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 andZO-1. Thesealterations were abolished byinhibition 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 barriercompromise, suggesting a therapeutic potential of statins in intestinal infections.     

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.     

Molecular epidemiology of Blastocystis infections in Turkey

Blastocystis is a very common unicellular intestinal parasite of ubiquitous occurrence. In order to describe the molecular epidemiology of Blastocystis infections in Turkey, 87 isolates from 69 symptomatic and 18 asymptomatic individuals were sequenced. Sequence data were phylogenetically analyzed and statistically tested against unmodifiable risk factors such as gender and age. Blastocystis-positive males were complaining mainly of gastroenteritis, whereas dyspepsia was the chief complaint among Blastocystis-positive females. Blastocystis sp. subtypes detected in the study included subtypes 1, 2, 3 and 4, subtype 3 being the most predominant (75.9%). No association was detected between Blastocystis sp. subtype and symptoms (p>0.365), or between infection intensity and symptoms (p>0.441). There was a tendency of subtype 2 isolates being more common among older study individuals, and subtype 2 isolates were significantly associated with higher parasite abundance (p=0.017). Compared to data from similar studies, the distribution of Blastocystis sp. isolates in Turkey was found to more or less reflect the one seen in other countries, and it was deduced that subtype 3 is generally by far the most common subtype infecting humans, followed by subtypes 1, 2 and 4.     

Blastocystis: past pitfalls and future perspectives

Blastocystis is a genetically heterogeneous protist found in the intestinal tract (IT) of many vertebrates, and although it is implicated in a variety of human intestinal disorders, data regarding the clinical relevance of Blastocystis is at best speculative. Several research issues, including a lack of standardization across studies, the potential for intrasubtype variation in pathogenicity, and difficulties associated with diagnostics for many idiopathic disorders of the human IT have led to conflicting reports in support of a role for Blastocystis pathogenicity. Here, several research areas and methodologies are reviewed that if integrated appropriately into a prospective study may prove useful and facilitate a better understanding of the role of Blastocystis in human health and disease.     

Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH

The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.     

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-κB 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.     

Blastocystis: subtyping isolates using pyrosequencing technology

Blastocystis is a prevalent single-celled enteric parasite of unresolved clinical significance. Efforts based on molecular methodologies to establish whether pathogenicity is linked to specific isolates of the genetically diverse genus of Blastocystis have been scarce and so far yielded ambiguous results which can be difficult to interpret. To alleviate some of the problems related to unravelling the molecular epidemiology of Blastocystis infections we developed and evaluated a simple and high-throughput sequence analysis (SQA) pyrosequencing technique based on the detection of genotype-specific nucleotide polymorphisms in the 18S small subunit rRNA gene for a rapid and cost-effective post-PCR screening of Blastocystis genotypes. The method was effectively capable of genotyping 48/48 isolates positive by nested PCR in approximately one hour, and in 94% of the cases the isolate detected by PCR and pyrosequencing was also detected by one of two different PCR assays with subsequent dideoxy sequencing.     

Biochemical aspects of egg hatch in endo- and ectoparasites: potential for rational drug design.

Control of parasites through rational drug design requires a thorough understanding of the parasite's lifecycle encompassing the biochemical and physiological processes which contribute to normal parasite homeostasis. The hatching of parasite eggs for example, represents an important process in the development of a parasitic infection. Previous studies in helminths have indicated that secreted enzymes often facilitate successful endoparasite egg hatch. In contrast, there are relatively few examples demonstrating a role for secreted enzymes in the egg hatching process of insects. An analysis of this process in the ectoparasite Lucilia cuprina suggests a role for secreted enzymes in the hatching of sheep blowfly eggs. Characterisation of the proteases collected at the time of egg hatch indicates the presence of serine proteases. Further purification and characterisation of these proteases may enable the design of specific inhibitors to interfere with the egg hatch process and therefore provide a novel means of control.     

Terminology for Blastocystis subtypes--a consensus

Blastocystis is a ubiquitous enteric protistan parasite that has extensive genetic diversity and infects humans and many other animals. Distinct molecular methodologies developed to detect variation and obtain information about transmission patterns and clinical importance have resulted in a confusing array of terminologies for the identification and designation of Blastocystis subtypes. In this article, we propose a standardization of Blastocystis terminology to improve communication and correlate research results. Based primarily on published small-subunit ribosomal RNA gene analyses, we propose that all mammalian and avian isolates should be designated Blastocystis sp. and assigned to one of nine subtypes.     

Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum

Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting approximately 600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three beta-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.     

Characterization of proteases secreted by leek roots

Some characteristics of root-secreted proteases were studied. We measured their molecular weights and mechanism of BSA digestion in comparison to endogenous root proteases. We related these studies to culture medium N composition. The seedlings of Allium porrum L. (cv. Bartek) were cultivated on MS medium, MS without inorganic nitrogen (MS-IN), and MS without IN, but with 0.1% casein (MS-IN + 0.1% casein). Electrophoretic study showed that root-secreted proteases had one isoform with a mol wt of 45 kD, regardless of medium N composition. Difference in molecular weights of root-secreted proteases and endogenous root proteases active under used conditions (>66 kD) provide us another strong evidence that root-secreted proteases were not just leaking from the roots, but they were secreted. Proteases exuded by roots degraded BSA in a similar way as endogenous proteases, with only one SDS-PAGE-detectable product of degradation. Our results may be a powerful tool in the extraction and purification of these enzymes and also in proteomic studies.     

In Vitro Response of Blastocystis hominis to Antiprotozoal Drugs

Ten antiprotozoal drugs were tested in vitro against four axenic strains of the intestinal parasite Blastocystis hominis. Inhibitory drugs in order of effectiveness were emetine, metronidazole, furazolidone, trimethoprim sulfamethoxazole, 5-chloro-8-hydroxy-7-iodo-quinoline (Entero-Vioform), and pentamidine. Moderately inhibitory were two quinolines other than iodochlorhydroxquin. These were chloroquine and 5, 7-diiodo-8-hydroxy-quinoline(Floraquin). Diloxanide furoate was not inhibitory. Paromomycin and other antibiotics were not inhibitory. Entero-Vioform and metronidazole have been effective in human and higher primate diarrhea caused by B. hominis.     

Phylogenetic position of Blastocystis hominis and of stramenopiles inferred from multiple molecular sequence data

Blastocystis hominis, a parasite of the human intestine, has recently been positioned within stramenopiles by the small subunit rRNA phylogeny. To further confirm its phylogenetic position using multiple molecular sequence data, we determined the nucleotide sequences putatively encoding small subunit ribosomal RNA, cytosolic-type 70-kDa heat shock protein, translation elongation factor 2, and the non-catalytic 'B' subunit of vacuolar ATPase of B. hominis (HE87-1 strain). Moreover, we determined the translation elongation factor 2 sequence of an apicomplexan parasite, Plasmodium falciparum, that belongs to alveolates. The maximum likelihood analyses of small subunit rRNA and cytosolic-type 70-kDa heat shock protein clearly demonstrated that B. hominis (HE87-1 strain) is positioned within stramenopiles, being congruent with the previous small subunit rRNA analysis, including the sequences of B. hominis (Nand strain) and a Blastocystis isolate from guinea pig. Although no clear resolution among major eukaryotic groups was obtained by the individual phylogenies based on the four molecules analyzed here, a combined analysis of various molecules, including these, clearly indicated that Blastocystis/stramenopiles are the closest relatives of alveolates.     

Blastocystis hominis, a long-misunderstood intestinal parasite

For over 50 years, Blastocystis hominis has been held to be a harmless intestinal yeast-probably frequent in stool samples from man and other primates, but usually ignored except as a possible source of confusion with Entamoeba histolytica. More recently, its status as a protozoan parasite has been accepted, and it is now increasingly recognized as an agent of intestinal disease - usually self-limiting but occasionally fatal in monkeys. Here, Charles Zierdt reviews the status o f this intriguing protozoan, drawing attention to its unusual biochemistry.     

Metagenomics for broad and improved parasite detection: a proof-of-concept study using swine faecal samples

Efficient and reliable identification of emerging pathogens is crucial for the design and implementation of timely and proportionate control strategies. This is difficult if the pathogen is so far unknown or only distantly related with known pathogens. Diagnostic metagenomics – an undirected, broad and sensitive method for the efficient identification of pathogens – was frequently used for virus and bacteria detection, but seldom applied to parasite identification. Here, metagenomics datasets prepared from swine faeces using an unbiased sample processing approach with RNA serving as starting material were re-analysed with respect to parasite detection. The taxonomic identification tool RIEMS, used for initial detection, provided basic hints on potential pathogens contained in the datasets. The suspected parasites/intestinal protists (Blastocystis, Entamoeba, Iodamoeba, Neobalantidium, Tetratrichomonas) were verified using subsequently applied reference mapping analyses on the base of rRNA sequences. Nearly full-length gene sequences could be extracted from the RNA-derived datasets. In the case of Blastocystis, subtyping was possible with subtype (ST)15 discovered for the first known time in swine faeces. Using RIEMS, some of the suspected candidates turned out to be false-positives caused by the poor status of sequences in publicly available databases. Altogether, 11 different species/STs of parasites/intestinal protists were detected in 34 out of 41 datasets extracted from metagenomics data. The approach operates without any primer bias that typically hampers the analysis of amplicon-based approaches, and allows the detection and taxonomic classification including subtyping of protist and metazoan endobionts (parasites, commensals or mutualists) based on an abundant biomarker, the 18S rRNA. The generic nature of the approach also allows evaluation of interdependencies that induce mutualistic or pathogenic effects that are often not clear for many intestinal protists and perhaps other parasites. Thus, metagenomics has the potential for generic pathogen identification beyond the characterisation of viruses and bacteria when starting from RNA instead of DNA.     

Identification and characterization of a serpin from Eimeria acervulina

Serpins are serine protease inhibitors that are widely distributed in metazoans but have not been previously characterized in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed and characterized. Random screening of an E.acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame (ORF) consisting of 1,245 nts that encode a peptide 413 amino acids in length with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues in length. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55 kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 kDa and 48 kDa in sporozoites (SZ) and merozoites (MZ) respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa which may correspond to secreted serpin. By immuno-staining the serpin was located in granules distributed throughout both the SZ and MZ but granules appeared to be concentrated in the parasite's anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis the endogenous serpin in MZ extracts did not form the expected high molecular weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.     

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.     

Complete circular DNA in the mitochondria-like organelles of Blastocystis hominis

Blastocystis hominis is an anaerobic parasite of the human intestinal tract belonging to the Stramenopile group. Using genome sequencing project data, we describe here the complete sequence of a 29,270-bp circular DNA molecule that presents mitochondrial features (such as oxidative phosphorylation complex I subunits) but lacks complexes III, IV and V. Transmission electron microscopy analyses reveal that this molecule, as well as mitochondrial (NADH:ubiquinone oxidoreductase subunit 7 (NAD7), beta-succinyl-CoA synthetase (beta-SCS)) and hydrogenosomal (pyruvate ferredoxin oxido-reductase (PFOR), iron-hydrogenase) proteins, are located within double-membrane surrounded-compartments known as mitochondria-like organelles (MLOs). As there is no evidence for hydrogen production by this organism, we suggest that MLOs are more likely anaerobic mitochondria.