Publications by year
2023
McLaren M, Conners R, Isupov MN, Gil-Díez P, Gambelli L, Gold VAM, Walter A, Connell SR, Williams B, Daum B, et al (2023). CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state.
Nature MicrobiologyAbstract:
CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state
AbstractTranslational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.
Abstract.
2022
McLaren M, Gil-Diez P, Isupov M, Conners R, Gambelli L, Gold V, Walter A, Connell S, Williams B, Daum B, et al (2022). <i>In situ</i> structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen.
Abstract:
In situ structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen
Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of cryo-electron tomography (cryoET) and single particle cryoEM. We show that microsporidian spores contain ribosomes primed for host cell invasion and thus shed new light on the infection mechanism of this important pathogen. Prior to host infection, virtually all ribosomes are locked in the 100 S dimeric state, which appears to be formed by a unique dimerization mechanism that is distinct from its bacterial counterparts. Within the dimer, the hibernation factor MDF1 is bound within the E site, locking the L1 stalk in a closed conformation, and thus preventing the translation of mRNAs to polypeptides.
Abstract.
Williams B, Williams T, Trew J (2022). Comparative Genomics of Microsporidia. In Weiss L, Reinke A (Eds.) Microsporidia.
Bojko J, Reinke AW, Stentiford GD, Williams B, Rogers MSJ, Bass D (2022). Microsporidia: a new taxonomic, evolutionary, and ecological synthesis.
Trends Parasitol,
38(8), 642-659.
Abstract:
Microsporidia: a new taxonomic, evolutionary, and ecological synthesis.
Microsporidian diversity is vast. There is a renewed drive to understand how microsporidian pathological, genomic, and ecological traits relate to their phylogeny. We comprehensively sample and phylogenetically analyse 125 microsporidian genera for which sequence data are available. Comparing these results with existing phylogenomic analyses, we suggest an updated taxonomic framework to replace the inconsistent clade numbering system, using informal taxonomic names: Glugeida (previously clades 5/3), Nosematida (4a), Enterocytozoonida (4b), Amblyosporida (3/5), Neopereziida (1), and Ovavesiculida (2). Cellular, parasitological, and ecological traits for 281 well-defined species are compared with identify clade-specific patterns across long-branch Microsporidia. We suggest that future taxonomic circumscriptions of Microsporidia should involve additional markers (SSU/ITS/LSU), and that a comprehensive suite of phenotypic and ecological traits help to predict broad microsporidian functional and lineage diversity.
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Author URL.
Bessette E, Williams B (2022). Protists in the Insect Rearing Industry: Benign Passengers or Potential Risk?.
Insects,
13(5).
Abstract:
Protists in the Insect Rearing Industry: Benign Passengers or Potential Risk?
As the insects for food and feed industry grows, a new understanding of the industrially reared insect microbiome is needed to better comprehend the role that it plays in both maintaining insect health and generating disease. While many microbiome projects focus on bacteria, fungi or viruses, protists (including microsporidia) can also make up an important part of these assemblages. Past experiences with intensive invertebrate rearing indicate that these parasites, whilst often benign, can rapidly sweep through populations, causing extensive damage. Here, we review the diversity of microsporidia and protist species that are found in reared insect hosts and describe the current understanding of their host spectra, life cycles and the nature of their interactions with hosts. Major entomopathogenic parasite groups with the potential to infect insects currently being reared for food and feed include the Amoebozoa, Apicomplexa, Ciliates, Chlorophyta, Euglenozoa, Ichtyosporea and Microsporidia. However, key gaps exist in the understanding of how many of these entomopathogens affect host biology. In addition, for many of them, there are very limited or even no molecular data, preventing the implementation of molecular detection methods. There is now a pressing need to develop and use novel molecular tools, coupled with standard molecular diagnostic methods, to help unlock their biology and predict the effects of these poorly studied protist parasites in intensive insect rearing systems.
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ALDAMA-CANO D, Wiredu-Boakye D, THANSA K, PRACHUMWAT A, TAENGCHAIYAPHUM S, ITSATHITPHAISARN O, CHAIJARASPHONG T, Williams BAP, STENTIFORD GD, SRITUNYALUCKSANA K, et al (2022). THE MICROSPORIDIAN ENTEROCYTOZOON. HEPATOPENAEI IN SHRIMP CULTURE. In Alday-Sanz V (Ed)
The Shrimp Book II, 5M Books Ltd, 525-546.
Abstract:
THE MICROSPORIDIAN ENTEROCYTOZOON. HEPATOPENAEI IN SHRIMP CULTURE
Abstract.
2019
Stentiford GD, Bass D, Williams BAP (2019). Ultimate opportunists-The emergent Enterocytozoon group Microsporidia.
PLoS Pathog,
15(5).
Author URL.
2018
Bass D, Czech L, Williams BAP, Berney C, Dunthorn M, Mahé F, Torruella G, Stentiford GD, Williams TA (2018). Clarifying the Relationships between Microsporidia and Cryptomycota.
J Eukaryot Microbiol,
65(6), 773-782.
Abstract:
Clarifying the Relationships between Microsporidia and Cryptomycota.
Some protists with microsporidian-like cell biological characters, including Mitosporidium, Paramicrosporidium, and Nucleophaga, have SSU rRNA gene sequences that are much less divergent than canonical Microsporidia. We analysed the phylogenetic placement and environmental diversity of microsporidian-like lineages that group near the base of the fungal radiation and show that they group in a clade with metchnikovellids and canonical microsporidians, to the exclusion of the clade including Rozella, in line with what is currently known of their morphology and cell biology. These results show that the phylogenetic scope of Microsporidia has been greatly underestimated. We propose that much of the lineage diversity previously thought to be cryptomycotan/rozellid is actually microsporidian, offering new insights into the evolution of the highly specialized parasitism of canonical Microsporidia. This insight has important implications for our understanding of opisthokont evolution and ecology, and is important for accurate interpretation of environmental diversity. Our analyses also demonstrate that many opisthosporidian (aphelid+rozellid+microsporidian) SSU V4 OTUs from Neotropical forest soils group with the short-branching Microsporidia, consistent with the abundance of their protist and arthropod hosts in soils. This novel diversity of Microsporidia provides a unique opportunity to investigate the evolutionary origins of a highly specialized clade of major animal parasites.
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Williams BAP, Hamilton KM, Jones MD, Bass D (2018). Group-specific environmental sequencing reveals high levels of ecological heterogeneity across the microsporidian radiation.
Environ Microbiol Rep,
10(3), 328-336.
Abstract:
Group-specific environmental sequencing reveals high levels of ecological heterogeneity across the microsporidian radiation.
The description of diversity is a key imperative in current biological studies and has been revolutionised by the molecular era that allows easy access to microbial diversity not visible to the naked eye. Broadly targeted SSU rRNA gene amplicon studies of diverse environmental habitats continue to reveal new microbial eukaryotic diversity. However, some eukaryotic lineages, particularly parasites, have divergent SSU sequences, and are therefore undersampled or excluded by the methodologies used for SSU studies. One such group is the Microsporidia, which have particularly divergent SSU sequences and are rarely detected in even large-scale amplicon studies. This is a serious omission as microsporidia are diverse and important parasites of humans and other animals of socio-economic importance. Whilst estimates of other microbial diversity are expanding, our knowledge of true microsporidian diversity has remained largely static. In this work, we have combined high throughput sequencing, broad environmental sampling and microsporidian-specific primers to broaden our understanding of the evolutionary diversity of the Microsporidia. Mapping our new sequences onto a tree of known microsporidian diversity we uncover new diversity across all areas of the microsporidian tree and uncover clades dominated by novel sequences, with no close described relatives.
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Jaroenlak P, Boakye DW, Vanichviriyakit R, Williams BAP, Sritunyalucksana K, Itsathitphaisarn O (2018). Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP).
Parasites and Vectors,
11(1).
Abstract:
Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP)
Background: the microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp. Results: We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein. Conclusions: Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.
Abstract.
2017
Wiredu Boakye D, Jaroenlak P, Prachumwat A, Williams TA, Bateman KS, Itsathitphaisarn O, Sritunyalucksana K, Paszkiewicz KH, Moore KA, Stentiford GD, et al (2017). Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia.
Environmental Microbiology,
19(5), 2077-2089.
Abstract:
Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia
Glycolysis and oxidative phosphorylation are the fundamental pathways of ATP generation in eukaryotes. Yet in microsporidia, endoparasitic fungi living at the limits of cellular streamlining, oxidative phosphorylation has been lost: energy is obtained directly from the host or, during the dispersive spore stage, via glycolysis. It was therefore surprising when the first sequenced genome from the Enterocytozoonidae – a major family of human and animal-infecting microsporidians – appeared to have lost genes for glycolysis. Here, we sequence and analyse genomes from additional members of this family, shedding new light on their unusual biology. Our survey includes the genome of Enterocytozoon hepatopenaei, a major aquacultural parasite currently causing substantial economic losses in shrimp farming, and Enterospora canceri, a pathogen that lives exclusively inside epithelial cell nuclei of its crab host. Our analysis of gene content across the clade suggests that Ent. canceri's adaptation to intranuclear life is underpinned by the expansion of transporter families. We demonstrate that this entire lineage of pathogens has lost glycolysis and, uniquely amongst eukaryotes, lacks any obvious intrinsic means of generating energy. Our study provides an important resource for the investigation of host-pathogen interactions and reductive evolution in one of the most medically and economically important microsporidian lineages.
Abstract.
Stentiford GD, Sritunyalucksana K, Flegel TW, Williams BAP, Withyachumnarnkul B, Itsathitphaisarn O, Bass D (2017). New Paradigms to Help Solve the Global Aquaculture Disease Crisis. PLoS Pathogens, 13(2).
2016
Williams TA, Nakjang S, Campbell SE, Freeman MA, Eydal M, Moore K, Hirt RP, Embley TM, Williams BAP (2016). A Recent Whole-Genome Duplication Divides Populations of a Globally Distributed Microsporidian.
Molecular Biology and Evolution,
33(8), 2002-2015.
Abstract:
A Recent Whole-Genome Duplication Divides Populations of a Globally Distributed Microsporidian
The Microsporidia are a major group of intracellular fungi and important parasites of animals including insects, fish, and immunocompromised humans. Microsporidian genomes have undergone extreme reductive evolution but there are major differences in genome size and structure within the group: some are prokaryote-like in size and organisation (
Abstract.
Jaroenlak P, Sanguanrut P, Williams BAP, Stentiford GD, Flegel TW, Sritunyalucksana K, Itsathitphaisarn O (2016). A nested PCR assay to avoid false positive detection of the microsporidian enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms.
PLoS ONE,
11(11).
Abstract:
A nested PCR assay to avoid false positive detection of the microsporidian enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms
Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (104 plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (106 copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers.
Abstract.
Stentiford GD, Becnel -JJ, Weiss LM, Keeling PJ, Didier ES, Williams B-AP, Bjornson S, Kent M-L, Freeman MA, Brown MJF, et al (2016). Microsporidia – Emergent Pathogens in the Global Food Chain. Trends in Parasitology, 32(4), 336-348.
Bateman KS, Wiredu-Boakye D, Kerr R, Williams BAP, Stentiford GD (2016). Single and multi-gene phylogeny of Hepatospora (Microsporidia) – a generalist pathogen of farmed and wild crustacean hosts.
Parasitology (Cambridge)Abstract:
Single and multi-gene phylogeny of Hepatospora (Microsporidia) – a generalist pathogen of farmed and wild crustacean hosts
Almost half of all known microsporidian taxa infect aquatic animals. of these, many cause disease in arthropods. Hepatospora, a recently erected genus, infects epithelial cells of the hepatopancreas of wild and farmed decapod crustaceans. We isolated Hepatospora spp. from three different crustacean hosts, inhabiting different habitats and niches; marine edible crab (Cancer pagurus), estuarine and freshwater Chinese mitten crab (Eriocheir sinensis) and the marine mussel symbiont pea crab (Pinnotheres pisum). Isolates were initially compared using histology and electron microscopy revealing variation in size, polar filament arrangement and nuclear development. However, sequence analysis of the partial SSU rDNA gene could not distinguish between the isolates (~99% similarity). In an attempt to resolve the relationship between Hepatospora isolated from E. sinensis and C. pagurus, six additional gene sequences were mined from on-going unpublished genome projects (RNA polymerase, arginyl tRNA synthetase, prolyl tRNA synthetase, chitin synthase, beta tubulin and heat shock protein 70). Primers were designed based on the above gene sequences to analyse Hepatospora isolated from pea crab. Despite application of gene sequences to concatenated phylogenies, we were unable to discriminate Hepatospora isolates obtained from these hosts and concluded that they likely represent a single species or, at least subspecies thereof. In this instance, concatenated phylogenetic analysis supported the SSU-based phylogeny, and further, demonstrated that microsporidian taxonomies based upon morphology alone are unreliable, even at the level of the species. Our data, together with description of H. eriocheir in Asian crab farms, reveal a preponderance for microvariants of this parasite to infect the gut of a wide array of decapods crustacean hosts and the potential for Hepatospora to exist as a cline across wide geographies and habitats.
Abstract.
2015
Watson AK, Williams TA, Williams BAP, Moore KA, Hirt RP, Embley TM (2015). Transcriptomic profiling of host-parasite interactions in the microsporidian Trachipleistophora hominis.
BMC GenomicsAbstract:
Transcriptomic profiling of host-parasite interactions in the microsporidian Trachipleistophora hominis
© 2015 Watson et al. Background: Trachipleistophora hominis was isolated from an HIV/AIDS patient and is a member of a highly successful group of obligate intracellular parasites. Methods: Here we have investigated the evolution of the parasite and the interplay between host and parasite gene expression using transcriptomics of T. hominis-infected rabbit kidney cells. Results: T. hominis has about 30 % more genes than small-genome microsporidians. Highly expressed genes include those involved in growth, replication, defence against oxidative stress, and a large fraction of uncharacterised genes. Chaperones are also highly expressed and may buffer the deleterious effects of the large number of non-synonymous mutations observed in essential T. hominis genes. Host expression suggests a general cellular shutdown upon infection, but ATP, amino sugar and nucleotide sugar production appear enhanced, potentially providing the parasite with substrates it cannot make itself. Expression divergence of duplicated genes, including transporters used to acquire host metabolites, demonstrates ongoing functional diversification during microsporidian evolution. We identified overlapping transcription at more than 100 loci in the sparse T. hominis genome, demonstrating that this feature is not caused by genome compaction. The detection of additional transposons of insect origin strongly suggests that the natural host for T. hominis is an insect. Conclusions: Our results reveal that the evolution of contemporary microsporidian genomes is highly dynamic and innovative. Moreover, highly expressed T. hominis genes of unknown function include a cohort that are shared among all microsporidians, indicating that some strongly conserved features of the biology of these enormously successful parasites remain uncharacterised.
Abstract.
2014
Williams BAP, Dolgikh VV, Sokolova YY (2014). Microsporidian Biochemistry and Physiology. In (Ed)
Microsporidia, John Wiley & Sons, Inc. 245-260.
Author URL.
2013
Read BA, Kegel J, Klute MJ, Kuo A, Lefebvre SC, Maumus F, Mayer C, Miller J, Monier A, Salamov A, et al (2013). Pan genome of the phytoplankton Emiliania underpins its global distribution.
Nature,
499(7457), 209-213.
Abstract:
Pan genome of the phytoplankton Emiliania underpins its global distribution.
Coccolithophores have influenced the global climate for over 200 million years. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space. Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions.
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Campbell SE, Williams TA, Yousuf A, Soanes DM, Paszkiewicz KH, Williams BAP (2013). The Genome of Spraguea lophii and the Basis of Host-Microsporidian Interactions.
PLoS Genet,
9(8).
Abstract:
The Genome of Spraguea lophii and the Basis of Host-Microsporidian Interactions
Author SummaryMicrosporidia are unusual intracellular parasites that infect a broad range of animal cells. In comparison to their fungal relatives, microsporidian genomes have shrunk during evolution, encoding as few as 2000 proteins. This minimal molecular repertoire makes them a reduced model system for understanding host-parasite interactions. A number of microsporidian genomes have now been sequenced, but the lack of a system for genetic manipulation makes it difficult to translate these data into a better understanding of microsporidian biology. Here we present a deep sequencing project of Spraguea lophii, a fish-infecting microsporidian that is abundantly available from environmental samples. We use our sequence data combined with germination protocols and complex-mix proteomics to identify proteins released by the cell at the earliest stage of germination, representing potential pathogenicity factors. We profile the RNA expression pattern of germinating cells and identify a set of highly transcribed hypothetical genes. Our study provides new insight into the importance of uncharacterized, lineage-specific and/or fast evolving proteins in microsporidia and provides new leads for the investigation of virulence factors in these enigmatic parasites.
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2011
Williams BAP, Keeling PJ (2011). Microsporidia – Highly Reduced and Derived Relatives of Fungi. In Pöggeler S, Wöstemeyer J (Eds.) Evolution of Fungi and Fungal-Like Organisms, Springer Verlag.
2010
Williams BAP, Elliot C, Burri L, Kido Y, Kita K, Moore AL, Keeling PJ (2010). A broad distribution of the alternative oxidase in microsporidian parasites.
PLoS Pathogens,
6(2).
Abstract:
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 ironsulphur 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. © 2010 Williams et al.
Abstract.
2009
Williams BAP (2009). Unique physiology of host-parasite interactions in microsporidia infections.
Cellular Microbiology,
11(11), 1551-1560.
Abstract:
Unique physiology of host-parasite interactions in microsporidia infections
Microsporidia are intracellular parasites of all major animal lineages and have a described diversity of over 1200 species and an actual diversity that is estimated to be much higher. They are important pathogens of mammals, and are now one of the most common infections among immunocompromised humans. Although related to fungi, microsporidia are atypical in genomic biology, cell structure and infection mechanism. Host cell infection involves the rapid expulsion of a polar tube from a dormant spore to pierce the host cell membrane and allow the direct transfer of the spore contents into the host cell cytoplasm. This intimate relationship between parasite and host is unique. It allows the microsporidia to be highly exploitative of the host cell environment and cause such diverse effects as the induction of hypertrophied cells to harbour prolific spore development, host sex ratio distortion and host cell organelle and microtubule reorganization. Genome sequencing has revealed that microsporidia have achieved this high level of parasite sophistication with radically reduced proteomes and with many typical eukaryotic pathways pared-down to what appear to be minimal functional units. These traits make microsporidia intriguing model systems for understanding the extremes of reductive parasite evolution and host cell manipulation. © 2009 Blackwell Publishing Ltd.
Abstract.
2008
Lee RCH, Williams BAP, Brown AMV, Adamson ML, Keeling PJ, Fast NM (2008). Alpha- and beta-tubulin phylogenies support a close relationship between the microsporidia Brachiola algerae and Antonospora locustae.
J Eukaryot Microbiol,
55(5), 388-392.
Abstract:
Alpha- and beta-tubulin phylogenies support a close relationship between the microsporidia Brachiola algerae and Antonospora locustae.
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 alpha- and beta-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.
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Author URL.
Williams BAP, Haferkamp I, Keeling PJ (2008). An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae.
J Mol Biol,
375(5), 1249-1257.
Abstract:
An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae.
The mitochondrion is one of the defining characteristics of eukaryotic cells, and to date, no eukaryotic lineage has been shown to have lost mitochondria entirely. In certain anaerobic or microaerophilic lineages, however, the mitochondrion has become severely reduced that it lacks a genome and no longer synthesizes ATP. One example of such a reduced organelle, called the mitosome, is found in microsporidian parasites. Only a handful of potential mitosomal proteins were found to be encoded in the complete genome of the microsporidian Encephalitozoon cuniculi, and significantly no proteins of the mitochondrial carrier family were identified. These carriers facilitate the transport of solutes across the inner mitochondrial membrane, are a means of communication between the mitochondrion and cytosol, and are abundant in organisms with aerobic mitochondria. Here, we report the characterization of a mitochondrial carrier protein in the microsporidian Antonospora locustae and demonstrate that the protein is heterologously targeted to mitochondria in Saccharomyces cerevisiae. The protein is phylogenetically allied to the NAD(+) transporter of S. cerevisiae, but we show that it has high specificity for ATP and ADP when expressed in Escherichia coli. An ADP/ATP carrier may provide ATP for essential ATP-dependent mitosomal processes such as Hsp70-dependent protein import and export of iron-sulfur clusters to the cytosol.
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Williams BAP, Cali A, Takvorian PM, Keeling PJ (2008). Distinct localization patterns of two putative mitochondrial proteins in the microsporidian Encephalitozoon cuniculi.
J Eukaryot Microbiol,
55(2), 131-133.
Abstract:
Distinct localization patterns of two putative mitochondrial proteins in the microsporidian Encephalitozoon cuniculi.
Microsporidia were once considered amitochondriate, but have now been found to retain relict mitochondria called mitosomes. These organelles have been identified by immunolocalization in Trachipleistophora hominis, whereas most data on function have been inferred from the presence of mitochondrial protein-encoding sequences in the genome of Encephalitozoon cuniculi. Here we describe the localization of two such enzymes in E. cuniculi cells. Immunofluorescent localization of ferredoxin involved in mitochondrial iron-sulfur cluster assembly reveals a punctate distribution as expected for mitochondria. In contrast, localization of mitochondrial glycerol-3-phosphate dehydrogenase suggests a cytoplasmic distribution in E. cuniculi and possible relocalization of this typically mitochondrial protein.
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Author URL.
Williams BAP, Lee RCH, Becnel JJ, Weiss LM, Fast NM, Keeling PJ (2008). Genome sequence surveys of Brachiola algerae and Edhazardia aedis reveal microsporidia with low gene densities.
BMC Genomics,
9Abstract:
Genome sequence surveys of Brachiola algerae and Edhazardia aedis reveal microsporidia with low gene densities.
BACKGROUND: Microsporidia are well known models of extreme nuclear genome reduction and compaction. The smallest microsporidian genomes have received the most attention, but genomes of different species range in size from 2.3 Mb to 19.5 Mb and the nature of the larger genomes remains unknown. RESULTS: Here we have undertaken genome sequence surveys of two diverse microsporidia, Brachiola algerae and Edhazardia aedis. In both species we find very large intergenic regions, many transposable elements, and a low gene-density, all in contrast to the small, model microsporidian genomes. We also find no recognizable genes that are not also found in other surveyed or sequenced microsporidian genomes. CONCLUSION: Our results demonstrate that microsporidian genome architecture varies greatly between microsporidia. Much of the genome size difference could be accounted for by non-coding material, such as intergenic spaces and retrotransposons, and this suggests that the forces dictating genome size may vary across the phylum.
Abstract.
Author URL.
Lee RCH, Williams BAP, Brown AMV, Adamson ML, Keeling PJ, Fast NM (2008). α- and β-tubulin phylogenies support a close relationship between the microsporidia Brachiola algerae and Antonospora locustae.
Journal of Eukaryotic Microbiology,
55(5), 388-392.
Abstract:
α- and β-tubulin phylogenies support a close relationship between the microsporidia Brachiola algerae and Antonospora locustae
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. © 2008 the Author(s).
Abstract.
2007
Weiss, L.M. Feng, X. Williams, B.A. (2007). Analysis of the beta-tubulin genes from <em>Enterocytozoon bieneusi</em> isolates from a human and rhesus macaque. J Eukaryot Microbiol, 54, 38-41.
2006
Burri L, Williams BAP, Bursac D, Lithgow T, Keeling PJ (2006). Microsporidian mitosomes retain elements of the general mitochondrial targeting system.
Proc Natl Acad Sci U S A,
103(43), 15916-15920.
Abstract:
Microsporidian mitosomes retain elements of the general mitochondrial targeting system.
Microsporidia are intracellular parasites that infect a variety of animals, including humans. As highly specialized parasites, they are characterized by a number of unusual adaptations, many of which are manifested as extreme reduction at the molecular, biochemical, and cellular levels. One interesting aspect of reduction is the mitochondrion. Microsporidia were long considered to be amitochondriate, but recently a tiny mitochondrion-derived organelle called the mitosome was detected. The molecular function of this organelle remains poorly understood. The mitosome has no genome, so it must import all its proteins from the cytosol. In other fungi, the mitochondrial protein import machinery consists of a network series of heterooligomeric translocases and peptidases, but in microsporidia, only a few subunits of some of these complexes have been identified to date. Here, we look at targeting sequences of the microsporidian mitosomal import system and show that mitosomes do in some cases still use N-terminal and internal targeting sequences that are recognizable by import systems of mitochondria in yeast. Furthermore, we have examined the function of the inner membrane peptidase processing enzyme and demonstrate that mitosomal substrates of this enzyme are processed to mature proteins in one species with a simplified processing complex, Antonospora locustae. However, in Encephalitozoon cuniculi, the processing complex is lost altogether, and the preprotein substrate functions with the targeting leader still attached. This report provides direct evidence for presequencing processing in mitosomes and also shows how a complex molecular system has continued to degenerate throughout the evolution of microsporidia.
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2005
Williams BAP, Slamovits CH, Patron NJ, Fast NM, Keeling PJ (2005). A high frequency of overlapping gene expression in compacted eukaryotic genomes.
Proc Natl Acad Sci U S A,
102(31), 10936-10941.
Abstract:
A high frequency of overlapping gene expression in compacted eukaryotic genomes.
The gene density of eukaryotic nuclear genomes is generally low relative to prokaryotes, but several eukaryotic lineages (many parasites or endosymbionts) have independently evolved highly compacted, gene-dense genomes. The best studied of these are the microsporidia, highly adapted fungal parasites, and the nucleomorphs, relict nuclei of endosymbiotic algae found in cryptomonads and chlorarachniophytes. These systems are now models for the effects of compaction on the form and dynamics of the nuclear genome. Here we report a large-scale investigation of gene expression from compacted eukaryotic genomes. We have conducted EST surveys of the microsporidian Antonospora locustae and nucleomorphs of the cryptomonad Guillardia theta and the chlorarachniophyte Bigelowiella natans. In all three systems we find a high frequency of mRNA molecules that encode sequence from more than one gene. There is no bias for these genes to be on the same strand, so it is unlikely that these mRNAs represent operons. Instead, compaction appears to have reduced the intergenic regions to such an extent that control elements like promoters and terminators have been forced into or beyond adjacent genes, resulting in long untranslated regions that encode other genes. Normally, transcriptional overlap can interfere with expression of a gene, but these genomes cope with high frequencies of overlap and with termination signals within expressed genes. These findings also point to serious practical difficulties in studying expression in compacted genomes, because many techniques, such as arrays or serial analysis of gene expression will be misleading.
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Keeling PJ, Fast NM, Law JS, Williams BAP, Slamovits CH (2005). Comparative genomics of microsporidia.
Folia Parasitol (Praha),
52(1-2), 8-14.
Abstract:
Comparative genomics of microsporidia.
Microsporidia have been known for some time to possess among the smallest genomes of any eukaryote. There is now a completely sequenced microsporidian genome, as well as several other large-scale sequencing efforts, so the nature of these genomes is becoming apparent. This paper reviews some of the characteristics of microsporidian genomes in general, and some of the recent discoveries made through comparative genomic analyses. In general, microsporidian genomes are both reduced and compacted. Reduction takes place through gene loss, which is understandable in obligate intracellular parasites that rely on their host for many metabolites. Compaction is a more complex process, and is as yet not fully understood. It is clear from genomes surveyed thus far that the remaining genes are tightly packed and that there is little non-coding sequence, resulting in some extraordinary arrangements, including overlapping genes. Compaction also seems to affect certain aspects of genome evolution, like the frequency of rearrangements. The force behind this compaction is not known, and is especially interesting in light of the fact that surveys of genomes that are significantly different in size yield similar complements of protein-coding genes. There are some interesting exceptions, including catalase, photolyase and some mitochondrial proteins, but the rarity of these raises an interesting question as to what accounts for the significant differences seen in the genome sizes among microsporidia.
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Williams BAP, Keeling PJ (2005). Microsporidian mitochondrial proteins: expression in Antonospora locustae spores and identification of genes coding for two further proteins.
J Eukaryot Microbiol,
52(3), 271-276.
Abstract:
Microsporidian mitochondrial proteins: expression in Antonospora locustae spores and identification of genes coding for two further proteins.
Microsporidia are obligate intracellular parasites, phylogenetically allied to the fungi. Once considered amitochondriate, now a number of mitochondrion-derived genes have been described from various species, and the relict organelle was recently identified in Trachipleistophora hominis. We have investigated the expression of potential mitochondrial targeted proteins in the spore stage to determine whether the organelle is likely to have a role in the spore or early infection stage. To investigate whether the Antonospora locustae genome codes for a different complement of mitochondrial proteins than Encephalitozoon cuniculi an EST library was searched for putative mitochondrial genes that have not been identified in the E. cuniculi genome project. The spore is the infectious stage of microsporidia, but is generally considered to be metabolically dormant. Fourteen genes for putatively mitochondrion-targeted proteins were shown to be present in purified spore mRNA by 3'-rapid amplification of cDNA ends and EST sequencing. Pyruvate dehydrogenase E1alpha and mitochondrial glycerol-3-phosphate dehydrogenase proteins were also shown to be present in A. locustae and E. cuniculi spores, respectively, suggesting a role for these proteins in the early stages of infection, or within the spore itself. EST sequencing also revealed two mitochondrial protein-encoding genes in A. locustae that are not found in the genome of E. cuniculi. One encodes a possible pyruvate transporter, the other a subunit of the mitochondrial inner membrane peptidase. In yeast mitochondria, this protein is part of a trimeric complex that processes proteins targeted to the inner membrane and the intermembrane space, and its substrate in A. locustae is presently unknown.
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2004
Williams BAP, Hirt RP (2004). RACE and RAGE cloning in parasitic microbial eukaryotes.
Methods Mol Biol,
270, 151-172.
Abstract:
RACE and RAGE cloning in parasitic microbial eukaryotes.
Many gene-cloning strategies and gene survey often provide partial sequence data. To exploit the information from these partial sequences numerous PCR-based approaches have been developed to clone full-length open reading frames. These approaches can be successful using small quantities of cDNA or genomic DNA as starting material and avoid the need to go through the complex and tedious process of constructing and screening gene libraries. Here we present two of these approaches, called RACE and RAGE, we used to successfully clone partial and full-length ORFs from amitochondriate parasitic microbial eukaryotes. The RACE approach uses cDNA as template for PCR cloning whereas RAGE uses genomic DNA. These two approaches were used to complement each other to provide full-length genes. The amitochondriate microbial eukaryotes we are investigating are of interest from both evolutionary and biomedical perspectives. We have investigated genes of mitochondrial origins in the obligate intracellular parasite called microsporidia. In these organisms spores are the only source of material that can be isolated from host cells and typically yield small amount of mRNA and genomic DNA for cloning. A full-length mitochondrial Hsp70 could be cloned and sequenced and specific antibody raised against a fusion protein. The highly specific antibody allowed us to demonstrate for the first time the presence of mitochondrial-like organelles in microsporidia.
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Slamovits CH, Williams BAP, Keeling PJ (2004). Transfer of Nosema locustae (Microsporidia) to Antonospora locustae n. comb. based on molecular and ultrastructural data.
J Eukaryot Microbiol,
51(2), 207-213.
Abstract:
Transfer of Nosema locustae (Microsporidia) to Antonospora locustae n. comb. based on molecular and ultrastructural data.
Nosema locustae is a microsporidian parasite of grasshopper pests that is used as a biological control agent, and is one of the emerging model systems for microsporidia. Due largely to its diplokaryotic nuclei, N. locustae has been classified in the genus Nosema, a large genus with members that infect a wide variety of insects. However, some molecular studies have cast doubt on the validity of certain Nosema species, and on the taxonomic position of N. locustae. To clarify the affinities of this important insect parasite we sequenced part of the rRNA operon of N. locustae and conducted a phylogenetic analysis using the complete small subunit rRNA gene. Nosema locustae is only distantly related to the nominotypic N. bombycis, and is instead closely related to Antonospora scoticae, a recently described parasite of bees. We examined the ultrastructure of mature N. locustae spores, and found the spore wall to differ from true Nosema species in having a multi-layered exospore resembling that of Antonospora (one of the distinguishing features of that genus). Based on both molecular and morphological evidence, therefore, we propose transferring N. locustae to the genus Antonospora, as Antonospora locustae n. comb.
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2003
Fast NM, Law JS, Williams BAP, Keeling PJ (2003). Bacterial catalase in the microsporidian Nosema locustae: implications for microsporidian metabolism and genome evolution.
Eukaryot Cell,
2(5), 1069-1075.
Abstract:
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.
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Williams BAP, Keeling PJ (2003). Cryptic organelles in parasitic protists and fungi.
Adv Parasitol,
54, 9-68.
Abstract:
Cryptic organelles in parasitic protists and fungi.
A number of parasitic protists and fungi have adopted extremely specialised characteristics of morphology, biochemistry, and molecular biology, sometimes making it difficult to discern their evolutionary origins. One aspect of several parasitic groups that reflects this is their metabolic organelles, mitochondria and plastids. These organelles are derived from endosymbiosis with an alpha-proteobacterium and a cyanobacterium respectively, and are home to a variety of core metabolic processes. As parasites adapted, new demands, or perhaps a relaxation of demands, frequently led to significant changes in these organelles. At the extreme, the organelles are degenerated and transformed beyond recognition, and are referred to as "cryptic". Generally, there is no prior cytological evidence for a cryptic organelle, and its presence is only discovered through phylogenetic analysis of molecular relicts followed by their localisation to organelle-like structures. Since the organelles are derived from eubacteria, the genes for proteins and RNAs associated with them are generally easily recognisable, and since the metabolic activities retained in these organelles are prokaryotic, or at least very unusual, they often serve as an important target for therapeutics. Cryptic mitochondria are now known in several protist and fungal parasites. In some cases (e.g. Trichomonas), well characterised but evolutionarily enigmatic organelles called hydrogenosomes were shown to be derived from mitochondria. In other cases (e.g. Entamoeba and microsporidia), "amitochondriate" parasites have been shown to harbour a previously undetected mitochondrial organelle. Typically, little is known about the functions of these newly discovered organelles, but recent progress in several groups has revealed a number of potential functions. Cryptic plastids have now been found in a small number of parasites that were not previously suspected to have algal ancestors. One recent case is the discovery that helicosporidian parasites are really highly adapted green alga, but the most spectacular case is the discovery of a plastid in the Apicomplexa. Apicomplexa are very well-studied parasites that include the malaria parasite, Plasmodium, so the discovery of a cryptic plastid in Apicomplexa came as quite a surprise. The apicomplexan plastid is now very well characterised and has been shown to function in the biosynthesis of fatty acids, isopentenyl diphosphate and heme, activities also found in photosynthetic plastids.
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Hirt, R.P. Williams, B.A. Embley, T.M. (2003). Horizontal gene transfer and the evolution of parasitic protozoa. Protist, 154, 17-32.
2002
Williams BAP, Hirt RP, Lucocq JM, Embley TM (2002). A mitochondrial remnant in the microsporidian Trachipleistophora hominis.
Nature,
418(6900), 865-869.
Abstract:
A mitochondrial remnant in the microsporidian Trachipleistophora hominis.
Microsporidia are obligate intracellular parasites of several eukaryotes. They have a highly complex and unique infection apparatus but otherwise appear structurally simple. Microsporidia are thought to lack typical eukaryotic organelles, such as mitochondria and peroxisomes. This has been interpreted as support for the hypothesis that these peculiar eukaryotes diverged before the mitochondrial endosymbiosis, which would make them one of the earliest offshoots in eukaryotic evolution. But microsporidial nuclear genes that encode orthologues of typical mitochondrial heatshock Hsp70 proteins have been detected, which provides evidence for secondary loss of the organelle or endosymbiont. In addition, gene trees and more sophisticated phylogenetic analyses have recovered microsporidia as the relatives of fungi, rather than as basal eukaryotes. Here we show that a highly specific antibody raised against a Trachipleistophora hominis Hsp70 protein detects the presence, under light and electron microscopy, of numerous tiny ( approximately 50 x 90 nm) organelles with double membranes in this human microsporidial parasite. The finding of relictual mitochondria in microsporidia provides further evidence of the reluctance of eukaryotes to lose the mitochondrial organelle, even when its canonical function of aerobic respiration has been apparently lost.
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1999
Bray RA, Littlewood DTJ, Herniou EA, Williams B, Henderson RE (1999). Digenean parasites of deep-sea teleosts: a review and case studies of intrageneric phylogenies.
Parasitology,
119(SUPPL.).
Abstract:
Digenean parasites of deep-sea teleosts: a review and case studies of intrageneric phylogenies
Studies on the digenean parasites of deep-sea (> 200 m depth) teleosts are reviewed and two case study generic phylogenies are presented based on LSU rDNA and ND1 mtDNA sequences. The phylogeny of the lepocreadiid genus Lepidapedon, the most common deep-sea digenean genus, is not clearly resolved as the two gene trees are not compatible. It can be inferred, however, that the genus has radiated in the deeper waters off the continental shelf, mainly in fishes of the gadiform family Macrouridae. Steringophorus, a fellodistomid genus, is better resolved. In this case a deep-sea radiation is also indicated, but the pattern of host-specificity is not clear, with evidence of much host-switching. Results of studies of the parasites of the macrourid fish Coryphaenoides (Nematonurus) armatus from various depths have reinforced recent views on the lack of zoned depth-related communities in the deep-sea. The diversity of deep-sea digeneans is relatively low with only 18 families (of about 60) reported. Little, or nothing, is known from most deep-sea areas and nothing from trenches and mid-ocean ridge systems.
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