In Vitro Replication of Nosema algerae (Microsporidia), a Parasite of Anopheline Mosquitoes, in Human Cells above 36° C

Microsporidia form a large and ubiquitous group of obligately intracellular parasitic eukaryotes, increasingly recognized as pathogens in humans. Transmission of invertebrate microsporidia to mammals has been considered impossible because temperature seemed to be a limiting factor for development. Nosema algerae, a microsporidian of anopheline mosquitoes, was cultured in human muscle fibroblasts at temperatures of 31 degrees C and 38 degrees C. This is the first record of an invertebrate microsporidian developing in human cells at a temperature above 36 degrees C. The ultrastructure of N. algerae growing in human muscle fibroblasts is similar to that of Brachiola vesicularum, a microsporidian species previously described in the muscle of an AIDS patient.     

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.     

A Broad Distribution of the Alternative Oxidase in Microsporidian Parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.     

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。     

Genome evolution: the dynamics of static genomes

A random survey of a microsporidian genome has revealed some striking features. Although the genomes of microsporidians are among the smallest known for eukaryotes, their organisation appears to be well conserved.     

The Genome of the Obligate Intracellular Parasite Trachipleistophora hominis: New Insights into Microsporidian Genome Dynamics and Reductive Evolution

The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.     

Effect of Tetramicra brevifilum (Microspora) infection on respiratory-burst responses of turbot (Scophthalmus maximus L.) phagocytes

In vitro assays were performed to investigate microsporidian-induced intracellular and extracellular production of reactive oxygen species (ROS) by peritoneal-exudate adherent (PEA) cells from turbot. ROS production was quantified using the fluorescent reagents OxyBURST Green H2HFF BSA (extracellular) and OxyBURST Green H2DCFDA succinimidyl ester (intracellular). Five days before assay, the cells had been elicited in vivo by intraperitoneal injection of sodium thioglycollate or spores of Tetramicra brevifilum. Elicitation with spores led to a marked increase in the proportion of neutrophils among PEA cells. PEA cells from normal turbot showed considerable extracellular and intracellular ROS production in response to microsporidian spores. By contrast, PEA cells from microsporidian-infected turbot showed considerably reduced extracellular and intracellular ROS production in response to microsporidian spores. Extracellular ROS production was affected by the addition of infected turbot serum to the assay medium, regardless of whether the PEA cells had been obtained from normal or infected fish. The presence of microsporidian-infected turbot serum significantly reduced intracellular ROS production by PEA cells elicited with microsporidian spores. These results suggest that (a) microsporidian spores partially suppress the repiratory-burst response of turbot phagocytes; and (b) infected turbot serum contains substances capable of modulating the respiratory-burst response of turbot phagocytes to microsporidian spores.     

Bacterial catalase in the microsporidian Nosema locustae: implications for microsporidian metabolism and genome evolution

Microsporidia constitute a group of extremely specialized intracellular parasites that infect virtually all animals. They are highly derived, reduced fungi that lack several features typical of other eukaryotes, including canonical mitochondria, flagella, and peroxisomes. Consistent with the absence of peroxisomes in microsporidia, the recently completed genome of the microsporidian Encephalitozoon cuniculi lacks a gene for catalase, the major enzymatic marker for the organelle. We show, however, that the genome of the microsporidian Nosema locustae, in contrast to that of E. cuniculi, encodes a group II large-subunit catalase. Surprisingly, phylogenetic analyses indicate that the N. locustae catalase is not specifically related to fungal homologs, as one would expect, but is instead closely related to proteobacterial sequences. This finding indicates that the N. locustae catalase is derived by lateral gene transfer from a bacterium. The catalase gene is adjacent to a large region of the genome that appears to be far less compact than is typical of microsporidian genomes, a characteristic which may make this region more amenable to the insertion of foreign genes. The N. locustae catalase gene is expressed in spores, and the protein is detectable by Western blotting. This type of catalase is a particularly robust enzyme that has been shown to function in dormant cells, indicating that the N. locustae catalase may play some functional role in the spore. There is no evidence that the N. locustae catalase functions in a cryptic peroxisome.     

Characterization of a divergent Sec61beta gene in microsporidia

The general secretory (Sec) pathway is the main mechanism for protein secretion and insertion into endoplasmic reticulum and plasma membrane in prokaryotes and eukaryotes. However, the complete genome of the highly specialized microsporidian parasite Encephalitozoon cuniculi appears to lack a gene for Sec61beta, one of three universally conserved proteins that form the core of the Sec translocon. We have identified a putative, highly divergent homologue of Sec61beta in the genome of another microsporidian, Antonospora locustae, and used this to identify a previously unrecognized Sec61beta in E. cuniculi. The identity of these genes is supported by evidence from secondary structure prediction and gene order conservation. Their functional conservation is confirmed by expressing both microsporidian homologues in yeast, where they are localized to the endoplasmic reticulum and rescue a yeast Sec61beta deletion mutant.     

Microsporidian Diversity in Soil,Sand, and Compost of the Pacific Northwest

Microsporidia are intracellular parasites considered to be ubiquitous in the environment. Yet the true extent of their diversity in soils, sand, and compost remains unclear. We examined microsporidian diversity found in the common urban environments of soil, sand, and compost. We retrieved 22 novel microsporidian sequences and only four from described species. Their distribution was generally restricted to a single site and sample type. Surprisingly, one novel microsporidian showed a wide distribution, and high prevalence, as it was detected in five different compost samples and in soil samples collected over 200 km apart. These results suggest that the majority of Microsporidia appear to have a narrow distribution. Our phylogenetic analysis indicated that the Microsporidia detected in this study include representatives from four of the five major microsporidian groups. Furthermore, the addition of our new sequences calls into question the cohesiveness of microsporidian clade II. These results highlight the importance of increasing our knowledge of microsporidian diversity to better understand the phylogenetic relationships and evolutionary history of this important group of emerging parasites.     

Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.     

Uptake of Encephalitozoon spp. and Vittaforma corneae (Microsporidia) by different cells

Microsporidia are obligate intracellular parasites infecting a broad range of vertebrates and invertebrates. Various microsporidian species induce different clinical pictures in humans. The reason for this is not clear. It has been speculated that the different microsporidian species are transmitted by various routes, thus causing infections in different organs. Another possibility is that the diverse microsporidia have different tropisms to organ-specific cells, thus causing various diseases. In this study, we investigated the uptake of microsporidian spores by different cells with an immunofluorescence staining technique to investigate whether there is a difference between microsporidian species as well as between different cells. Using this technique, we were able to distinguish between intra- and extracellular microsporidian spores. All examined cell lines were able to internalize microsporidian spores, but the extent of internalization differed significantly between the cells. Although the results showed some patterns that correlate with the distribution of the parasites in humans, the different clinical pictures cannot be sufficiently explained by this phenomenon, so it seems more likely that the various clinical manifestations caused by the different microsporidian species are a consequence of different infection routes rather than of different affinities of the microsporidian species to different cells.     

Microsporidia: a journey through radical taxonomical revisions

Microsporidia are obligate intracellular parasites of medical and commercial importance, characterized by a severe reduction, or even absence, of cellular components typical of eukaryotes such as mitochondria, Golgi apparatus and flagella. This simplistic cellular organization has made it difficult to infer the evolutionary relationship of Microsporidia to other eukaryotes, because they lack many characters historically used to make such comparisons. Eventually, it was suggested that this simplicity might be due to Microsporidia representing a very early eukaryotic lineage that evolved prior to the origin of many typically eukaryotic features, in particular the mitochondrion. This hypothesis was supported by the first biochemical and molecular studies of the group. In the last decade, however, contrasting evidence has emerged, mostly from molecular sequences, that show Microsporidia are related to fungi, and it is now widely acknowledged that features previously recognized as primitive are instead highly derived adaptations to their obligate parasitic lifestyle. The various sharply differing views on microsporidian evolution resulted in several radical reappraisals of their taxonomy. Here we will chronologically review the causes and consequences for these taxonomic revisions, with a special emphasis on why the unique cellular and genomic features of Microsporidia lured scientists towards the wrong direction for so long.     

Respiratory burst responses of rat macrophages to microsporidian spores.

This study investigated the respiratory burst responses of rat resident peritoneal macrophages and of peritoneal macrophages stimulated 5 days previously with viable spores of the fish infecting microsporidian Microgemma caulleryi. Nitric oxide production by resident macrophages and prestimulated macrophages in response to viable microsporidian spores was significantly lower than in response to Escherichia coli lipopolysaccharide (LPS) (nitrite concentration in medium 57 +/- 1 microM for resident macrophages stimulated with LPS versus 31 +/- 1 microM for resident macrophages stimulated with microsporidian spores and 36 +/- 4 microM for M. caulleryi prestimulated macrophages; P < 0.05). Extracellular release of reactive oxygen species (ROS) by resident macrophages in response to microsporidian spores was similar to that in response to Kluyveromyces lactis yeast cells and to that in response to phorbol myristate (a stimulator of protein C kinase). Intracellular ROS production by resident macrophages in response to microsporidian spores was similar to that produced in response to yeast cells. Both extracellular ROS production and intracellular ROS production (in response to all stimuli) were significantly lower after in vivo prestimulation of macrophages with microsporidian spores. These results demonstrate that microsporidian spores of species other than those that habitually infect mammals are capable of modulating the respiratory burst of rat peritoneal macrophages. Such modulation may contribute to avoidance by the microsporidian of cytotoxic responses associated with the respiratory burst.     

Peculiarities of glycosylation of proteins in spores of microsporidia Paranosema (Antonospora) grylli

The long-term adaptation of microsporidia, a large group of fungi-related unicellular microorganisms, to intracellular parasitism has led to extreme minimization of the cell functional apparatus. For instance, diversity of carbohydrates in the composition of parasite glycoproteins and proteoglycans seems to be restricted to the presence of O-bound chains composed of mannose residues. This suggestion is based on the discovery in the genome of the human microsporidian Encephalitozoon cuniculi of three genes responsible for the O-mannosylation of proteins with a lack of enzymes participating in N-glycosylation. In the present work, peculiarities of protein glycosylation in spores of the microsporidian Paranosema grylli infecting the fat body of the Mediterranean field cricket Gryllus bimaculatus was studied. SDS-PAGE analysis of spore proteins with subsequent staining by periodate and Schiff reagent has shown that individual glycoproteins of P. grylli are highly glycosylated, while the maximal stain intensity was seen in the major polar-tube protein PTP1. Treatment of the extracted material with N-glycosidase F and hybridization with WGA lectin conjugated with horseradish peroxidase showed no presence of glycosylated proteins in the P. grylli spores. At the same time, the selectively extracted major protein of the exospore p40 was specifically recognized by lectin GNA conjugated with agarose balls. Pretreatment of p40 with α-and β-mannosidases decreased considerably the efficiency of binding. Since lectin GNA is specific towards mannose terminal residues, this indicates the O-mannosylation of the microsporidial exospore major protein. In spite of the intensive PTP1 glycosylation, extracted proteins of the P. grylli polar-tube had no specific binding with GNA-agarose, so the issue of peculiarities of their glycosylation remains an open question. Comparison of the obtained data with results of deciphering of the E. cuniuculi genome allows for the conclusion to be made that the minimization of the glycosylation apparatus of microsporidial proteins is the common peculiarity of this group of parasites.     

The Prediction and Validation of Small CDSs Expand the Gene Repertoire of the Smallest Known Eukaryotic Genomes

The proper prediction of the gene catalogue of an organism is essential to obtain a representative snapshot of its overall lifestyle, especially when it is not amenable to culturing. Microsporidia are obligate intracellular, sometimes hard to culture, eukaryotic parasites known to infect members of every animal phylum. To date, sequencing and annotation of microsporidian genomes have revealed a poor gene complement with highly reduced gene sizes. In the present paper, we investigated whether such gene sizes may have induced biases for the methodologies used for genome annotation, with an emphasis on small coding sequence (CDS) gene prediction. Using better delineated intergenic regions from four Encephalitozoon genomes, we predicted de novo new small CDSs with sizes ranging from 78 to 255 bp (median 168) and corroborated these predictions by RACE-PCR experiments in Encephalitozoon cuniculi. Most of the newly found genes are present in other distantly related microsporidian species, suggesting their biological relevance. The present study provides a better framework for annotating microsporidian genomes and to train and evaluate new computational methods dedicated at detecting ultra-small genes in various organisms.     

Revisiting the phylogeny of microsporidia

Canonical microsporidians are a group of obligate intracellular parasites of a wide range of hosts comprising ~1,300 species of >220 genera. Microsporidians are related to fungi, and many characterised and uncharacterized groups closely related to them have been discovered recently, filling the knowledge gaps between them. These groups assigned to the superphylum Opisthosporidia have provided several important insights into the evolution of diverse intracellular parasitic lineages within the tree of eukaryotes. The most studied among opisthosporidians, canonical microsporidians, were known to science more than 160 years ago, however, the classification of canonical Microsporidia has been challenging due to common morphological homoplasy, and accelerated evolutionary rates. Instead of morphological characters, ssrRNA sequences have been used as the primary data for the classification of canonical microsporidians. Previous studies have produced a useful backbone of the microsporidian phylogeny, but provided only some nodal support, causing some confusion. Here, we reconstructed phylogenetic trees of canonical microsporidians using Bayesian and Maximum Likelihood inferences. We included rRNA sequences of 126 described/named genera, by far the broadest taxon coverage to date. Overall, our trees show similar topology and recovered four of the five main clades demonstrated in previous studies (Clades 1, 3, 4 and 5). Family level clades were well resolved within each major clade, but many were discordant with the recently revised classification. Therefore, revision and some reshuffling, especially within and between Clades 1 and 3 are required. We also reconstructed phylogenetic trees of Opisthosporidia to better integrate the evolutionary history of canonical microsporidians in a broader context. We discuss several traits shared only by canonical microsporidians that may have contributed to their striking ecological success in diverse metazoans. More targeted studies on the neglected host groups will be of value for a better understanding of the evolutionary history of these interesting intracellular parasites.     

Mechanisms of Microsporidial Cell Division: Ultrastructural Study on Encephalitozoon hellem

The mitotic process in microsporidian Encephalitozoon hellem. a known human pathogen, has been studied with the aim of elucidating some ultrastructural aspects of its nuclear division. The presence of a nuclear spindle, of "electrondense spindle plaques" associated with the nuclear envelope and of cytoplasmic double walled vesicles are reported. We suggest that these "electrondense spindle plaques" serve as foci for intranuclear and cytoplasmic microtubule arrangements, similar to the microtubule organizing centers within the centrosomes of animal cells. The extent to which the microsporidial division process is comparable with that of more familiar eukaryotes such as yeast cells is discussed.