Association of Blastocystis ST6 with higher protease activity among symptomatic subjects.

BACKGROUND: Blastocystis sp. is an anaerobic intestinal protozoan parasite of humans and a wide range of animals worldwide. In the current study the correlation between the cysteine protease activity of clinical samples of Blastocystis sp. ST1-3 and 6 with the levels of pro-inflammatory cytokines was evaluated. METHODS: Stool samples were collected from subjects with or without clinical symptoms. All samples were cultivated in DMEM medium. The bacteria were eliminated or reduced in Blastocystis sp. positive samples subtypes 1-3 and 6 by a variety of antibiotics and consecutive sub-cultures. To prepare parasite lysate, 1 × 105 Blastocystis sp. from each isolate were harvested and lysed using freeze-thaw. Protease activity of each isolate was measured and the gene expression of pro-inflammatory biomarkers in HT-29 cell line sensed by isolates was investigated using quantitative Real-time PCR. RESULTS: Protease activity assay showed inter- and intra-subtype variations among subtypes regarding the presence of symptoms, while the protease activity of symptomatic isolates was higher than asymptomatic isolates. The highest and lowest levels of protease activity were seen in ST6 and ST2, respectively. However, patterns of the expression of pro-inflammatory biomarkers in HT-29 cell line was different regarding the presence of symptoms and time points. There was no significant correlation between protease activity of different subtypes with the expression levels of pro-inflammatory biomarkers. CONCLUSIONS: Our study indicated a higher protease activity among isolates from symptomatic compared to asymptomatic subjects, suggesting functional role for proteases in clinical symptoms due to Blastocystis sp. The lack of correlation between the levels of expression of pro-inflammatory biomarkers with subtypes regarding the presence of clinical symptoms proposes the importance of host-related factors in presentation of clinical symptoms.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers.

Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases.

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r)) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton and ZO-1. These alterations were abolished by inhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barrier-compromise, suggesting a therapeutic potential of statins in intestinal infections.

Blastocystis legumain is localized on the cell surface, and specific inhibition of its activity implicates a pro-survival role for the enzyme.

Programmed cell death (PCD) is crucial for cellular growth and development in multicellular organisms. Although distinct PCD features have been described for unicellular eukaryotes, homology searches have failed to reveal clear PCD-related orthologues among these organisms. Our previous studies revealed that a surface-reactive monoclonal antibody (mAb) 1D5 could induce multiple PCD pathways in the protozoan Blastocystis. In this study, we identified, by two-dimensional gel electrophoresis and mass spectrometry, the target of mAb 1D5 as a surface-localized legumain, an asparagine endopeptidase that is usually found in lysosomal/acidic compartments of other organisms. Recombinant Blastocystis legumain displayed biphasic pH optima in substrate assays, with peaks at pH 4 and 7.5. Activity of Blastocystis legumain was greatly inhibited by the legumain-specific inhibitor carbobenzyloxy-Ala-Ala-AAsn-epoxycarboxylate ethyl ester (APE-RR) (where AAsn is aza-asparagine) and moderately inhibited by mAb 1D5, cystatin, and caspase-1 inhibitor. Interestingly, inhibition of legumain activity induced PCD in Blastocystis, observed by increased externalization of phosphatidylserine residues and in situ DNA fragmentation. In contrast to plants, in which legumains have been shown to play a pro-death role, legumain appears to display a pro-survival role in Blastocystis.

A metronidazole-resistant isolate of Blastocystis spp. is susceptible to nitric oxide and downregulates intestinal epithelial inducible nitric oxide synthase by a novel parasite survival mechanism.

Blastocystis, one of the most common parasites colonizing the human intestine, is an extracellular, noninvasive, luminal protozoan with controversial pathogenesis. Blastocystis infections can be asymptomatic or cause intestinal symptoms of vomiting, diarrhea, and abdominal pain. Although chronic infections are frequently reported, Blastocystis infections have also been reported to be self-limiting in immunocompetent patients. Characterizing the host innate response to Blastocystis would lead to a better understanding of the parasite's pathogenesis. Intestinal epithelial cells produce nitric oxide (NO), primarily on the apical side, in order to target luminal pathogens. In this study, we show that NO production by intestinal cells may be a host defense mechanism against Blastocystis. Two clinically relevant isolates of Blastocystis, ST-7 (B) and ST-4 (WR-1), were found to be susceptible to a range of NO donors. ST-7 (B), a metronidazole-resistant isolate, was found to be more sensitive to nitrosative stress. Using the Caco-2 model of human intestinal epithelium, Blastocystis ST-7 (B) but not ST-4 (WR-1) exhibited dose-dependent inhibition of Caco-2 NO production, and this was associated with downregulation of inducible nitric oxide synthase (iNOS). Despite its higher susceptibility to NO, Blastocystis ST-7 (B) may have evolved unique strategies to evade this potential host defense by depressing host NO production. This is the first study to highlight a strain-to-strain variation in the ability of Blastocystis to evade the host antiparasitic NO response.

The nucleotide specificity of succinyl-CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone.

Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl-CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild-type), generated by refolding of the individual P. falciparum SCSß and Blastocystis SCSα subunits, demonstrated ADP-forming activity (KmATP  = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSß subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required.

Characterisation of novel functionality within the Blastocystis tryptophanase gene.

In recent years, the human gut microbiome has been recognised to play a pivotal role in the health of the host. Intestinal homeostasis relies on this intricate and complex relationship between the gut microbiota and the human host. While much effort and attention has been placed on the characterization of the organisms that inhabit the gut microbiome, the complex molecular cross-talk between the microbiota could also exert an effect on gastrointestinal conditions. Blastocystis is a single-cell eukaryotic parasite of emerging interest, as its beneficial or pathogenic role in the microbiota has been a subject of contention even to-date. In this study, we assessed the function of the Blastocystis tryptophanase gene (BhTnaA), which was acquired by horizontal gene transfer and likely to be of bacterial origin within Blastocystis. Bioinformatic analysis and phylogenetic reconstruction revealed distinct divergence of BhTnaA versus known bacterial homologs. Despite sharing high homology with the E. coli tryptophanase gene, we show that Blastocystis does not readily convert tryptophan into indole. Instead, BhTnaA preferentially catalyzes the conversion of indole to tryptophan. We also show a direct link between E. coli and Blastocystis tryptophan metabolism: In the presence of E. coli, Blastocystis ST7 is less able to metabolise indole to tryptophan. This study examines the potential for functional variation in horizontally-acquired genes relative to their canonical counterparts, and identifies Blastocystis as a possible producer of tryptophan within the gut.

Staurosporine-induced programmed cell death in Blastocystis occurs independently of caspases and cathepsins and is augmented by calpain inhibition.

Previous studies have shown that the protozoan parasite Blastocystis exhibits apoptotic features with caspase-like activity upon exposure to a cytotoxic monoclonal antibody or the anti-parasitic drug metronidazole. The present study reports that staurosporine (STS), a common apoptosis inducer in mammalian cells, also induces cytoplasmic and nuclear features of apoptosis in Blastocystis, including cell shrinkage, phosphatidylserine (PS) externalization, maintenance of plasma membrane integrity, extensive cytoplasmic vacuolation, nuclear condensation and DNA fragmentation. STS-induced PS exposure and DNA fragmentation were abolished by the mitochondrial transition pore blocker cyclosporine A and significantly inhibited by the broad-range cysteine protease inhibitor iodoacetamide. Interestingly, the apoptosis phenotype was insensitive to inhibitors of caspases and cathepsins B and L, while calpain-specific inhibitors augmented the STS-induced apoptosis response. While the identities of the proteases responsible for STS-induced apoptosis warrant further investigation, these findings demonstrate that programmed cell death in Blastocystis is complex and regulated by multiple mediators.

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.

Blastocystis ratti contains cysteine proteases that mediate interleukin-8 response from human intestinal epithelial cells in an NF-kappaB-dependent manner.

Blastocystis is a ubiquitous enteric protozoan found in the intestinal tracts of humans and a wide range of animals. Evidence accumulated over the last decade suggests association of Blastocystis with gastrointestinal disorders involving diarrhea, abdominal pain, constipation, nausea, and fatigue. Clinical and experimental studies have associated Blastocystis with intestinal inflammation, and it has been shown that Blastocystis has potential to modulate the host immune response. Blastocystis is also reported to be an opportunistic pathogen in immunosuppressed patients, especially those suffering from AIDS. However, nothing is known about the parasitic virulence factors and early events following host-parasite interactions. In the present study, we investigated the molecular mechanism by which Blastocystis activates interleukin-8 (IL-8) gene expression in human colonic epithelial T84 cells. We demonstrate for the first time that cysteine proteases of Blastocystis ratti WR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial cells. Furthermore, we show that NF-kappaB activation is involved in the production of IL-8. In addition, our findings show that treatment with the antiprotozoal drug metronidazole can avert IL-8 production induced by B. ratti WR1. We also show for the first time that the central vacuole of Blastocystis may function as a reservoir for cysteine proteases. Our findings will contribute to an understanding of the pathobiology of a poorly studied parasite whose public health importance is increasingly recognized.

Can the pyruvate: ferredoxin oxidoreductase (PFOR) gene be used as an additional marker to discriminate among Blastocystis strains or subtypes?

BACKGROUND: Blastocystis spp. are the most prevalent intestinal eukaryotes identified in humans, with at least 17 genetic subtypes (ST) based on genes coding for the small-subunit ribosomal RNA (18S). It has been argued that the 18S gene should not be the marker of choice to discriminate between STs of these strains because this marker exhibits high intra-genomic polymorphism. By contrast, pyruvate:ferredoxin oxidoreductase (PFOR) is a relevant enzyme involved in the core energy metabolism of many anaerobic microorganisms such as Blastocystis, which, in other protozoa, shows more polymorphisms than the 18S gene and thus may offer finer discrimination when trying to identify Blastocystis ST. Therefore, the objective of the present study was to assess the suitability of the PFOR gene as an additional marker to discriminate among Blastocystis strains or subtypes from symptomatic carrier children. METHODS: Faecal samples from 192 children with gastrointestinal symptoms from the State of Mexico were submitted for coprological study. Twenty-one of these samples were positive only for Blastocystis spp.; these samples were analysed by PCR sequencing of regions of the 18S and PFOR genes. The amplicons were purified and sequenced; afterwards, both markers were assessed for genetic diversity. RESULTS: The 18S analysis showed the following frequencies of Blastocystis subtypes: ST3 = 43%; ST1 = 38%; ST2 = 14%; and ST7 = 5%. Additionally, using subtype-specific primer sets, two samples showed mixed Blastocystis ST1 and ST2 infection. For PFOR, Bayesian inference revealed the presence of three clades (I-III); two of them grouped different ST samples, and one grouped six samples of ST3 (III). Nucleotide diversity (π) and haplotype polymorphism (θ) for the 18S analysis were similar for ST1 and ST2 (π = ~0.025 and θ = ~0.036); remarkably, ST3 showed almost 10-fold lower values. For PFOR, a similar trend was found: clade I and II had π = ~0.05 and θ = ~0.05, whereas for clade III, the values were almost 6-fold lower. CONCLUSIONS: Although the fragment of the PFOR gene analysed in the present study did not allow discrimination between Blastocystis STs, this marker grouped the samples in three clades with strengthened support, suggesting that PFOR may be under different selective pressures and evolutionary histories than the 18S gene. Interestingly, the ST3 sequences showed lower variability with probable purifying selection in both markers, meaning that evolutionary forces drive differential processes among Blastocystis STs.

Mitochondrial Glycolysis in a Major Lineage of Eukaryotes.

The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.

Higher Caspase-like activity in symptomatic isolates of Blastocystis spp.

BACKGROUND: Biochemical evidence of a caspase-like execution pathway has been demonstrated in a variety of protozoan parasites, including Blastocystis spp. The distinct differences in the phenotypic characterization reported previously have prompted us to compare the rate of apoptosis in Blastocystis spp. isolated from individuals who were symptomatic and asymptomatic. In the current study, we analysed the caspase activation involved in PCD mediated by a cytotoxic drug, (metronidazole) in both symptomatic & asymptomatic isolates. METHODS: Apoptosis was induced in Blastocystis spp. by treating cultures of symptomatic and asymptomatic isolates of 3 sub-types namely 1, 3 and 5 with two different concentrations, 0.1 and 0.0001 mg/ml of metronidazole (with and without pre-treatment with a pan-caspase inhibitor, zVAD.fmk). The experiment was repeated to assess the number of apoptotic cells in all the isolates of both conditions. RESULTS: Symptomatic isolates of subtype 3 (without pre-treatment with a pan-caspase inhibitor, zVAD.fmk) showed high fluorescence intensity for active caspase-like proteases [0.0001 mg/ml, 88% (p < 0.001) at 0.1 mg/ml, 70% (p < 0.001)] at the 72nd hour in vitro culture in comparison with asymptomatic isolates [0.0001 mg/ml, 65%, at 0.1 mg/ml, 55%]. The number of apoptotic cells was higher [0.0001 mg/ml, 89% (p < 0.001) and at 0.1 mg/ml, 70% (p < 0.001)] at the 72nd hour of in vitro culture in comparison with asymptomatic isolates [0.0001 mg/ml, 66% (p < 0.001) and at 0.1 mg/ml, 45% (p < 0.01)]. Cells treated with metronidazole in the presence of zVAD.fmk showed less than 10% caspase activation. CONCLUSION: The high number of symptomatic cells expressing active caspase-like proteases and becoming apoptotic compared to asymptomatic cells clearly demonstrates that the response to metronidazole treatment is isolate dependent. Hence this justifies the conflicting reports on the curative success rates when treated with this drug. The study has also created a need to identify apoptosis effectors in Blastocystis spp of different isolates especially as it was shown that apoptosis was sub-typed related. These findings can be exploited for the development of diagnostic markers and novel therapeutic drugs to enhance the effectiveness of the diagnosis and treatment of the patients infected with Blastocystis spp.

Amoebic forms of Blastocystis spp. - evidence for a pathogenic role.

BACKGROUND: Blastocystis spp. are one of the most prevalent parasites isolated from patients suffering from diarrhea, flatulence, constipation and vomiting. It's pathogenicity and pathophysiology remains controversial to date. Protease activity and amoebic forms have been reported previously in symptomatic isolates but there has been no conclusive evidence provided to correlate the protease activity and any specific life cycle stage of the parasite thus far. METHODS: Symptomatic isolates with amoebic form were tested for protease activity and compared with symptomatic and asymptomatic isolates without amoebic form for 10 days culture period. RESULTS: The present study demonstrates an elevated protease activity in cultures having a higher percentage of amoebic forms seen in symptomatic isolates. The growth curve demonstrated a significantly (p < 0.05) higher average number of parasite counts in asymptomatic compared to symptomatic isolates. Symptomatic isolates showed amoebic forms with percentages ranging from 5% to 17%. Elevated protease activity was demonstrated in isolates that had higher percentages of amoebic forms with intense bands at higher molecular weight proteases (60 - 100 kDa). As days of culture proceeded, the protease quantification also showed a steady increase. CONCLUSION: This study elucidates a correlation between protease activity and percentage of amoebic forms. The finding implies that these forms could play a role in exacerbation of intestinal symptoms during Blastocystis spp. infection.

Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite.

Blastocystis spp. are unicellular anaerobic intestinal parasites of both humans and animals and the most prevalent ones found in human stool samples. Their association with various gastrointestinal disorders raises the questions of its pathogenicity and of the molecular mechanisms involved. Since secreted proteases are well-known to be implicated in intestinal parasite virulence, we intended to determine whether Blastocystis spp. possess such pathogenic factors. In silico analysis of the Blastocystis subtype 7 (ST7) genome sequence highlighted 22 genes coding proteases which were predicted to be secreted. We characterized the proteolytic activities in the secretory products of Blastocystis ST7 using specific protease inhibitors. Two cysteine proteases, a cathepsin B and a legumain, were identified in the parasite culture supernatant by gelatin zymographic SDS-PAGE gel and MS/MS analysis. These proteases might act on intestinal cells and disturb gut function. This work provides serious molecular candidates to link Blastocystis spp. and intestinal disorders.

Blastocystis exhibits inter- and intra-subtype variation in cysteine protease activity.

Blastocystis is an enteric protistan parasite of zoonotic potential and poorly understood pathogenesis. We have previously reported that Blastocystis cysteine proteases can degrade human secretory IgA and are also responsible for the induction of IL-8 response in colonic epithelial cells in vitro. Differences in virulence between Blastocystis subtypes have been reported recently in both animal models and clinical studies, although cellular mechanisms for these differences are currently unknown. Parasites such as Giardia intestinalis and Entamoeba histolytica have distinct virulent and non-virulent strains which may be attributable to variations in their cysteine proteases. In the present study, variations in cysteine protease activity was observed between avian (subtype 7) and rodent (subtype 4) isolates of Blastocystis with avian isolates exhibiting approximately two times higher peak cysteine protease activity than rodent isolates. Cysteine protease activity and parasite cell size varied over time within cultures of the same isolate. An association between parasite cell size and protease activity was observed.

Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7.

A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml(-1). Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP(+) oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD(+) or FMN(+) as electron acceptor but is inactive with NAD(+), Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.

Degradation of human secretory immunoglobulin A by Blastocystis.

Microbial immunoglobulin A (IgA) proteases cleave human secretory IgA, promoting the mucosal adhesion of pathogens. To investigate if the enteric protozoan Blastocystis degrades human secretory IgA, cell lysate and conditioned medium from two species were exposed to immunoglobulin A. Secretory IgA was cleaved by both cell lysate and conditioned medium with mainly cysteine proteinase activity in B. hominis B isolate and aspartic proteinase activity in B. ratii WR1 isolate. These findings suggest that Blastocystis proteases may play a role in parasite survival in vivo.