The ins and outs of host‐microsporidia interactions during invasion,proliferation and exit

Microsporidia are a large group of fungal‐related obligate intracellular parasites. They are responsible for infections in humans as well as in agriculturally and environmentally important animals. Although microsporidia are abundant in nature, many of the molecular mechanisms employed during infection have remained enigmatic. In this review, we highlight recent work showing how microsporidia invade, proliferate and exit from host cells. During invasion, microsporidia use spore wall and polar tube proteins to interact with host receptors and adhere to the host cell surface. In turn, the host has multiple defence mechanisms to prevent and eliminate these infections. Microsporidia encode numerous transporters and steal host nutrients to facilitate proliferation within host cells. They also encode many secreted proteins which may modulate host metabolism and inhibit host cell defence mechanisms. Spores exit the host in a non‐lytic manner that is dependent on host actin and endocytic recycling proteins. Together, this work provides a fuller picture of the mechanisms that these fascinating organisms use to infect their hosts.     

Microsporidiosis in mammals

Microsporidia are small, single-celled, obligately intracellular parasites that have caused significant agricultural losses and interference with biomedical research. Interest in the microsporidia is growing, as these organisms are recognized as agents of opportunistic infections in persons with AIDS and in organ transplant recipients. Microsporidiosis is also being recognized in children and travelers, and furthermore, concern exists about the potential of zoonotic and waterborne transmission of microsporidia to humans. This article reviews the basic biology and epidemiology of microsporidiosis in mammals.     

Microsporidia and acquired immunodeficiency syndrome

Microsporidia is a common term that has been used to refer to a group of eukaryotic, obligate intracellular protozoan parasites belonging to the phylum Microspora. They are important agricultural parasites, contaminating commercial insects; they are also important by infecting laboratory rodents, rabbits and primates. Ever since the early cases found by Magarino Torres, who reported the presence of Encephalitozoon in a patient suffering of a meningoencephalomyelitis, some human pathology caused by microsporidia has been described. However, only after the acquired immunodeficiency syndrome outbreak have these organisms appeared as significant etiological agents in different pathologies. Even so, they remain underestimated. In the present article, the importance of microsporidia for the human pathology in immunocompromised host has been stressed.     

微孢子虫生物多样性研究的述评

微孢子虫作为一类专营细胞内寄生的低等的原生动物,有着比较悠久的进化历史。微孢子虫是一种既具有真核生物特征又具有原核生物特征的生物,同样具有生物多样性的本质,文章尝试用生物多样性的概念和原理,阐述原始的真核寄生物微孢子虫的物种多样性、遗传多样性和生态系统多样性的研究概况。     

A new monoclonal antibody enzyme-linked immunosorbent assay to measure in vitro multiplication of the microsporidium Encephalitozoon intestinalis

Microsporidia are obligate intracellular eukaryotic parasites that infect a wide range of hosts, including invertebrates and vertebrates. Microsporidia have emerged as important opportunistic pathogens of humans with the onset of the AIDS pandemic. The potential impact of these infections in human pathology has required the development of antiparasitic strategies, based on the search for molecules having an effect on the development and/or the multiplication of microsporidia. This creates a demand for a simple and reliable in vitro technique for measuring the multiplication of microsporidia. We developed a new monoclonal antibody (MAb) enzyme-linked immunosorbent assay (ELISA) technique and measured the growth of Encephalitozoon intestinalis in an in vitro culturing system using this method. The monoclonal antibody is specific for a coat protein of E. intestinalis sporogonic stages produced in parasitophorous vacuole. An anti-mouse antibody labeled with peroxidase was used as conjugate. This ELISA is a suitable, specific and semiquantitative technique for measuring the spread of E. intestinalis. It is easy to perform and required 5 h from start to end. A good correlation was observed when the ELISA data were compared with the manual microscopic counts of parasitophorous vacuoles obtained after immunofluorescent assay (IFA). Moreover, the ELISA method proved more accurate than the immunofluorescent assay. In summary, the ELISA system described in this study provides a simple reliable assay for measuring the spread of microsporidia in vitro and may prove valuable for the screening of putative interesting antimicrosporidial compounds.     

Phylogenetic Analysis of the Complete Genome Sequence of <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis> Supports the Fungal Origin of Microsporidia and Reveals a High Frequency of Fast-Evolving Genes

Microsporidia are unicellular eukaryotes living as obligate intracellular parasites. Lacking mitochondria, they were initially considered as having diverged before the endosymbiosis at the origin of mitochondria. That microsporidia were primitively amitochondriate was first questioned by the discovery of microsporidial sequences homologous to genes encoding mitochondrial proteins and then refuted by the identification of remnants of mitochondria in their cytoplasm. Various molecular phylogenies also cast doubt on the early divergence of microsporidia, these organisms forming a monophyletic group with or within the fungi. The 2001 proteins putatively encoded by the complete genome of Encephalitozoon cuniculi provided powerful data to test this hypothesis. Phylogenetic analysis of 99 proteins selected as adequate phylogenetic markers indicated that the E. cuniculi sequences having the lowest evolutionary rates preferentially clustered with fungal sequences or, more rarely, with both animal and fungal sequences. Because sequences with low evolutionary rates are less sensitive to the long-branch attraction artifact, we concluded that microsporidia are evolutionarily related to fungi. This analysis also allowed comparing the accuracy of several phylogenetic algorithms for a fast-evolving lineage with real rather than simulated sequences.This article contains online supplementary material.Reviewing Editor: Dr. Wen-Hsiung LiSupplementary material is available at     

Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi

Microsporidia are obligate intracellular parasites that were thought to be an ancient eukaryotic lineage based on molecular phylogenies using ribosomal RNA and translation elongation factors. However, this ancient origin of microsporidia has been contested recently, as several other molecular phylogenies suggest that microsporidia are closely related to fungi. Most of the protein trees that place microsporidia with fungi are not well sampled, however, and it is impossible to resolve whether microsporidia evolved from a fungus or from a protistan relative of fungi. We have sequenced beta-tubulins from 3 microsporidia, 4 chytrid fungi, and 12 zygomycete fungi, expanding the representation of beta-tubulin to include all four fungal divisions and a wide diversity of microsporidia. In phylogenetic trees including these new sequences, the overall topology of the fungal beta-tubulins generally matched the expected relationships among the four fungal divisions, although the zygomycetes were polyphyletic in some analyses. The microsporidia consistently fell within this fungal diversification, and not as a sister group to fungi. Overall, beta-tubulin phylogeny suggests that microsporidia evolved from a fungus sometime after the divergence of chytrids. We also found that chytrid alpha- and beta-tubulins are much less divergent than are tubulins from other fungi or microsporidia. In trees in which the only fungal representatives were the chytrids, microsporidia still branched with fungi (i.e., with chytrids), suggesting that the affiliation between microsporidian and fungal tubulins is not an artifact of long-branch attraction.     

Innate immune responses to Encephalitozoon species infections

Microsporidia are obligate intracellular, eukaryotic fungi, which have gained recognition as opportunistic parasites in immunocompromised patients. Resistance to lethal microsporidia infections requires a Th1 immune response; how this protection is initiated against Encephalitozoon species is the focus of this review article.     

A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria

Microsporidia are small (1–20 μm) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2] and [3], and are reported to lack mitochondria [2] and [4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2] and [4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5], [6], [7], [8] and [9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.     

Using average nucleotide identity (ANI) to evaluate microsporidia species boundaries based on their genetic relatedness

Microsporidia are obligatory intracellular parasites related to fungi and since their discovery their classification and origin has been controversial due to their unique morphology. Early taxonomic studies of microsporidia were based on ultrastructural spore features, characteristics of their life cycle and transmission modes. However, taxonomy and phylogeny based solely on these characteristics can be misleading. SSU rRNA is a traditional marker used in taxonomical classifications, but the power of SSU rRNA to resolve phylogenetic relationships between microsporidia is considered weak at the species level, as it may not show enough variation to distinguish closely related species. Overall genome relatedness indices (OGRI), such as average nucleotide identity (ANI), allows fast and easy-to-implement comparative measurements between genomes to assess species boundaries in prokaryotes, with a 95% cutoff value for grouping genomes of the same species. Due to the increasing availability of complete genomes, metrics of genome relatedness have been applied for eukaryotic microbes taxonomy such as microsporidia. However, the distribution of ANI values and cutoff values for species delimitation have not yet been fully tested in microsporidia. In this study we examined the distribution of ANI values for 65 publicly available microsporidian genomes and tested whether the 95% cutoff value is a good estimation for circumscribing species based on their genetic relatedness.     

α‐ and β‐Tubulin Phylogenies Support a Close Relationship Between the Microsporidia Brachiola algerae and Antonospora locustae

ABSTRACT. Microsporidia are a large and diverse group of intracellular parasites related to fungi. Much of our understanding of the relationships between microsporidia comes from phylogenies based on a single gene, the small subunit (SSU) rRNA, because only this gene has been sampled from diverse microsporidia. However, SSUrRNA trees are limited in their ability to resolve basal branches and some microsporidian affiliations are inconsistent between different analyses. Protein phylogenies have provided insight into relationships within specific groups of microsporidia, but have rarely been applied to the group as a whole. We have sequenced α‐ and β‐tubulins from microsporidia from three different subgroups, including representatives from what have previously been inferred to be the basal branches, allowing the broadest sampled protein‐based phylogenetic analysis to date. Although some relationships remain unresolved, many nodes uniting subgroups are strongly supported and consistent in both individual trees as well as a concatenate of both tubulins. One such relationship that was previously unclear is between Brachiola algerae and Antonospora locustae, and their close association with Encephalitozoon and Nosema. Also, an uncultivated microsporidian that infects cyclopoid copepods is shown to be related to Edhazardia aedis.     

On the use of FTA technology for collection, archieving, and molecular analysis of microsporidia dna from clinical stool samples

The FTA technology was applied for sampling, archiving, and molecular analysis of the DNA isolated from stool samples to diagnose and identify microsporidia, the intracellular opportunistic parasites which induce malabsortion syndrome in immunosuppressed humans, particularly in patients with AIDS. Microsporidia DNA was successfully amplified in 6 of 50 stool samples of HIV-positive patients of the S. P. Botkin Memorial Infectious Disease Hospital (St. Petersburg) applied to FTA cards (FTA-Cars, Whatman Inc. Florham Park, NJ, USA). Amplicons (the fragments of rDNA) were directly sequenced, and microsporidia species--Encephalitozoon intestinalis, E. cuniculi, E. hellem, and Enterocytozoon bieneusi--were identified in Genbank by NCBI BLAST program. The FTA method of DNA immobilization is especially promising for epidemiological and field population studies which involve genotyping of microsporidia species and isolates.     

鳞翅目昆虫病原微孢子虫研究进展

微孢子虫广泛存在于鳞翅目昆虫中,是一类重要的病原微生物。微孢子虫病一方面影响野外昆虫种群的自然平衡,另一方面对家蚕、柞蚕等经济和资源昆虫造成了严重的危害。微孢子虫分子生物学研究基础相对薄弱,再加上微孢子虫表面坚厚的孢壁,无疑增加了研究难度。随着核酸、蛋白质等生物大分子分离制备方法和高通量测序技术的不断更新发展,基于各种组学(Omics)研究微孢子虫的工作方兴未艾,并且有了一些重要的发现。本文综述了微孢子虫与鳞翅目昆虫寄主的相互作用及寄生于鳞翅目昆虫的病原微孢子虫基因组、转录组和蛋白质组进展情况,以期为微孢子虫的深入研究提供参考。这些昆虫微生物研究将为鳞翅目害虫生物防治提供新的思路,并对家蚕等经济昆虫微粒子病的诊断、防控及治疗产生积极影响。     

Microsporidia: accumulating molecular evidence that a group of amitochondriate and suspectedly primitive eukaryotes are just curious fungi

Microsporidia are obligate intracellular parasites that have long been considered to be primitive eukaryotes, both on the basis of morphological features and on the basis of molecular, mainly ribosomal RNA-based, phylogenies. However, accumulating sequence data and the use of more sophisticated tree construction methods now seem to suggest that microsporidia share a common origin with fungi and are therefore most probably just curious fungi. In this paper, we describe the current views on the phylogenetic position of the microsporidia and present additional evidence for a close relationship between fungi and microsporidia on the basis of reanalyzed ribosomal RNA data. In this respect, the importance of incorporating detailed knowledge of the substitution pattern of sequences into phylogenetic methods is discussed.     

Peculiarities of metabolism of the microsporidia Nosema grylli during the intracellular development

A long adaptation of Microsporidia to intracellular development supposes the host-derived ATP dependence of merogony and sporogony stages. To prove this assumption the activities of ten carbohydrate and energy metabolism enzymes were compared in the microsporidia Nosema grylli intracellular stages and mature spores. This species infects the fat body of crickets Gryllus bimaculatus. We have demonstrated lower activities of glycolytic enzymes, phosphoglucomutase and glucose-6-PhDH in the metabolically active meronts and sporonts than in the dormant mature spores. Low glycolysis level indicates that carbohydrate catabolism is not a principal mechanism of ATP supply in the N. grylli intracellular stages. Furthermore, we have not revealed a preferable expenditure of glycogen in comparison with triglycerides in infected cricket fat bodies. The N. grylli infection causes an equal reduction of glycogen and lipid content approximately in 2-3 times. Microsporidia have not mitochondria, Krebs cycle and electron-transport chain. Therefore they are not able to utilise fat reserves for ATP production. It seems to be proposed that microsporidia consume exogenous ATP which is produced by host cell metabolic system. The N. grylli infection provokes an increase of ATP content and ratio of ATP/ADP concentrations in cricket fat bodies approximately in 4 times. These data indicates a rise of host cell energy metabolism rate during the infection.     

Immunocytochemical identification of the major exospore protein and three polar-tube proteins of the microsporidia Paranosema grylli

Microsporidia are intracellular eukaryotic parasites that can infect a wide range of animal hosts with several genera causing opportunistic infections in immunodeficient patients. Their spore wall and their unique extrusion apparatus, which has the form of a long polar tube, confer resistance of these parasites against the environment and during host-cell invasion. In contrast to parasites of vertebrates, the spore-wall and polar-tube proteins of many microsporidia species still remain to be characterized, even though a great number of microsporidia infect invertebrates. Here, we have identified one spore-wall protein and three polar-tube proteins of the microsporidia Paranosema grylli that infects the cricket Gryllus bimaculatus. Incubation of intact spores with an alkaline-saline solution resulted in the selective extraction of a major 40 kDa protein. A wash of the discharged (or destroyed) spores with SDS and the following solubilization of their polar tubes with 50-75% 2-mercaptoethanol extracted a major protein of ca. 56 kDa. When the polar tubes were solubilized in the presence of SDS, two additional proteins of 46 and 34 kDa were extracted. Antibodies specific for these extracted proteins were generated and isolated by incubation of immune sera with the protein bands that had been transferred to nitrocellulose. Western blotting demonstrated the cross-reactivity of the anti-p46 and anti-p34 antibodies. Immuno-electron microscopy with the anti-p40 antibody revealed specific decoration of the microsporidia exospore. The 56, 46 and 34 kDa proteins were characterized as polar-tube components due to the clear antibody labeling of the polar filament.     

人类微孢子虫检测方法研究进展

微孢子虫(microsporidia)是一类专性细胞内寄生的单细胞真核生物。是引起微孢子虫病的真菌类病原。在已知并被命名的1500多种微孢子虫中,共有9个属中的17个虫种可以感染人。人类微孢子虫可侵染包括肠道、肝、肺、脑等部位,引起慢性腹泻、肝炎、角膜炎、脑炎、血液系统性感染等,严重影响人类健康。研究开发快速高效的人类微孢子虫诊断方法成为当前病原微生物检测领域研究的热点。人类微孢子虫的发现历史实际上是伴随检测方法的不断进步而逐渐进行的。这些检测方法包括,透射电镜(transmission electron microscopy)、苏木精-伊红染色(hematoxylin-eosin stain,HE)、亚甲蓝染色(methylene blue)、吉姆萨染色(giemsa)、革兰氏染色(gram stain)、韦伯氏改良三色染色法(Weber’s chromotrope-based staining)、荧光增白剂染色法(calcofluor white staining)、抗原检测、抗体检测、实时荧光定量PCR (quantitative real-time PCR,q PCR)、环介导等温扩增(loop-mediated isothermal amplification,LAMP)、DNA点杂交模型等。随着技术的进步以及更多微孢子虫的检出,使人类能够更好地认识微孢子虫、并制定微孢子虫准确快速检测方法和防控策略。