Proteogenomic Insights into the Intestinal Parasite Blastocystis sp. Subtype 4 Isolate WR1.

Blastocystis sp. is known for years as a highly prevalent anaerobic eukaryotic parasite of humans and animals. Several monophyletic clades have been delineated based on molecular data, and the occurrence of each subtype in humans and/or animal hosts has been documented. The genome of several representatives has been sequenced revealing specific traits such as an intriguing 3'-end processing of primary transcripts. Here, a first high-throughput proteomics dataset acquired on this difficult-to-cultivate parasite is presented for the zoonotic subtype T4 isolate WR1. Amongst the 2766 detected proteins, we highlighted the role of a small ADP ribosylation factor GTP-binding protein involved in intracellular traffic as major regulator of vesicle biogenesis and a voltage-dependent anion-selective channel protein because both were unexpectedly highly abundant. We show how these data may be used for gaining proteogenomics insights into Blastocystis sp. specific molecular mechanisms. We evidenced for the first time by proteogenomics a functional termination codon derived from transcript polyadenylation for seven different key cellular components.

A large number of nuclear genes in the human parasite blastocystis require mRNA polyadenylation to create functional termination codons.

Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However, in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3'-ends of hundreds of genes. From sequence data we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus, the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3'-untranslated regions to the point that, in many genes, only one or two nucleotides of the termination codon are left.

Subtype determination of Blastocystis isolates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

The pathogenic role of the enteric parasite Blastocystis remains controversial. Recent studies have suggested that various subtypes (STs) found in human samples could be correlated to the presence or absence and variability of clinical manifestations, and that STs can differ with respect to drug sensitivity. Polymerase chain reaction (PCR) techniques used to determine these STs are expensive and are usually restricted to research laboratory settings. This study evaluates the potential application of the inexpensive matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) technique to discriminate Blastocystis STs. A database of parasitic protein signatures was constructed for five Blastocystis STs, and the reference spectra were challenged with those from 19 axenic cultures of ST1, ST2, ST3, ST4 and ST8 and those from nine xenic liquid cultures of ST3 and ST4. Samples from axenic cultures were prepared using standard formic acid extraction and direct deposition procedures. The reference spectra revealed five distinct spectral profiles, and the database library allowed for discrimination between all of the cultures with reliability indices ranging from 2.038 to greater than 2.8 when an extraction was performed. The direct deposition procedure resulted in greater variability in the discrimination and direct MALDI-TOF MS identification from xenic liquid cultures was effective in 3 out of 9 samples. MALDI-TOF MS proved to be an effective technology for efficiently discriminating Blastocystis STs in axenic cultures.

Blastocystis surface antigen is stable in chemically preserved stool samples for at least 1 year.

Blastocystis spp. refer to a group of prevalent enteric protists found in humans and animals. Detection of Blastocystis spp. in fecal samples is often performed by clinicians with direct microscopy, which provides low sensitivity, or with culture and polymerase chain reaction testing, a method which is problematic when used with formalin-preserved stool samples. Prior study of Blastocystis and other enteric protists suggests that immunofluorescence antibody (IFA) stain could provide sensitivity and compatibility with formalin, but no information is available on the longevity of Blastocystis sp.'s surface antigens in formalin. We collected fecal samples from animals at a country fair held in the summer of 2009 in Oregon, USA. Samples were tested for the presence of Blastocystis infection using an IFA stain shortly after collection, and again after 1 year, with samples stored refrigerated at 4-8 °C. Most samples collected from steer, pigs, and goats were found to be Blastocystis positive. All fecal samples that were Blastocystis positive initially remained positive after 1 year. Blastocystis-negative samples remained negative. Minimal degradation was observed in stained slides. Blastocystis surface antigens detected by a polyclonal stain remained stable in formalin for a period of at least 1 year.

Localization and nucleotide specificity of Blastocystis succinyl-CoA synthetase.

The anaerobic lifestyle of the intestinal parasite Blastocystis raises questions about the biochemistry and function of its mitochondria-like organelles. We have characterized the Blastocystis succinyl-CoA synthetase (SCS), a tricarboxylic acid cycle enzyme that conserves energy by substrate-level phosphorylation. We show that SCS localizes to the enigmatic Blastocystis organelles, indicating that these organelles might play a similar role in energy metabolism as classic mitochondria. Although analysis of residues inside the nucleotide-binding site suggests that Blastocystis SCS is GTP-specific, we demonstrate that it is ATP-specific. Homology modelling, followed by flexible docking and molecular dynamics simulations, indicates that while both ATP and GTP fit into the Blastocystis SCS active site, GTP is destabilized by electrostatic dipole interactions with Lys 42 and Lys 110, the side-chains of which lie outside the nucleotide-binding cavity. It has been proposed that residues in direct contact with the substrate determine nucleotide specificity in SCS. However, our results indicate that, in Blastocystis, an electrostatic gatekeeper controls which ligands can enter the binding site.

Freeze-fracture and cytochemical studies on the in vitro cyst form of reptilian Blastocystis pythoni.

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.

Characterization of protein profiles and cross-reactivity of Blastocystis antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis.

The protein profiles of Blastocystis hominis, B. lapemi, and B. ratti were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and species could be differentiated by this means as well as by Western-blot analysis with polyclonal antibodies. No intraspecies difference could be distinguished between the two B. hominis isolates or the three B. ratti isolates. Western-blot analysis showed extensive cross-reactivity of B. lapemi and B. hominis antigens with anti-B. ratti serum. Some of the cross-reactive antigens were glycoproteins as determined on the basis of their sensitivity to periodate treatment.

In vitro encystation of Blastocystis hominis: a kinetics and cytochemistry study.

The kinetics of in vitro encystation of Blastocystis hominis was studied over 9 days. The differentiation between trophic (TF) and cyst forms (CF) was determined by differential counts before and after treatment with distilled water. A cytochemistry study using acridine orange and Calcofluor white wet-mount preparations of CF was carried out. The growth curves of TF and CF were related because the decrease in TF was followed by an increase in CF, and vice versa. The maximum of CF counts was obtained on the 6th or 7th day. Using the differential acridine orange stain, two subpopulations of CF, yellow-orange fluorescent cells or precysts and green fluorescent cells or cysts, were detected and their curves were also related. CF was stained by Calcofluor white, which suggested the existence of beta-(1-4)-glycosyl residues in the wall cysts of B. hominis.