Osmoregulation in the parasitic protozoan Tritrichomonas foetus

Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K(+) concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K(+) concentrations and was inhibited when K(+) and/or Cl(-) was absent from the medium. The results suggested that the well-documented Na(+)-K(+)-2Cl(-) cotransport system was responsible for the K(+) influx activated during the RVI. However, inhibitors of Na(+)-K(+)-2Cl(-) cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K(+) influx in T. foetus.     

The role of interferons in parasitic protozoan infection

Interferons are well-known for their role as a first line defence against viral attack, but they also modulate the host immune system in responses to protozoan infections. In this article, John Kelly explains what interferons are, and discusses their potential role against parasitic infection.     

Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum

We have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum , a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH₂ terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C . parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.     

Molecular systematics of the parasitic protozoan Giardia intestinalis.

The long-standing controversy regarding whether Giardia intestinalis is a single species prevalent in both human and animal hosts or a species complex consisting of morphologically similar organisms that differ in host range and other biotypic characteristics is an issue with important medical, veterinary, and environmental management implications. In the past decade, highly distinct genotypes (some apparently confined to particular host groups) have been identified by genetic analysis of samples isolated from different host species. The aim of this study was to undertake a phylogenetic analysis of G. intestinalis that were representative of all known major genetic groups and compare them with other Giardia species, viz. G. ardeae, G. muris, and G. microti. Segments from four "housekeeping" genes (specifying glutamate dehydrogenase, triose phosphate isomerase, elongation factor 1 alpha, and 18S ribosomal RNA) were examined by analysis of 0.48-0.69-kb nucleotide sequences determined from DNA amplified in polymerase chain reactions from each locus. In addition, isolates were compared by allozymic analysis of electrophoretic data obtained for 21 enzymes representing 23 gene loci. The results obtained from these independent techniques and different loci were essentially congruous. Analyses using G. ardeae and/or G. muris as outgroups supported the monophyly of G. intestinalis and also showed that this species includes genotypes that represent at least seven deeply rooted lineages, herein designated assemblages A-G. Inclusion of G. microti in the analysis of 18S rRNA sequence data demonstrated the monophyly of Giardia with the same median body morphology but did not support the monophyly of G. intestinalis, instead placing G. microti within G. intestinalis. The findings support the hypothesis that G. intestinalis is a species complex and suggest that G. microti is a member of this complex.     

Cellular defence mechanism of the clam Tapes semidecussatus against infection by the protozoan Perkinsus sp.

The cellular defence mechanism of the clam Tapes semidecussatus (Mollusca, Bivalvia) against infection by the parasite protozoan Perkinsus sp. (Apicomplexa, Perkinsea) was studied in the gill filaments. The parasites, localized in the connective tissue, induced a cellular reaction involving infiltrated granulocytes. These showed a secretory aspect, with the cytoplasm being filled by membrane-bound granules with internal membranes. The holocrine secretion, which was proteic and slightly glycosylated, by the granulocytes gave rise to the encapsulation of the parasites. After incubation with lectins from Canavalia ensiformis, Triticum vulgaris, Helix pomatia, Glycine max, Arachis hypogaea, Ricinus communis (agglutinin), Ulex europeus I and Limax flavus, a lack of specific and/or main sugars was observed in the plasma membrane of parasite and granulocyte, and in the wall of the former. Furthermore, GalNac1,3GalNac and -d-gal residues were only detected in association with the internal membranes and dense regions of both granules and capsule, respectively. Blood granulocytes were observed at the periphery of the cellular reaction, close to blood vessels, and these appeared to re-differentiate to give the granulocytes of the cellular reaction. The data reported here suggest that this parasite induces the infiltration and re-differentiation of specialized cells in the host mollusc. In addition, a polarized secretion of a specific defence product is described for the first time.     

Vesicles as carriers of virulence factors in parasitic protozoan diseases

Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell–cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function.     

Regulatory properties of carbamoyl-phosphate synthetase II from the parasitic protozoan Crithidia fasciculata

1. At the lowered concentrations of 0.5 mM ATP and 1.5 mM MgCl2, 2.0 mM UTP, UDP and UMP inhibited the activity of Crithidia fasciculata carbamoyl-phosphate synthetase II by about 65, 80 and 40% respectively. 2. The result suggests that feedback inhibition of the activity by uridine nucleotides is a mechanism of regulation of the de novo pyrimidine biosynthetic pathway in C. fasciculata. 3. ADP, AMP and CDP inhibited the activity (about 70, 40 and 40%). 4. Excess Mg2+ at around 1 mM, relative to the ATP concentration, was required for the maximum activity. 5. 5-Phosphoribosyl 1-pyrophosphate had no significant effect on the activity under various conditions examined.     

A novel high-affinity arginine transporter from the human parasitic protozoan Leishmania donovani

We describe the first functional and molecular characterization of an amino acid permease (LdAAP3) from the human parasitic protozoan Leishmania donovani, the causative agent of visceral leishmaniasis in humans. This permease contains 480 amino acids with 11 predicted trans-membrane domains. Expressing LdAAP3 in Saccharomyces cerevisiae mutants revealed that LdAAP3 codes for a high-affinity arginine transporter (Km 1.9 microM). LdAAP3 is highly specific for arginine as its transport was not inhibited by other amino acids or arginine-related compounds. Using green fluorescence protein (GFP) fused to the N-terminus of LdAAP3, this transporter was localized to the surface membrane of promastigotes. The GFP-LdAAP3 chimera mediated a threefold increase in arginine transport in promastigotes, indicating that it is active and confirmed that LdAAP3 codes for an arginine transporter in parasite cells as well. LdAAP3 is novel as it shares a high level of homology with amino acid permeases from other trypanosomatidae but almost none with permeases from other phyla. The results of this work suggest that LdAAP3 might play a role in host-parasite interaction.     

An isolation method of DNA from Pneumocystis carinii: a quantitative comparison to known parasitic protozoan DNA

1. A method for isolating DNA from Pneumocystis carinii is described. 2. The DNA content per nucleus is 0.22-0.34 pg. 3. This finding is consistent with other parasitic protozoa DNA content per nuclei.     

An unusual mechanism of sustained right atrial tachycardia

Lower loop re-entry (LLR) flutter is a rare type of atypical right atrial flutter. Most of the reported cases occurred in association with typical flutter patterns as a transient arrhythmia. Our case is unique in the fact the LLR was sustained and persisted independently.     

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.     

An unusual strain of Desulfovibrio sp. from an Antarctic lake

Abstract An unusual sulphate-reducing bacterium was isolated from Lake Fryxell, Antarctica. Designated strain FESu, it illustrates the difficulty in assigning some isolates of Desulfovibrio spp. to a described species. FESu was characterized by its relatively low mol% G + C (45%), its rod-shaped morphology, inability to grow on lactate, pyruvate, malate or choline in the absence of sulphate, and by its lack of desulfoviridin.     

Purification and properties of a secondary alcohol dehydrogenase from the parasitic protozoan Tritrichomonas foetus

A novel secondary alcohol dehydrogenase has been isolated from Tritrichomonas foetus, the protozoan parasite which is responsible for bovine trichomonal abortion. The enzyme has been obtained in apparently homogeneous form after a 120-fold purification from cell homogenates, thus indicating that this activity constitutes an unusually high 1% of the total cytosolic protein. The native Mr = 115,000, determined by polyacrylamide gel electrophoresis. Mobility on sodium dodecyl sulfate gels suggests that the enzyme is composed of 6-8 subunits, identical as to molecular size (Mr = 17,000). The enzyme catalyzes the reversible oxidation of 2-propanol to acetone, using NADP+ (and not NAD+) as the redox-active co-substrate. Other small secondary alcohols, such as 2-butanol, 2- and 3-pentanol, cyclobutanol, and cyclopentanol are substrates, as are the corresponding ketones of these alcohols. Primary alcohols, such as ethanol and 1-propanol, are oxidized at rates less than 5% of that observed for 2-propanol. Product inhibition studies demonstrate an ordered kinetic mechanism, wherein the co-substrate (NADP+/NADPH) binds to the enzyme prior to binding of the substrate (alcohol/ketone).     

Viral RNA-based transfection and expression of enhanced green fluorescent protein in the parasitic protozoan Eimeria stiedae

Eimeria stiedae(E.stiedae),the most common protozoan pathogen of rabbits,causes coccidiosis.This disease is problematic for rabbit breeders [1].There is little information about the cellular and molecular biology of this pathogen and a lack of genetic tools.Genetic manipulation is necessary to advance our understanding of parasite biology and ultimately develop vaccines and treat diseases.DNA-mediated and viral RNA-mediated transfection systems have successfully been established in many protozoan parasites [2,3].It was discovered that E.stiedae possesses a dsRNA virus(EsRV1),and more recently,the genome of this virus has been sequenced.To date,however,there is no report on the use of viral RNA-mediated transfection for E.stiedae.In this study,we report a transient viral RNA-mediated transfection of E.stiedae using enhanced green fluorescent protein(EGFP)as a reporter gene.Our study provides a valuable genetic tool for studying the biology of E.stiedae.     

Identification of a surface membrane proton-translocating ATPase in promastigotes of the parasitic protozoan Leishmania donovani.

ATPase activities were measured in surface membranes and mitochondria isolated from promastigotes of the parasitic protozoan Leishmania donovani. The two enzymes were differentiated on the basis of pH optima, inhibitor sensitivity and by immunochemical methods. The surface-membrane (SM-) ATPase had an activity of 100 nmol/min per mg of protein, which was optimal at pH 6.5. The enzyme was Mg2+-dependent, partially inhibited by Ca2+, and unaffected by Na+ or K+. The SM-ATPase was inhibited by orthovanadate, NN'-dicyclohexylcarbodi-imide, and N-ethylmaleimide [IC50 (concentration causing half-maximal inhibition) 7.5, 25 and 520 microM respectively]; however, it was unaffected by ouabain, azide or oligomycin. The SM-ATPase demonstrated a Km of 1.05 mM and a Vmax. of 225 nmol/min per mg of protein. Moreover, fine-structure cytochemical results demonstrated that the SM-ATPase was localized to the cytoplasmic lamina of the parasite SM. A method was devised for the isolation of SM-derived vesicles. These were used to demonstrate the proton-pumping capacity of the SM-ATPase. Cumulatively, these results constitute the first demonstration of a surface-membrane proton-translocating ATPase in a parasitic protozoan.     

IMP dehydrogenase from the intracellular parasitic protozoan Eimeria tenella and its inhibition by mycophenolic acid

Mycophenolic acid (MA) was demonstrated to be an effective inhibitor of the growth of the intracellular parasitic protozoan Eimeria tenella in tissue culture and guanine was shown to reverse this inhibition as expected for an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). A high performance liquid chromatography study of the intracellular nucleotide pools labeled with [3H]hypoxanthine was carried out in host cells lacking hypoxanthine-guanine phosphoribosyltransferase, and the depletion of guanine nucleotides demonstrated that the intracellular parasite enzyme was being inhibited by the drug. Kinetic studies carried out on the enzyme derived from E. tenella oocysts demonstrated substrate inhibition by NAD and mycophenolic acid inhibition similar to that found for mammalian enzymes, but different from that for bacterial enzymes. The inhibition by mycophenolic acid was not time-dependent and was immediately reversed upon dilution. As found previously for other IMP dehydrogenases, an Ordered Bi-Bi mechanism prevails with IMP on first followed by NAD, NADH off first, and then XMP. The kinetic patterns are consistent with substrate inhibition at high concentrations of NAD due to the formation of an E X XMP X NAD complex. Uncompetitive inhibition by MA versus IMP, NAD, and K+ was found and this was interpreted as evidence for the formation of an E X XMP X MA complex. A speculative mechanism for the inhibition of the enzyme is offered which is consistent with the fact that E X XMP X MA readily forms, whereas E X IMP X MA does not.