorismate synthase (CS), an enzyme thought to be involved in folate synthesis, to examine the requirement for this enzyme for parasite growth. Growth decreased (P<0.001) though less markedly than by dsRNA encoding DHODH. These results demonstrate the utility of this assay in assessing requirements for gene products, and their potential as chemotherapeutic targets.     

Drug resistance and P-glycoprotein gene amplification in the protozoan parasite Leishmania

Amplification of the H circle is often associated with methotrexate (MTX) selection in Leishmania species. We have shown that the H circle of Leishmania tarentolae contains an open reading frame, ItpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). H region amplification was also noted in some mutants selected for resistance to arsenite and vinblastine. Mutants having the complete 68-kb circles were cross-resistant to MTX, but two arsenite mutants having only part of the H region amplified, but including ItpgpA, were not cross-resistant to MTX. These results suggest that the putative determinant for MTX resistance present on the H circle is not ItpgpA. We have also determined how ItpgpA-containing plasmids were generated from the chromosomal copy. The H circle contains a 30-kb inverted duplication separated by two unique DNA segments. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We have shown that the two unique segments in chromosomal DNA are flanked by inverted repeats suggesting that H circles could be formed by a foldback mechanism (see fig. 2). Unexpectedly, a plasmid present in cells selected for arsenite resistance lacked part of the H region and the long inverted repeats. It appears to have been formed by intrachromosomal recombination between two P-glycoprotein genes, ItpgpA and ItpgpB, located adjacent to the H region. Our results show that under drug pressure, the same P-glycoprotein-encoding region in Leishmania may be amplified by very different mechanisms and yield different amplicons.     

Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania

Folates are polyglutamylated in most organisms by the enzyme folylpolyglutamate synthetase (FPGS). The Leishmania tarentolae FPGS gene was isolated. Its predicted product contains 538 amino acids and shows 33 and 30% identity with the human and yeast FPGS proteins, respectively. The level of folate polygtutamylation was studied in L. tarentolae promastigotes and in Leishmania infantum promastigotes and axenic amastigotes. In all species examined, folates were found predominantly as pentaglutamates, although monoglutamates were found in higher proportion in L. infantum axenic amastigote cells. Leishmania cells transfected with a FPGS containing plasmid (FPGS transfectant) exhibited a 6-fold increase in FPGS activity (32.7 pmol mg(-1) h(-1)) compared with wild-type cells (4.7 pmol mg(-1) h(-1)). HPLC analysis of the polyglutamylated forms of folates indicated a 2-fold increase of hexaglutamates in the FPGS transfectant compared with wild-type cells, while cells with one FPGS allele interrupted showed a higher proportion of short chain glutamates. The long-term accumulation of folates was greatly increased in the FPGS transfectant. Overall, this work indicates that FPGS activity is expressed in all forms of the parasite, and modulates the retention of folate, thereby possibly playing an important role in physiology.     

Functional analysis and complex gene rearrangements of the folate/biopterin transporter (FBT) gene family in the protozoan parasite Leishmania

The protozoan parasite Leishmania is a folic acid auxotroph. Previous work has led to the characterization of the main folate transporter FT1. FT1 is part of the folate/biopterin transporter (FBT) family and Leishmania with its 14 members is, of all sequenced organisms, the one with the most FBTs. We developed a real-time TaqMan RT-PCR assay to follow the expression of these FBT genes during growth phases, life cycles and in methotrexate-resistant mutants of Leishmania infantum. FT1 is expressed preferentially in the logarithmic phase which is consistent with the higher accumulation of folate in that stage. FT1 RNA levels even seemed to be related to folate concentration in the medium. Surprisingly, several of the FBT genes were expressed preferentially in the stationary phase of growth, a stage with minimal folate accumulation. It suggests that these FBT members may transport other related substrates. Resistance to methotrexate is associated with FT1 inactivation and upregulation of other FBT genes. Inactivation of FT1 is due either to a gene deletion mediated by homologous recombination between conserved FBT sequences or by segmental gene conversion. This study highlighted the multiplicity of FBT genes in Leishmania, their complex RNA expression, and novel gene rearrangements associated with FT1 inactivation and antifolate resistance.     

Development of RNA interference (RNAi) as potential antiviral strategy against enterovirus 70

Enterovirus 70 (EV70) is recognized as the main causative agent of acute hemorrhagic conjunctivitis (AHC), a highly contagious viral infection of the eye. Currently, there is no available treatment for EV70 infections. In this study, we developed a potential intervention strategy using RNA interference (RNAi) against EV70 infection in an in vitro system. Two synthetic 19-mer siRNAs, si-3D1 and si-3D2, were designed to target the 3Dpol region of the EV70 genome. Significant dosage dependent inhibition of EV70 in rhabdomyosarcoma cell line, as shown by reduction of viral RNA and VP1 production, was observed. Both siRNAs prevented EV70 replication in RD cells when transfected into these cells 48 hr prior to virus infection. Introduction of these siRNAs into RD cells 1-3 hr after infection with EV70 reduced production of viral RNA by approximately 60%. Thus, RNAi is a promising strategy to prevent EV70 infections and may have therapeutic potential.     

Delivery systems for the direct application of siRNAs to induce RNA interference (RNAi) in vivo

RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies. It is mediated through small interfering RNAs (siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor is the ability to deliver intact siRNAs into target cells/organs in vivo. This review highlights the mechanism of RNAi and the guidelines for the design of optimal siRNAs. It gives an overview of studies based on the systemic or local application of naked siRNAs or the use of various nonviral siRNA delivery systems. One promising avenue is the the complexation of siRNAs with the polyethylenimine (PEI), which efficiently stabilizes siRNAs and, upon systemic administration, leads to the delivery of the intact siRNAs into different organs. The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described.     

Viral RNA silencing suppressors (RSS): novel strategy of viruses to ablate the host RNA interference (RNAi) defense system

Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recent studies of mammalian viruses exhibiting RNA silencing suppressor (RSS) activity have further advanced our understanding of RNAi in terms of host-virus interactions. Viral proteins and non-coding viral RNAs can inhibit the RNAi (miRNA/siRNA) pathway through different mechanisms. Mammalian viruses having dsRNA-binding regions and GW/WG motifs appear to have a high chance of conferring RSS activity. Although, RSSs of plant and invertebrate viruses have been well characterized, mammalian viral RSSs still need in-depth investigations to present the concrete evidences supporting their RNAi ablation characteristics. The information presented in this review together with any perspective research should help to predict and identify the RSS activity-endowed new viral proteins that could be the potential targets for designing novel anti-viral therapeutics.     

Toll-like receptor 4 contributes to efficient control of infection with the protozoan parasite Leishmania major

The essential role of Toll-like receptors (TLR) in innate immune responses to bacterial pathogens is increasingly recognized, but very little is known about the role of TLRs in host defense against infections with eukaryotic pathogens. For the present study, we investigated whether TLRs contribute to the innate and acquired immune response to infection with the intracellular protozoan parasite Leishmania major. Our results show that TLR4 contributes to the control of parasite growth in both phases of the immune response. We also addressed the mechanism that results in killing or growth of the intracellular parasites. Control of parasite replication correlates with the early induction of inducible nitric oxide synthase in TLR4-competent mice, whereas increased parasite survival in host cells from TLR4-deficient mice correlates with a higher activity of arginase, an enzyme known to promote parasite growth. This is the first study showing that TLR4 contributes to the effective control of Leishmania infection in vivo.     

Stage specific gene expression and cellular localization of two isoforms of the serine hydroxymethyltransferase in the protozoan parasite Leishmania

Serine hydroxymethyltransferase (SHMT) catalyses the reversible conversion of serine and tetrahydrofolate to glycine and methylene-tetrahydrofolate. The recent completion of the genome sequence of Leishmania major revealed the presence of two genes coding for two isoforms of this protein. In silico analysis showed that one isoform had an extension at its N-terminus and was predicted to localize to the mitochondrion. The situation is different in other kinetoplastid parasites with only one SHMT encoding gene in Trypanosoma cruzi and no SHMT encoding gene in Trypanosoma brucei. The two L. major SHMT genes were cloned in frame with the green fluorescent protein and the resulting fusion proteins showed differential localization: the short form (SHMT-S) was found in the cytosol while the long one (SHMT-L) was found in an organelle that has hallmarks of the parasite mitochondrion. Indeed, SHMT-L had a similar cellular fractionation pattern as the mitochondrial HSP60 as determined by digitonin fractionation. Both SHMT-S and SHMT-L genes were expressed preferentially in the amastigote stage of the parasite and the RNA levels of SHMT-L could be modulated by glycine, serine, and folate. Overexpression of SHMT-S increased resistance to the antifolate methotrexate and to a lower level to the inhibitor thiosemicarbazide in a rich folate containing medium. These findings suggest that folate metabolism is compartmentalised in Leishmania and that SHMT RNA levels are responsive to environmental conditions.     

Ultrastructural observations of a microsporidian protozoan parasite in Libinia dubia (Decapoda)

Summary A microsporidian parasite, Nosema sp., has been found in the epithelium of the vas deferens of the spider crab, Libinia dubia. Cells were heavily infected with developing and mature spores. The spore mother cell possesses a dikaryon. The nuclear membrane shows a simple form of nuclear pore. Within the nucleus microtubules of the intercellular mitotic spindle are observed. The sporont undergoes cytokinesis to form the early sporoblast. The sporoblasts possess a dikaryon., and a centriolar plaque may be observed at the nuclear surface. The polar filament develops in association with a modified Golgi apparatus, consisting of numerous cisternae frequently filled with electron dense material, sometimes surrounded by several layers of membrane. The polar filament is formed as two hollow tubes, the inner tube being eccentrically placed within the outer one. Preparations treated with the periodic acid—silver methenamine technique for the detection of complex polysaccharides show a positive reaction in the Golgi, the sperical masses and associated membranes and in the polar filament. The developing spore wall also shows a positive reaction.Work supported by a grant (F-70-UM-6A) from the Florida Division, American Cancer Society.Contribution No. 196 from the Institute of Molecular Evolution.NIH Career Development Awardee.     

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.     

RNA interference (RNAi) for the silencing of extracellular serine proteases genes in Acanthamoeba: molecular analysis and effect on pathogenecity

Silencing of extracellular serine protease genes was undertaken by interference RNA (RNAi). Chemically synthesized, small interfering RNA (siRNA) were highly specific and efficient in silencing the catalytic domain of extracellular serine proteases of Acanthamoeba. In order to confirm the silencing phenomenon, the extracellular serine protease activities in RNAi-treated parasites were compared to non-treated parasites, using zymography profiles, Acanthamoeba-conditioned medium (ACM) protease activity, cytotoxicity assays and extracellular serine protease mRNA levels analysis. Zymography profiles showed a decrease in the extracellular protease levels in the moderate pathogenic and pathogenic strains, after treatment with siRNA. These results were supported after the ACM protease activity and CPE assays were performed in all studied isolates, showing a lower protease activity or cytotoxicity both in the pathogenic and moderate pathogenic strains treated with RNAi. These results support that extracellular serine proteases are directly involved in the pathogenesis and virulence of Acanthamoeba.     

Taxonomic position of the human intestinal protozoan parasite Isospora belli as based on ribosomal RNA sequences

The taxonomic positions of Isospora belli and other members of the genus Isospora are controversial. We determined the small-subunit ribosomal RNA of I. belli and used this sequence in combination with other coccidian RNA sequences for analysis of the taxonomic position of I. belli. The phylogenetic trees we obtained provide molecular evidence for three clades within a monophyletic group that represents the suborder Eimeriina. The clade containing I. belli consists of tissue-cyst-forming coccidia (Toxoplasma and Neospora) and members of the genus Isospora (I. ohioensis, I. suis, I. belli). The second clade, representing a sister clade of that containing the Isospora species, contains members of the genus Sarcocystis. The third one consists of members of the family Eimeriidae, including Eimeria and Cyclospora species. This shows that although I. belli as well as other members of the genus Isospora belong to the suborder Eimeriina, the family to which they belong is not Eimeriidae but rather Sarcocystidae. We suggest that the genus Isospora should be removed from the family Eimeriidae and placed into the family Sarcocystidae within the suborder Eimeriina. Received: 10 November 1999 / Accepted: 24 February 2000     

Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.     

N-linked glycosylation of proteins in the protozoan parasite Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite of animal cells. Infection of humans is common and may result in devastating disease, especially in immunocompromised individuals. Despite previous reports that N-glycosylation of proteins may be a rare post-translational modification in this and related organisms, we demonstrate that it is actually quite prevalent in Toxoplasma. N-Glycosylation is completely inhibited by treatment of parasites with tunicamycin, but this does not appear to exert its major effect on the parasites until they have egressed from their host cells. Although the tunicamycin-treated parasites appear structurally normal at this time they are not motile and mostly incapable of invading new host cells. The few tunicamycin-treated parasites that do invade are severely affected in their ability to replicate and accumulate with a distended endoplasmic reticulum, deformed nuclei, and without recognizable late secretory organelles. We provide experimental evidence that indicate that Toxoplasma N-glycans differ structurally from those in other eukaryotes.     

Lipophosphoglycan of the protozoan parasite Leishmania: stage- and species-specific importance for colonization of the sandfly vector, transmission and virulence to mammals

Leishmaniasis is a major health problem to the human population of the tropics, subtropics and Mediterranean regions. This disease is caused by the parasitic protozoa Leishmania, which have adapted to survive in several hostile environments such as the vector insect midgut, blood and the mammalian macrophage phagolysosome. Several Leishmania glycoconjugates have been implicated as key molecules for these remarkable capabilities. This review summarizes the current knowledge on potential and proven functions of the most prominent of the Leishmania glycoconjugates, the lipophosphoglycan.     

Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major

Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.