Protists: Eukaryotic single-celled organisms and the functioning of their organelles.

Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the diversity and function of protistan organelles has increased in recent years, providing a complex panoply of structure/function relationships. This is particularly noticeable with the description of hydrogenosomes, and the diverse array of structures that followed, having hybrid hydrogenosome/mitochondria attributes.

Commitment to cyst formation in Giardia.

Giardia trophozoites differentiate into infectious cysts (encystment) in response to physiological stimuli; encystment is crucial for Giardia's transmission, survival and pathogenesis. In vitro, Giardia encysts when bile sequesters lipids necessary for this lipid auxotroph, and in vivo they encyst to infect new hosts. In this study, we investigated, for the first time, commitment to encystment in Giardia using both molecular and cellular techniques.

Metabolomics and protozoan parasites.

In this review, we examine the state-of-the-art technologies (gas and liquid chromatography, mass spectroscopy and nuclear magnetic resonance, etc.) in the well-established area of metabolomics especially as they relate to protozoan parasites.

Balamuthia mandrillaris: role of galactose in encystment and identification of potential inhibitory targets.

Balamuthia mandrillaris is a causative agent of granulomatous encephalitis that almost always proves fatal. A major concern during the course of therapy is that B. mandrillaris can transform into cysts.

Carbohydrate analysis of Acanthamoeba castellanii.

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting "cyst wall" material was subsequently lyophilized and subjected to glycosyl composition analysis.

Balamuthia mandrillaris: staining properties of cysts and trophozoites and the effect of 2,6-dichlorobenzonitrile and calcofluor white on encystment.

Here, we determined the staining properties of Balamuthia mandrillaris cysts, and assessed the effect of 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, and calcofluor white, a brightening agent, on its encystment. Periodic acid-Schiff reagent stained the inner wall intensely and middle and outer walls weakly suggesting that the cyst wall of B. mandrillaris may contain glycans.

The cyst wall carbohydrate composition of Balamuthia mandrillaris.

Balamuthia mandrillaris is an opportunistic cyst-producing amoeba that can cause rare, but fatal, Balamuthia amoebic encephalitis (BAE). Cysts are resistant to harsh environmental conditions and many antimicrobial compounds and thus can contribute to BAE recurrence. However, little is known of cyst wall synthesis, cyst wall composition, or how encystment is induced.

UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity.

The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a beta (1-3) polymer of N-acetylgalactosamine.

Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis.

Background: Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined.

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts.

Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall.

Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall of Giardia and is synthesized by inducible enzymes in the cytosol of encysting trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encysting Giardia is reported, and this activity exclusively incorporates [1-(14)C]GalNAc (from UDP-[(14)C]GalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall of GIARDIA: This ethanol precipitate exhibits characteristics of Giardia cyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS).

Menadione kills trophozoites and cysts of Giardia intestinalis.

Production of reactive oxygen species by redox cycling in the presence of low levels of oxygen has been studied as a possible approach to anti-protozoal chemotherapeutic strategy. Incubation of the diplomonad flagellate Giardia intestinalis with 2-methy-1,4-naphthoquinone (menadione), under anaerobic conditions, gave UV absorption changes characteristic of reduction to menadiol; partial reversal was observed on admitting O(2). Under microaerobic conditions, similar to those on the surface of the jejunal mucosa, trophozoites consumed O(2) rapidly in the presence of menadione; reaction products included singlet O(2) (monitored by single photon counting of O(2)-dependent low-level chemiluminescence) and H(2)O(2) (measured by the formation of Complex I of microperoxidase).

Amino sugar phosphate levels in Giardia change during cyst wall formation.

The parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique beta(1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates.

Potential drug targets in cyst-wall biosynthesis by intestinal protozoa.

Given that resistance to antiprotozoal drugs exists and is likely to increase and given that currently no reliable treatments exist for some of these infections, efforts to find new targets for chemotherapy must be continued. In the case of cyst-forming pathogenic protozoa, one such target might be encystment pathways and cyst-wall assembly. Information is increasing on protozoan encystment and, as it does, we can begin to answer the question of whether targeting it for chemotherapy is a viable drug strategy.

Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis.

The cyst wall of Giardia intestinalis contains proteins and a novel N-acetylgalactosamine (GalNAc) polysaccharide, which is its major constituent. GalNAc is not present in growing trophozoites, but is synthesized during encystment via an inducible pathway of enzymes that produce UDP-GalNAc from fructose 6-phosphate. This report focuses on the regulation of these enzymes and thus the genes for glucosamine 6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (AGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and UDP-N-acetylglucosamine 4-epimerase (UAE) were cloned and expressed in Escherichia coli.

Molecular and physiological differentiation between pathogenic and nonpathogenic Acanthamoeba.

In this study, 14 isolates of Acanthamoeba from both clinical and environmental sources belonging to seven different species were assayed for tolerance of high osmotic pressure, temperature tolerance, extracellular proteases, and cytopathic effects (CPE) on immortalized rabbit corneal epithelial cells. On the basis of the results, amoeba isolates were divided into pathogenic and nonpathogenic groups. Ribosomal DNA sequencing was performed on these isolates.

The Giardia intestinalis filamentous cyst wall contains a novel beta(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study.

Assembly of a protective cyst wall by Giardia is essential for the survival of the parasite outside the host intestine and for transmission among susceptible hosts. The structure of the G. intestinalis filamentous cyst wall was studied by chemical methods, mass spectrometry, and (1)H nuclear magnetic resonance spectroscopy.

Acanthamoeba can be differentiated by the polymerase chain reaction and simple plating assays.

Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells.

Regulation of carbohydrate metabolism during Giardia encystment.

Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-D-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake.

Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba.

Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens.

UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated.

Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 microm). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine 6-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below.

Detection of Giardia Trophozoites in Archival Pathology Specimens of Human Small Intestine.

: The examination of archival pathology specimens of human small intestine by light microscopy, field emission scanning electron microscopy (FESEM), and confocal scanning laser microscopy using fluorescent in situ hybridization (FISH) techniques was undertaken to better understand the epidemiology of Giardia. Giardia trophozoites were tentatively identified in the light microscope after hematoxylin and eosin (H&E) staining. The organisms were adherent to the intestinal epithelium where they were also associated with strands of mucus within the lumen.

Cloning of two putative Giardia lamblia glucosamine 6-phosphate isomerase genes only one of which is transcriptionally activated during encystment.

The biosynthesis of the carbohydrate component of the cyst wall of the protozoan parasite Giardia lamblia, a polymer of N-acetylgalactosamine (GalNac), is by a pathway that is initiated with the conversion of fructose 6-phosphate to glucosamine 6-phosphate by an aminating isomerase, glucose 6-phosphate isomerase. This enzyme appears only after Giardia trophozoites are induced to start the production of cyst wall components after bile is added. To investigate whether induction of glucosamine 6-phosphate isomerase is by protein modification or by transcription activation, its gene was cloned and sequenced.