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.     

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.     

肠道菌群与相关疾病

近年来肠道菌群的研究发展迅速,肠道菌群对宿主消化、代谢和免疫功能的影响逐渐被人们所熟知并重视。大量研究提示,肠道菌群的改变可能引发代谢、肝脏和肠道等方面的多种相关疾病。因此,研究肠道菌群对宿主健康及疾病的影响尤为重要,也能为预防和治疗肠道菌群相关疾病提供建议。     

基于肠道微生态的白术散作用机制研究进展

肠道作为人体消化吸收的重要场所,是人体最大的免疫器官,在维持正常生理活动中发挥着非常重要的作用.随着人类饮食的多元化,肠道菌群对健康的影响吸引了越来越多的关注.肠道微生态与免疫、代谢系统疾病和肿瘤等50多种疾病相关,同时疾病也会影响肠道菌群,通过饮食调节和用药会影响肠道菌群,进而影响健康.白术散是以食药两用药材为主的方...     

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.     

肠道菌群与抑郁症相关性的研究进展

正常人体内的肠道菌群数量可达100万亿,可参与人体的多项生理活动,包括营养物质的吸收与代谢、免疫系统的发育与成熟、抵挡外来病原体的入侵等,对人类的健康有着至关重要的作用。近年来发现肠道中的定植微生物与抑郁症、自闭症、焦虑症和帕金森病等一系列的神经精神疾病密切相关。最新研究表明,肠道菌群是通过神经、体液、代谢和免疫多种途径双向调节肠道和中枢神经系统的。目前,随着医学技术和医学理论的的提高,抑郁症与肠道菌群间的关系受到极大地重视。本文从肠道菌群对抑郁症的影响机制以及益生菌对抑郁症的改善作用两方面来综述肠道菌群与抑郁症相关性的最新进展。     

多糖对肠道功能调节作用的研究进展

肠道是机体重要的消化器官,亦是共生微生物群的主要寄居场所,在维持机体正常生命活动如免疫和内分泌功能中发挥着重要作用。 肠道功能紊乱与疾病的发生以及发展过程密切相关。近年来,多项研究结果显示,多糖具有肠道功能调节作用,包括通过作用于肠道黏膜 参与机体免疫过程、保护肠道屏障结构和功能的完整性、调节肠道菌群组成以及刺激肠道内分泌。从伴随疾病过程中的肠道功能紊乱的角度, 对多糖调节肠道功能的作用机制进行综述。     

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.     

微生物在精神分裂症发病机制的研究现状

脑和肠道微生物群之间的相互作用逐渐被揭示。目前已经提出脑-肠轴失调和异常与各种中枢神经系统疾病有关。精神分裂症是一种病因不明的严重精神障碍。最近研究表明,肠道微生物的组成和数量变化会通过肠道菌群-肠-脑轴影响人类的认知和社会行为,这意味着肠道菌群在精神分裂症患者中可能起着重要的作用,并有望成为精神分裂症新的治疗靶点。本文综述了肠道菌群与精神分裂症相关性的研究进展,为预防和治疗精神分裂症等精神障碍类疾病提供了理论依据。     

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.     

Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns

A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.     

Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes

Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.     

肠道菌群失调与高尿酸血症关系的研究进展

高尿酸血症（hyperuricemia,HUA）是一种涉及肝、肾、肠等多个器官的代谢性疾病,因尿酸代谢异常而引起代谢障碍。尿酸在肝脏和肾脏中的代谢途径目前已经被阐明,但在肠道内的代谢途径尚未完全清晰。肠道菌群在人体肠道中定植,与宿主存在互惠共生的关系,在宿主的代谢和免疫调节中起着至关重要的作用。肠道菌群结构的变化可能引起代谢紊乱,肠道菌群参与嘌呤代谢酶的合成和炎症因子的释放,与HUA的发生发展密切相关。肠道菌群作为探讨HUA发病机制的切入点,已成为新的研究热点。本综述主要阐述HUA与肠道菌群之间的关系,探讨肠道菌群抗HUA的机制,如肠道菌群促进嘌呤和尿酸分解代谢,影响尿酸排泄,以及HUA引起的肠道炎症反应等,以期为通过调节肠道菌群来治疗HUA提供一定的依据。

     

肠道菌群与神经精神疾病

人体肠道中存在着数量庞大和种类繁多的细菌,这些细菌及其代谢产物在代谢、免疫、内分泌、神经等方面起着重要作用,对于人类的健康有着重要影响。近年来越来越多的研究表明,正常的肠道菌群在维持大脑的发育与功能方面扮演着重要角色,而肠道菌群的失调与一些神经精神疾病密切相关,例如帕金森症、多发性硬化、抑郁、自闭症等。本文就肠道菌群与神经精神疾病的关系作一综述。     

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.     

Recent advances in Blastocystis hominis research: hot spots in terra incognita

Despite being discovered more than 80 years ago, progress in Blastocystis research has been gradual and challenging, due to the small number of laboratories currently working on this protozoan parasite. To date, the morphology of Blastocystis hominis has been extensively studied by light and electron microscopy but all other aspects of its biology remain little explored areas. However, the availability of numerous and varied molecular tools and their application to the study of Blastocystis has brought us closer to understanding its biology. The purpose of this review is to describe and discuss recent advances in B. hominis research, with particular focus on new, and sometimes controversial, information that has shed light on its genetic heterogeneity, taxonomic links, mode of transmission, in vitro culture and pathogenesis. We also discuss recent observations that B. hominis has the capacity to undergo programmed cell death; a phenomenon similarly reported for many other unicellular organisms. There are still many gaps in our knowledge of this parasite. Although there is a growing body of evidence suggesting that B. hominis can be pathogenic under specific conditions, there are also other studies that indicated otherwise. Indeed, more studies are warranted before this controversial issue can be resolved. There is an urgent need for the identification and/or development of an animal model so that questions on its pathogenesis can be better answered. Another area that requires attention is the development of methods for the transfection of foreign/altered genes into B. hominis in order to facilitate genetic experiments.     

LEEways: tales of EPEC,ATEC and EHEC

Intestinal pathogenic Escherichia coli are a major cause of worldwide morbidity and mortality. Currently seven intestinal pathovars are recognized causing a wide range of intestinal disorders that are sometimes associated with severe and even lethal complications. The arsenal of virulence factors is used to subvert cellular functions of the host thereby enhancing adaptation, virulence and pathogenicity. Virulence factor profiles are largely the result of the acquisition of mobile genetic elements such as prophages and pathogenicity islands. A group of highly adapted intestinal pathogenic E. coli that are characterized by the induction of ‘attaching‐and‐effacing (A/E) lesions’ have acquired a decisive pathogenicity island, the ‘locus of enterocyte effacement – LEE’ by horizontal gene transfer. This review focuses on recent advances in our understanding of A/E E. coli. It highlights novel functions of effector proteins, addresses the LEE flanking regions where additional genetic elements such as the LifA/Efa1 region have been identified, and points to implications for diagnostics and therapy due to the putative interconversion of A/E E. coli during infection.     

消化道微生态与胰腺疾病

消化道微生态参与人体多种生理及病理过程,是消化领域的研究热点。消化道微生态可能与急慢性胰腺炎和胰腺癌等胰腺疾病关系密切,但学术界很少关注。慢性胰腺炎患者存在肠道菌群结构失衡,并易伴发小肠细菌过度生长。肠道菌群可能与自身免疫性胰腺炎等IgG4相关性疾病关系密切。急性胰腺炎患者存在肠道菌群结构变化,肠道屏障功能受损和细菌移位在急性胰腺炎疾病进展中起重要作用,但不同类型和疾病程度的急性胰腺炎患者肠道菌群结构和功能特征仍不清楚。牙周疾病和口腔微生态失衡会增加罹患胰腺癌的风险,但胰腺癌时肠道菌群的具体变化及作用仍不明确。本文就各类胰腺疾病背景下的消化道微生态研究现状及未来可能的研究方向进行阐述     

浅析肠道菌群干预治疗癫痫的潜在应用前景

肠道菌群是存在于人体内的庞大而复杂的微生物群落,菌群的变化会影响大脑的生理、行为和认知功能,而大脑可以通过免疫、内分泌和神经通路等途径调节神经生理行为,与很多神经和精神疾病(如癫痫、脱髓鞘疾病、阿尔茨海默症、自闭症等)的发生发展有关。近年来诸多证据表明自身免疫机制在癫痫进展中发挥重要作用,肠道菌群可通过调节免疫反应来影响疾病的进展。本文从肠道菌群与大脑间关系的认知、相互间功能影响及与癫痫的关系等方面,探讨通过肠道菌群干预治疗癫痫的机制与潜在应用前景,希望为菌群干预治疗与预防一些神经及精神疾病提供参考。     

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.