微孢子虫系统进化研究的变迁与展望

微孢子虫(Microsporidia)是一类专性细胞内寄生的单细胞真核生物,在科研、医疗、农业、商业等领域具有重要影响。由于其不具有某些典型的真核生物细胞结构,如线粒体、过氧化物酶体、高尔基体、鞭毛,曾将其归属于古真核生物谱系,认为其进化历程先于这些细胞器的起源,该假说也得到了一些生物化学和分子生物学研究证据的支持。然而,在最近十年里,通过更深入的研究,尤其是基于分子序列的系统进化分析,表明微孢子虫和真菌具有一定亲缘关系,并认为其结构的简约性恰好体现了微孢子虫营寄生生活的高度退化现象。目前对微孢子虫的系统进化仍存在各种不同意见,对其进化研究历史进行探讨有着重要意义。本文将按照时间顺序回顾微孢子虫进化分类研究过程中的各种研究成果,并讨论为什么微孢子虫独特的细胞和基因组特性会导致众多的学者在其进化分类问题上争执这么久。     

Microsporidia: emerging advances in understanding the basic biology of these unique organisms

Microsporidia are long-known parasites of a wide variety of invertebrate and vertebrate hosts. The emergence of these obligate intracellular organisms as important opportunistic pathogens during the AIDS pandemic and the discovery of new species in humans renewed interest in this unique group of organisms. This review summarises recent advances in the field of molecular biology of microsporidia which (i) contributed to the understanding of the natural origin of human-infecting microsporidia, (ii) revealed unique genetic features of their dramatically reduced genome and (iii) resulted in the correction of their phylogenetic placement among eukaryotes from primitive protozoans to highly evolved organisms related to fungi. Microsporidia might serve as new intracellular model organisms in the future given that gene transfer systems will be developed.     

A kingdom's progress: Archezoa and the origin of eukaryotes

The taxon Archezoa was proposed to unite a group of very odd eukaryotes that lack many of the characteristics classically associated with nucleated cells, in particular the mitochondrion. The hypothesis was that these cells diverged from other eukaryotes before these characters ever evolved, and therefore they represent ancient and primitive eukaryotic lineages. The kingdom comprised four groups: Metamonada, Microsporidia, Parabasalia, and Archamoebae. Until recently, molecular work supported their primitive status, as they consistently branched deeply in eukaryotic phylogenetic trees. However, evidence has now emerged that many Archezoa contain genes derived from the mitochondrial symbiont, revealing that they actually evolved after the mitochondrial symbiosis. In addition, some Archezoa have now been shown to have evolved more recently than previously believed, especially the Microsporidia for which considerable evidence now indicates a relationship with fungi. In summary, the mitochondrial symbiosis now appears to predate all Archezoa and perhaps all presently known eukaryotes. BioEssays 20:87–95, 1998. © 1998 John Wiley & Sons, Inc.     

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.     

Cell biology and invasion of the microsporidia

Microsporidia are amitochondrial eukaryotic obligate intracellular parasites. They are reported to infect every animal group from protists to vertebrates, including humans. Microsporidia are of interest as opportunistic pathogens in humans and for certain characteristics which raise questions about their evolution and phylogenetic position. This review describes the basic biology and invasion mechanisms of microsporidian species infecting humans.     

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。     

Seeking the determinants of the elusive functions of Sco proteins

Sco proteins are present in all types of organisms, including the vast majority of eukaryotes and many prokaryotes. It is well established that Sco proteins in eukaryotes are involved in the assembly of the Cu(A) cofactor of mitochondrial cytochrome c oxidase; however their precise role in this process has not yet been elucidated at the molecular level. In particular, some but not all eukaryotes including humans possess two Sco proteins whose individual functions remain unclear. There is evidence that eukaryotic Sco proteins are also implicated in other cellular processes such as redox signalling and regulation of copper homeostasis. The range of physiological functions of Sco proteins appears to be even wider in prokaryotes, where Sco-encoding genes have been duplicated many times during evolution. While some prokaryotic Sco proteins are required for the biosynthesis of cytochrome c oxidase, others are most likely to take part in different processes such as copper delivery to other enzymes and protection against oxidative stress. The detailed understanding of the multiplicity of roles ascribed to Sco proteins requires the identification of the subtle determinants that modulate the two properties central to their known and potential functions, i.e. copper binding and redox properties. In this review, we provide a comprehensive summary of the current knowledge on Sco proteins gained by genetic, structural and functional studies on both eukaryotic and prokaryotic homologues, and propose some hints to unveil the elusive molecular mechanisms underlying their functions.     

Unique characteristics of the energy metabolism in Microsporidia as a result of durational adaptation to the intracellular development

Microsporidia is a large group of fungi-related unicellular eukaryotes with obligate intracellular lifestyle infecting a wide range of invertebrate and vertebrate hosts. Long adaptation of the parasites to intracellular development resulted in extraordinary minimization of their metabolic system. The paper summarizes the original results and literature data on the study of microsporidian carbohydrate and energy metabolism. On the basis of the material, it is concluded that minimization of microsporidian cell machinery was accompanied by the acquisition of a number of unique characteristics, which were not found in other eukaryotes.     

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.     

A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems

Microsporidia are obligate intracellular eukaryotic parasites known to parasitize many species of the animal kingdom as well as some protists. However, their diversity is underestimated, in part as a consequence of the failure of ‘universal’ primers to detect them in metabarcoding studies. Besides, due to the inconsistency between taxonomy and phylogenetic data, available databases may assign incorrectly sequences obtained with high-throughput sequencing. In this work, we developed a comprehensive reference database which positions microsporidian SSU rRNA gene sequences within a coherent ranked phylogenetic framework. We used this phylogenetic framework to study the microsporidian diversity in lacustrine ecosystems, focusing on < 150 μm planktonic size fractions. Our analysis shows a high diversity of Microsporidia, with the identification of 1531 OTUs distributed within seven clades, of which 76% were affiliated to clade IV2 and 20% to clade I (nomenclature presented hereby). About a quarter of the obtained sequences shared less than 85% identity to the closest known species, which might represent undescribed genera or families infecting small hosts. Variations in the abundance of Microsporidia were recorded between the two lakes sampled and across the sampling period, which might be explained by spatio-temporal variations of their potential hosts such as microeukaryotes and metazooplankton.     

The Genome of Spraguea lophii and the Basis of Host-Microsporidian Interactions

Microsporidia are obligate intracellular parasites with the smallest known eukaryotic genomes. Although they are increasingly recognized as economically and medically important parasites, the molecular basis of microsporidian pathogenicity is almost completely unknown and no genetic manipulation system is currently available. The fish-infecting microsporidian Spraguea lophii shows one of the most striking host cell manipulations known for these parasites, converting host nervous tissue into swollen spore factories known as xenomas. In order to investigate the basis of these interactions between microsporidian and host, we sequenced and analyzed the S. lophii genome. Although, like other microsporidia, S. lophii has lost many of the protein families typical of model eukaryotes, we identified a number of gene family expansions including a family of leucine-rich repeat proteins that may represent pathogenicity factors. Building on our comparative genomic analyses, we exploited the large numbers of spores that can be obtained from xenomas to identify potential effector proteins experimentally. We used complex-mix proteomics to identify proteins released by the parasite upon germination, resulting in the first experimental isolation of putative secreted effector proteins in a microsporidian. Many of these proteins are not related to characterized pathogenicity factors or indeed any other sequences from outside the Microsporidia. However, two of the secreted proteins are members of a family of RICIN B-lectin-like proteins broadly conserved across the phylum. These proteins form syntenic clusters arising from tandem duplications in several microsporidian genomes and may represent a novel family of conserved effector proteins. These computational and experimental analyses establish S. lophii as an attractive model system for understanding the evolution of host-parasite interactions in microsporidia and suggest an important role for lineage-specific innovations and fast evolving proteins in the evolution of the parasitic microsporidian lifecycle.     

Parasitic systems of Microsporidia: descriptions and terminology questions

Three parasitic systems of Microsporidia are described: the system of monoxenic Vairimorpha mesnili with paraxenic hosts presented lepidopteran and hymenopteran species; the system of dixenic Amblyospora sp. with metaxenic hosts presented bloodsucking mosquitoes and crustaceans and the system of Metchnikovella sp. as parasite of other obligate parasite. The last case is characterized by very intimate interrelations between hyperparasite (microsporidian species), obligate parasite--host of Microsporidia (gregarine) and hyperhost--host of gregarine (polychaeta). This hyperparasite system is exclusive case of parasitic systems. Parasitic and hyperparasitic systems reflects a population level of host-parasite interactions. On biocenotic level many other organisms such as predators, vectors and reservators of invasion stages of Microsporidia affect parasitic systems giving a chance to one of the members of the system (to the host or to the parasite). These organisms form epiparasitic system. In all cases of the parasitic systems there are two-way communications between parasites and their hosts. In systems on biocenotic level--parasitic consortium--members of epiparasitic systems acts on parasitic systems, but members of parasitic systems don't affect epiparasitic systems.     

Evidence from small-subunit ribosomal RNA sequences for a fungal origin of Microsporidia

The phylum Microsporidia comprises a species-rich group of minute, single-celled, and intra-cellular parasites. Lacking normal mitochondria and with unique cytology, microsporidians have sometimes been thought to be a lineage of ancient eukaryotes. Although phylogenetic analyses using small-subunit ribosomal RNA (SSU-rRNA) genes almost invariably place the Microsporidia among the earliest branches on the eukaryotic tree, many other molecules suggest instead a relationship with fungi. Using maximum likelihood methods and a diverse SSU-rRNA data set, we have re-evaluated the phylogenetic affiliations of Microsporidia. We demonstrate that tree topologies used to estimate likelihood model parameters can materially affect phylogenetic searches. We present a procedure for reducing this bias: "tree-based site partitioning," in which a comprehensive set of alternative topologies is used to estimate sequence data partitions based on inferred evolutionary rates. This hypothesis-driven approach appears to be capable of utilizing phylogenetic information that is not available to standard likelihood implementations (e.g., approximation to a gamma distribution); we have employed it in maximum likelihood and Bayesian analysis. Applying our method to a phylogenetically diverse SSU-rRNA data set revealed that the early diverging ("deep") placement of Microsporidia typically found in SSU-rRNA trees is no better than a fungal placement, and that the likeliest placement of Microsporidia among non-long-branch eukaryotic taxa is actually within fungi. These results illustrate the importance of hypothesis testing in parameter estimation, provide a way to address certain problems in difficult data sets, and support a fungal origin for the Microsporidia.     

The phylogeny and evolution of deoxyribonuclease II: an enzyme essential for lysosomal DNA degradation

Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH, localized within the lysosomes of higher eukaryotes. The origin of this enzyme remains in dispute, and its phylogenetic distribution leaves many questions about its subsequent evolutionary history open. Earlier studies have documented its presence in various metazoans, as well as in Dictyostelium, Trichomonas and, anomalously, a single genus of bacteria (Burkholderia). This study makes use of searches of the genomes of various organisms against known DNase II query sequences, in order to determine the likely point of origin of this enzyme among cellular life forms. Its complete absence from any other bacteria makes prokaryotic origin unlikely. Convincing evidence exists for DNase II homologs in Alveolates such as Paramecium, Heterokonts such as diatoms and water molds, and even tentative matches in green algae. Apparent absences include red algae, plants, fungi, and a number of parasitic organisms. Based on this phylogenetic distribution and hypotheses of eukaryotic relationships, the most probable explanation is that DNase II has been subject to multiple losses. The point of origin is debatable, though its presence in Trichomonas and perhaps in other evolutionarily basal "Excavate" protists such as Reclinomonas, strongly support the hypothesis that DNase II arose as a plesiomorphic trait in eukaryotes. It probably evolved together with phagocytosis, specifically to facilitate DNA degradation and bacteriotrophy. The various absences in many eukaryotic lineages are accounted for by loss of phagotrophic function in intracellular parasites, in obligate autotrophs, and in saprophytes.     

Temporal variations of Microsporidia diversity and discovery of new host–parasite interactions in a lake ecosystem

Microsporidia are a large group of obligate intracellular eukaryotic parasites related to Fungi. Recent studies suggest that their diversity has been greatly underestimated and little is known about their hosts other than metazoans, and thus about their impact on the communities at the base of the food web. In this work, we therefore studied the diversity of Microsporidia over one year and identified potential new hosts in small-sized fractions (<150 μm) in a lake ecosystem using a metabarcoding approach coupled with co-occurrence networks and tyramide signal amplification-fluorescent in situ hybridization. Our analysis shows a great Microsporidia diversity (1 472 OTUs), with an important part of this diversity being unknown. Temporal variations of this diversity have been observed, which might follow temporal variations of their potential hosts such as protists and microzooplankton. New hosts among them were identified as well as associations with phytoplankton. Indeed, repeated infections were observed in Kellicottia (rotifers) with a prevalence of 38% (infected individuals). Microsporidia inside a Stentor (ciliate) were also observed. Finally, potential infections of the diatom Asterionella were identified (prevalence <0.1%). The microsporidian host spectrum could be therefore even more important than previously described, and their role in the functioning of lake ecosystems is undoubtedly largely unknown.     

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.     

A structural analysis of the group II intron active site and implications for the spliceosome

Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site.