Evolution of microsporidia: An extremely successful group of eukaryotic intracellular parasites

Evolution of microsporidia: An extremely successful group of eukaryotic intracellular parasites PEARLS Evolution of microsporidia: An extremely successful group of eukaryotic intracellular parasites Lina Wadi , Aaron W. Reinke * ID ID Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada * aaron.reinke@utoronto.ca Overview Microsporidia are obligate intracellular parasites that can infect a wide range of hosts. They cause death and disease in both humans and agriculturally important animals. They have also become a powerful model for understanding the evolution of intracellular parasites. Recent work has explored how microsporidia genomes have evolved, the evolutionary origins of a1111111111 microsporidia, and how microsporidia adapt to interact with their hosts. a1111111111 a1111111111 What do microsporidia have in common? a1111111111 Microsporidia can only exist outside of their host as environmentally resistant, chitin-contain- a1111111111 ing spores. These spores contain a unique infectious apparatus known as the polar tube. Dur- ing infection, the polar tube is rapidly discharged and can pierce a host cell, depositing the parasite’s sporoplasm within the host cell [1]. The sporoplasm then proliferates, eventually producing spores that then exit the host to cause subsequent infections. Microsporidia are OPENACCESS thought to reproduce mostly asexually, although most are likely diploid based on genomic het- Citation: Wadi L, Reinke AW (2020) Evolution of erozygosity and conservation of meiotic genes [2–5]. microsporidia: An extremely successful group of Because of the intimate relationship between microsporidia and their hosts, they are heavily eukaryotic intracellular parasites. PLoS Pathog 16 dependent on host resources and have undergone extensive genomic reduction [6,7]. Analysis (2): e1008276. https://doi.org/10.1371/journal. of sequenced microsporidia genomes (Fig 1A) has revealed a wide loss of protein families that ppat.1008276 are present in other eukaryotes, leaving only approximately 800 conserved microsporidia pro- Editor: Laura J. Knoll, University of Wisconsin teins [6]. The proteins that have been retained function in essential core cellular processes Medical School, UNITED STATES such as DNA replication, transcription, and translation [6,8,9]. Proteins that have been lost Published: February 13, 2020 include many metabolic enzymes, regulatory pathways, and proteins involved in vesicular transport such as TOR (Target of Rapamycin) and clathrin [8–10]. In addition, the proteins Copyright:© 2020 Wadi, Reinke. This is an open access article distributed under the terms of the that are retained are shorter than their fungal orthologs, with Encephalitozoon cuniculi proteins Creative Commons Attribution License, which being on average only 85% as long as their yeast counterparts [3,11]. One such instance of pro- permits unrestricted use, distribution, and teins that have decreased in size are the aminoacyl-tRNA synthetases, which have lost many reproduction in any medium, provided the original regulatory regions, including a domain in leucyl-tRNA synthetase (LeuRS) that edits mis- author and source are credited. charged tRNAs. The microsporidia Vavraia culicis was shown to have mistranslation error Funding: This project was funded by the Canadian rates of 5.9% at positions that coded for leucine—although valine, which has a corresponding Institutes of Health Research grant PJT-159479 tRNA synthetases with an intact editing domain, has a mistranslation rate of 7.5%, suggesting and the Alfred P. Sloan Research Fellowship FG- that there are other factors responsible for the high levels of mistranslated proteins [12]. 2019-12040. The funders had no role in study design, data collection and analysis, decision to Microsporidia also have reduced noncoding RNAs, including the 18s RNA, which is only publish, or preparation of the manuscript. approximately 2/3 as large as in other eukaryotes and results in the smallest eukaryotic ribo- somes [13]. Although this reduction has led to several binding sites being eliminated, struc- Competing interests: The authors have declared that no competing interests exist. tural and mass spectrometry analysis of ribosomes from Vairimorpha necatrix demonstrated PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 1 / 8 Fig 1. Phylogeny, hosts, and characteristics of microsporidia species. (A) Phylogenetic tree of whole-genome sequenced microsporidia and related species. Tree was generated as described previously using single-copy orthologs present in all species [19,35]. Predicted proteins for each genome were obtained from https://www.ncbi. nlm.nih.gov, except for Metchnikovella incurvata and Enterospora canceri, which were predicted from genome assemblies using Prodigal 2.6.3. Orthologous gene −5 families were identified using OrthoMCL 2.0.9 using an inflation index of 1.5 and a BLAST E-value cutoff of 10 . Phylogeny was constructed from 43 single-copy orthologs present in all 28 species. Proteins from each orthogroup were aligned using MUSCLE 3.8.31. These alignments were trimmed using trimAl 1.4 with the option-gappyout. Each orthogroup alignment was then concatenated into a single alignment using FASconCAT 1.11. Using ProtTest 3.4.2, it was determined that PROTGAMMAIWAGF was the best fitting model for the data. Phylogeny was then inferred using RAxML 8.2.12 with the best fitting model and 1,000 bootstrap replicates. Scale bar indicates changes per site. To the right of each species are illustrations of the host(s) that each species has been reported to infect. This phylogenetic tree is largely in agreement with recent previously published phylogenies [3,4,8,9,19,23,27], with the exception of Anncaliia algerae or Edhazardia aedis, which have different placements in several reports [3,8,23,27]. (B) Comparision of protein content for each species. (C) Comparision of genome size for each species. (D) Morphological and molecular characteristics of each species. E-value, expect value; TOR, target of rapamycin. https://doi.org/10.1371/journal.ppat.1008276.g001 that most conserved ribosomal proteins can still bind [14]. Microsporidia also have reduced organelles, including mitochondrial remnants known as mitosomes [15]. These mitosomes do not encode a mitochondrial genome and are incapable of carrying out oxidative phosphoryla- tion; microsporidia must instead import ATP from their host [16]. Interestingly, there are 32 conserved protein families in microsporidia that are not found in any other eukaryote [6]. Sev- eral of these proteins function as part of the polar tube or spore wall, and the function of most of these proteins is unknown [6]. How do microsporidia genomes differ from one another? Microsporidia genome sizes vary significantly. The largest is Edhazardia aedis at 51.3 Mb (encoding approximately 4,200 proteins), and Encephalitozoon genomes are the smallest, with PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 2 / 8 some being only 2.3 MB (encoding approximately 1,800 proteins) [17] (Fig 1B and 1C). Much of this size disparity results from noncoding DNA, with only 9% of the E. aedis genome coding for proteins, compared to approximately 90% for E. cuniculi. The regions between genes can become very compact, with the intergenic regions averaging only 115 base pairs in Encephali- tozoon intestinalis [17,18]. Though microsporidia have much fewer proteins overall compared to other eukaryotes, genomic reduction was followed by a large expansion in lineage-specific protein families [6]. For example, Nematocida parisii comprises 2,661 proteins, of which only 1,074 are conserved with other eukaryotes or with other microsporidia species outside of its genus. This means that over half of the proteins in N. parisii are unique to the Nematocida genus, with 318 not even present in N. ironsii, a closely related sister species [19]. Many of these unique genes are found in subtelomeric regions and likely arose through local duplica- tion events [19]. Additional mechanisms that have changed the protein content of microspori- dia species include whole-genome duplications and genes arising from noncoding regions [20]. Several key eukaryotic processes have been lost in some, but not all, microsporidia. The ability to splice mRNA transcripts has been lost multiple times in microsporidia evolution, including in E. aedis, Nematocida species, and Enterocytozoon species, which no longer contain functional spliceosome machinery [21] (Fig 1D). This has resulted in some genomes having no detectable introns and the loss of most conserved splicing proteins [3]. Although many micro- sporidia genomes have retained Dicer and Argonaute, the two key effectors of the RNA inter- ference (RNAi) pathway, both of these proteins have been lost several times, including in all of the species in Nematocida and Encephalitozoon [4,22]. Although all microsporidia species have lost many metabolic enzymes, there are important distinctions such as differential losses in lipid biosynthesis genes [3]. Additionally, Enterocytozoon species have lost genes necessary for glycolysis, leaving them completely dependent on their host for energy production [23]. What did the last common ancestor of microsporidia look like? Microsporidia were once considered the earliest diverging eukaryotes, but sequencing of a large number of microsporidia genomes in the last several decades has made it clear that they belong to a group of the earliest diverging fungi [2,24]. More recently, the discovery and subse- quent genome sequencing of related basal species has shed light on the evolutionary origin of microsporidia. Microsporidia are closely related to a group of obligate intracellular parasites called the Cryptomycota or Rozellomycota [25] (Fig 1A). The only one of these species to have its genome sequenced so far, Rozella allomycis, infects water mold using motile flagellated spores [26]. Different than microsporidia, R. allomycis possesses mitochondria and encodes a much larger set of proteins that is more conserved with other eukaryotes. To date, the two most early diverging microsporidian species that have been sequenced are Paramicrospori- dium saccamoebae (classified as a Rozellomycota), an intranuclear parasite of amoeba, and Mitosporidium daphniae (classified as a microsporidia), which infects daphnia [27,28]. Although there has been debate about whether these species should be classified as microspori- dia, recent analysis of environmental samples supports the existence of an expanded micro- sporidia that includes a diversity of early diverging species [29]. P. Saccamoebae and M. daphniae differ from R. allomycis in that their spores have lost flagella and contain a polar fila- ment, although invasion by P. saccamoebae is carried out through host phagocytosis [30]. While P. saccamoebae and M. daphnia have a reduced gene set compared to other fungi, they have undergone less genome reduction than canonical microsporidia. Both species also have mitochondrial genomes, although P. saccamoebae has a conserved electron-transport chain whereas both R. allomycis and M. daphnia have lost Complex I and have different nuclear- PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 3 / 8 encoded genes that are thought to facilitate ATP generation [26–28]. Additionally, P. sacca- moebae has more genes in common with distantly related fungi than with R. allomycis or M. daphnia, suggesting that these species have undergone independent gene loss during coevolu- tion with their hosts [27]. Another early diverging group of microsporidia are the metchnikovellids, which parasitize apicomplexan gregarines. Recent genome sequencing of two of these species, Metchnikovella incurvata and Amphiamblys sp., has revealed that this group is most similar to the canonical microsporidia, as these species have undergone dramatic gene loss and do not contain mito- chondrial genomes [8,9] (Fig 1). One of the most striking differences in metchnikovellids is the presence of clathrin, which has been lost in the canonical microsporidia [8,9]. Additionally, the 32 conserved protein families that were found to be specific to the canonical microsporidia are not present in Amphiamblys sp. [9], indicating that the canonical microsporidia have a number of proteins that differentiate themselves from the metchnikovellids. Taken together, these genomic analyses suggest that the earliest steps in microsporidia evo- lution were the development of the defining polar filament and the loss of flagellum. This was followed by genomic reduction, including loss of the mitochondrial genome and subsequent expansion of microsporidia-specific and genus-specific gene families. Recent environmental sampling has revealed a great diversity of early diverging microsporidia species, and it is expected that additional whole-genome sequencing of these species will provide even greater clarity into the evolution of microsporidia [29]. Are there species that have evolved polar fila- ments while maintaining a flagella apparatus? Was the mitochondria genome lost in indepen- dent lineages? In species that have retained the mitochondrial genome, are there proteins besides Complex I that are commonly lost? Have other branches of early diverging microspori- dia undergone as dramatic of gene reduction as in canonical microsporidia? Are there features associated with the transition to infecting animals? Was the last common ancestor of Rozella and microsporidia a parasite, or did this ancestor have a free-living lifestyle? Additional com- parative studies of microsporidia and related species will help in answering these and other questions of how microsporidia evolved. What is the host specificity of microsporidia? Microsporidia infections are extremely common in animal species. Fig 1A shows a variety of invertebrate and vertebrate species that microsporidia infect. Microsporidia also infect protists such as free-living ciliates and other parasites, including paramyxids, which infect bivalve mol- lusks [29]. Although most microsporidia have a narrow host range and infect only one or sev- eral closely related host species, several microsporidia species are generalists and have broad host ranges [3,31–33]. Examples of generalists include Enterocytozoon bieneusi and the Ence- phalitozoon species, which can infect a wide variety of birds and mammals [34]. Many genera of microsporidia show specialization to a certain group of animals, such as the nine known Nematocida species that parasitize nematodes [19,31,35]. Species within a clade have also been observed to have different host specificity. For example, Encephalitozoon romaleae infects grasshoppers, unlike the other vertebrate-infecting members of the genus. This suggests that host specificity can rapidly switch between vertebrates and insects [36]. There are also several examples in which microsporidia have independently evolved to infect the same species, including humans, mosquitos, and nematodes [31]. Interestingly, host ranges can be extended when microsporidia encounter immunocompromised hosts [37]. This is thought to be the case for several species that infect immunocompromised humans, such as Trachipleistophora hominis, whose natural host is thought to be an insect, and Anncaliia algerae, whose natural host is a mosquito [38,39]. Tissue specificities can also differ within a host [2]. Of two different PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 4 / 8 species of Nematocida that infect Caenorhabditis elegans, N. parisii only infects the intestine, whereas N. displodere proliferates in several tissues including the muscle and epidermis. Differ- ences in subcellular localization have also been observed, such as with the related crab-infect- ing species Hepatospora eriocheir and Enterospora canceri, which infect the cytoplasm and nuclei, respectively [23]. Although approximately 1,400 microsporidia species have been described, there is likely a large amount of undiscovered species diversity [7,40]. For example, a study sampling terres- trial nematodes identified 12 distinct microsporidia species, and C. elegans alone has been reported to be infected by seven different species [19,31,35]. Additionally, environmental sam- pling has identified dozens of novel species [41,42]. Based on the wide diversity of known ani- mal hosts and the specificity of microsporidia species to infect one or several closely related hosts, it has been suggested that the number of microsporidia species equals the number of animal species. This has led to estimates of over 100 million microsporidia species [43]. How have microsporidia adapted to their hosts? Microsporidia use many proteins to directly interact with and manipulate their hosts. These host-exposed proteins contain targeting signals that direct them for secretion into the host or to the membrane of the parasite. Microsporidia species are predicted to have approximately 100 to 1,300 host-exposed proteins, which includes lineage-specific, expanded gene families [6,19]. These large gene families are particularly interesting as they can make up over 10% of a single microsporidia genome [35]. Although the function of these families is unknown, many family members contain protein–protein interaction domains, suggesting that they may inter- act with host proteins [19]. Because canonical microsporidia cannot undergo oxidative phosphorylation, they rely on their hosts for ATP and other metabolites. The most extensively studied microsporidia protein that directly interacts with hosts is hexokinase. Microsporidia hexokinases have gained a signal peptide and were experimentally shown to be secreted in several species [19,44–46]. This enzyme potentially increases host metabolism to provide nutrients for the developing para- sites, and consistent with this idea, knockdown of the enzyme reduces microsporidia prolifera- tion [44]. To obtain ATP from their hosts, microsporidia encode a nucleotide transport protein that was likely acquired via horizontal gene transfer from bacteria. These proteins allow for the transport of ATP into the parasite and have been diversified to acquire other sub- strates [16]. A second family of nucleotide transporters, the major facilitator superfamily, was recently shown to transport ATP and was found in many eukaryotes and in Rozella and all of the sequenced microsporidia [47]. Microsporidia can interact with host mitochondria, which is thought to increase the transport of ATP into the parasite. Recent work identified a protein from Encephalitozoon hellem, sporoplasm surface protein 1 (SSP1), that is on the sporoplasm that is involved in both invasion of host cells and association with the host mitochondria [48]. Future perspective Microsporidia were discovered over 160 years ago, but our understanding of these organisms has lagged compared to many other eukaryotic parasites of humans [24]. The use of genomic sequencing technologies has allowed for rapid advances in our understanding of how these cryptic parasites function and how they have evolved. Many other technologies are now being successfully applied to microsporidia. The application of whole-genome amplification to single infected host cells has allowed for the sequencing of species that are challenging to culture [8]. The use of ancestral gene reconstruction has revealed how nucleotide transporter genes evolved [16]. Proteomic technologies such as mass spectrometry have been used to study the PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 5 / 8 localization of microsporidia proteins as well as their translational fidelity [12,14,19]. Cryo- electron microscopy has been used to determine the structures of microsporidia ribosomes. This work allowed the identification of a conserved, microsporidia-specific ribosomal protein (msL1) and also identified two conserved microsporidia dormancy factors, one that is con- served throughout eukaryotes (MDF1) and the other that was only found in several species of microsporidia (MDF2) [14]. Although it is currently not possible to genetically modify micro- sporidia, using RNAi to knockdown genes provides a powerful approach to directly study microsporidia protein function in the context of infection [44,49]. Finally, the discovery of microsporidia that infect model organisms such as C. elegans and Drosophila melanogaster provides easily cultured, genetically tractable hosts for studying how microsporidia function [2,19,35,50]. Continued use of the technological advances highlighted here is likely to provide additional insight into the function and evolution of these fascinating pathogens. Acknowledgments We thank Alexandra R. Willis for the illustrations in Fig 1 and Calvin A. Mok, Hala Tamim El Jarkass, and Alexandra R. Willis for helpful comments on the manuscript. References 1. Han B, Weiss LM. Microsporidia: Obligate Intracellular Pathogens Within the Fungal Kingdom. Microbiol Spectr. 2017; 5. https://doi.org/10.1128/microbiolspec.FUNK-0018-2016 PMID: 28944750 2. Cuomo CA, Desjardins CA, Bakowski MA, Goldberg J, Ma AT, Becnel JJ, et al. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome Res. 2012; 22: 2478– 2488. https://doi.org/10.1101/gr.142802.112 PMID: 22813931 3. Desjardins CA, Sanscrainte ND, Goldberg JM, Heiman D, Young S, Zeng Q, et al. Contrasting host- pathogen interactions and genome evolution in two generalist and specialist microsporidian pathogens of mosquitoes. Nat Commun. 2015; 6: 7121. https://doi.org/10.1038/ncomms8121 PMID: 25968466 4. Ndikumana S, Pelin A, Williot A, Sanders JL, Kent M, Corradi N. Genome Analysis of Pseudoloma neu- rophilia: A Microsporidian Parasite of Zebrafish (Danio rerio). J Eukaryot Microbiol. 2017; 64: 18–30. https://doi.org/10.1111/jeu.12331 PMID: 27230544 5. Selman M, Sak B, Kvac M, Farinelli L, Weiss LM, Corradi N. Extremely reduced levels of heterozygosity in the vertebrate pathogen Encephalitozoon cuniculi. Eukaryot Cell. 2013; 12: 496–502. https://doi.org/ 10.1128/EC.00307-12 PMID: 23376943 6. Nakjang S, Williams TA, Heinz E, Watson AK, Foster PG, Sendra KM, et al. Reduction and expansion in microsporidian genome evolution: new insights from comparative genomics. Genome Biol Evol. 2013; 5: 2285–2303. https://doi.org/10.1093/gbe/evt184 PMID: 24259309 7. Keeling PJ, Fast NM. Microsporidia: biology and evolution of highly reduced intracellular parasites. Annu Rev Microbiol. 2002; 56: 93–116. https://doi.org/10.1146/annurev.micro.56.012302.160854 PMID: 12142484 8. Galindo LJ, Torruella G, Moreira D, Timpano H, Paskerova G, Smirnov A, et al. Evolutionary Genomics of Metchnikovella incurvata (Metchnikovellidae): An Early Branching Microsporidium. Genome Biol Evol. 2018; 10: 2736–2748. https://doi.org/10.1093/gbe/evy205 PMID: 30239727 9. Mikhailov KV, Simdyanov TG, Aleoshin VV. Genomic Survey of a Hyperparasitic Microsporidian Amphiamblys sp. (Metchnikovellidae). Genome Biol Evol. 2017; 9: 454–467. https://doi.org/10.1093/ gbe/evw235 PMID: 27694476 10. Shertz CA, Bastidas RJ, Li W, Heitman J, Cardenas ME. Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom. BMC Genomics. 2010; 11: 510. https://doi.org/10.1186/1471- 2164-11-510 PMID: 20863387 11. Katinka MD, Duprat S, Cornillot E, Metenier G, Thomarat F, Prensier G, et al. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature. 2001; 414: 450–453. https://doi.org/10.1038/35106579 PMID: 11719806 12. Melnikov SV, Rivera KD, Ostapenko D, Makarenko A, Sanscrainte ND, Becnel JJ, et al. Error-prone protein synthesis in parasites with the smallest eukaryotic genome. Proc Natl Acad Sci U S A. 2018; 115: E6245–E6253. https://doi.org/10.1073/pnas.1803208115 PMID: 29915081 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 6 / 8 13. Melnikov SV, Manakongtreecheep K, Rivera KD, Makarenko A, Pappin DJ, Soll D. Muller’s Ratchet and Ribosome Degeneration in the Obligate Intracellular Parasites Microsporidia. Int J Mol Sci. 2018;19. https://doi.org/10.3390/ijms19124125 PMID: 30572624 14. Barandun J, Hunziker M, Vossbrinck CR, Klinge S. Evolutionary compaction and adaptation visualized by the structure of the dormant microsporidian ribosome. Nat Microbiol. 2019. https://doi.org/10.1038/ s41564-019-0514-6 PMID: 31332387 15. Williams BAP, Hirt RP, Lucocq JM, Embley TM. A mitochondrial remnant in the microsporidian Trachi- pleistophora hominis. Nature. 2002; 418: 865–869. https://doi.org/10.1038/nature00949 PMID: 16. Dean P, Sendra KM, Williams TA, Watson AK, Major P, Nakjang S, et al. Transporter gene acquisition and innovation in the evolution of Microsporidia intracellular parasites. Nat Commun. 2018; 9: 1709. https://doi.org/10.1038/s41467-018-03923-4 PMID: 29703975 17. Corradi N, Pombert J-F, Farinelli L, Didier ES, Keeling PJ. The complete sequence of the smallest known nuclear genome from the microsporidian Encephalitozoon intestinalis. Nat Commun. 2010; 1: 77. https://doi.org/10.1038/ncomms1082 PMID: 20865802 18. Pombert J-F, Xu J, Smith DR, Heiman D, Young S, Cuomo CA, et al. Complete genome sequences from three genetically distinct strains reveal high intraspecies genetic diversity in the microsporidian Encephalitozoon cuniculi. Eukaryot Cell. 2013; 12: 503–511. https://doi.org/10.1128/EC.00312-12 PMID: 23291622 19. Reinke AW, Balla KM, Bennett EJ, Troemel ER. Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins. Nat Commun. 2017; 8: 14023. https://doi.org/10.1038/ ncomms14023 PMID: 28067236 20. Williams TA, Nakjang S, Campbell SE, Freeman MA, Eydal M, Moore K, et al. A Recent Whole- Genome Duplication Divides Populations of a Globally Distributed Microsporidian. Mol Biol Evol. 2016; 33: 2002–2015. https://doi.org/10.1093/molbev/msw083 PMID: 27189558 21. Whelan TA, Lee NT, Lee RCH, Fast NM. Microsporidian Introns Retained against a Background of Genome Reduction: Characterization of an Unusual Set of Introns. Genome Biol Evol. 2019; 11: 263– 269. https://doi.org/10.1093/gbe/evy260 PMID: 30496512 22. Huang Q. Evolution of Dicer and Argonaute orthologs in microsporidian parasites. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2018; 65: 329–332. https://doi.org/10.1016/j.meegid.2018.08.011 PMID: 30142384 23. Wiredu Boakye D, Jaroenlak P, Prachumwat A, Williams TA, Bateman KS, Itsathitphaisarn O, et al. Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia. Environ Microbiol. 2017; 19: 2077–2089. https://doi.org/10.1111/1462-2920.13734 PMID: 28345194 24. Keeling P. Five questions about microsporidia. PLoS Pathog. 2009; 5: e1000489. https://doi.org/10. 1371/journal.ppat.1000489 PMID: 19779558 25. Jones MDM, Forn I, Gadelha C, Egan MJ, Bass D, Massana R, et al. Discovery of novel intermediate forms redefines the fungal tree of life. Nature. 2011; 474: 200–203. https://doi.org/10.1038/nature09984 PMID: 21562490 26. James TY, Pelin A, Bonen L, Ahrendt S, Sain D, Corradi N, et al. Shared signatures of parasitism and phylogenomics unite Cryptomycota and microsporidia. Curr Biol CB. 2013; 23: 1548–1553. https://doi. org/10.1016/j.cub.2013.06.057 PMID: 23932404 27. Quandt CA, Beaudet D, Corsaro D, Walochnik J, Michel R, Corradi N, et al. The genome of an intranuc- lear parasite, Paramicrosporidium saccamoebae, reveals alternative adaptations to obligate intracellu- lar parasitism. eLife. 2017; 6. https://doi.org/10.7554/eLife.29594 PMID: 29171834 28. Haag KL, James TY, Pombert J-F, Larsson R, Schaer TMM, Refardt D, et al. Evolution of a morphologi- cal novelty occurred before genome compaction in a lineage of extreme parasites. Proc Natl Acad Sci U S A. 2014; 111: 15480–15485. https://doi.org/10.1073/pnas.1410442111 PMID: 25313038 29. Bass D, Czech L, Williams BAP, Berney C, Dunthorn M, Mahe F, et al. Clarifying the Relationships between Microsporidia and Cryptomycota. J Eukaryot Microbiol. 2018; 65: 773–782. https://doi.org/10. 1111/jeu.12519 PMID: 29603494 30. Corsaro D, Walochnik J, Venditti D, Steinmann J, Muller K-D, Michel R. Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota. Parasitol Res. 2014; 113: 1909–1918. https://doi.org/10.1007/s00436-014-3838-4 PMID: 24652444 31. Zhang G, Sachse M, Prevost M-C, Luallen RJ, Troemel ER, Felix M-A. A Large Collection of Novel Nematode-Infecting Microsporidia and Their Diverse Interactions with Caenorhabditis elegans and Other Related Nematodes. PLoS Pathog. 2016; 12: e1006093. https://doi.org/10.1371/journal.ppat. 1006093 PMID: 27942022 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 7 / 8 32. Shaw RW, Kent ML, Brown AM, Whipps CM, Adamson ML. Experimental and natural host specificity of Loma salmonae (Microsporidia). Dis Aquat Organ. 2000; 40: 131–136. https://doi.org/10.3354/ dao040131 PMID: 10782347 33. Solter LF, Pilarska DK, McManus ML, Zubrik M, Patocka J, Huang W-F, et al. Host specificity of micro- sporidia pathogenic to the gypsy moth, Lymantria dispar (L.): field studies in Slovakia. J Invertebr Pathol. 2010; 105: 1–10. https://doi.org/10.1016/j.jip.2010.04.009 PMID: 20435042 34. Hinney B, Sak B, Joachim A, Kvac M. More than a rabbit’s tale—Encephalitozoon spp. in wild mammals and birds. Int J Parasitol Parasites Wildl. 2016; 5: 76–87. https://doi.org/10.1016/j.ijppaw.2016.01.001 PMID: 28560162 35. Luallen RJ, Reinke AW, Tong L, Botts MR, Felix M-A, Troemel ER. Discovery of a Natural Microspori- dian Pathogen with a Broad Tissue Tropism in Caenorhabditis elegans. PLoS Pathog. 2016; 12: e1005724. https://doi.org/10.1371/journal.ppat.1005724 PMID: 27362540 36. Johny S, Larson TM, Solter LF, Edwards KA, Whitman DW. Phylogenetic characterization of Encephali- tozoon romaleae (Microsporidia) from a grasshopper host: relationship to Encephalitozoon spp. infect- ing humans. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2009; 9: 189–195. https://doi.org/ 10.1016/j.meegid.2008.10.010 PMID: 19027883 37. Stentiford GD, Bass D, Williams BAP. Ultimate opportunists-The emergent Enterocytozoon group Microsporidia. PLoS Pathog. 2019; 15: e1007668. https://doi.org/10.1371/journal.ppat.1007668 PMID: 38. Watson AK, Williams TA, Williams BAP, Moore KA, Hirt RP, Embley TM. Transcriptomic profiling of host-parasite interactions in the microsporidian Trachipleistophora hominis. BMC Genomics. 2015; 16: 983. https://doi.org/10.1186/s12864-015-1989-z PMID: 26589282 39. Watts MR, Chan RCF, Cheong EYL, Brammah S, Clezy KR, Tong C, et al. Anncaliia algerae microspor- idial myositis. Emerg Infect Dis. 2014; 20: 185–191. https://doi.org/10.3201/eid2002.131126 PMID: 40. Vavra J, Lukes J. Microsporidia and “the art of living together”. Adv Parasitol. 2013; 82: 253–319. https://doi.org/10.1016/B978-0-12-407706-5.00004-6 PMID: 23548087 41. Ardila-Garcia AM, Raghuram N, Sihota P, Fast NM. Microsporidian diversity in soil, sand, and compost of the Pacific Northwest. J Eukaryot Microbiol. 2013; 60: 601–608. https://doi.org/10.1111/jeu.12066 PMID: 23869987 42. Williams BAP, Hamilton KM, Jones MD, Bass D. Group-specific environmental sequencing reveals high levels of ecological heterogeneity across the microsporidian radiation. Environ Microbiol Rep. 2018; 10: 328–336. https://doi.org/10.1111/1758-2229.12642 PMID: 29578312 43. Larsen BB, Miller EC, Rhodes MK, Wiens JJ. Inordinate Fondness Multiplied and Redistributed: the Number of Species on Earth and the New Pie of Life. Q Rev Biol. 2017; 92: 229–265. https://doi.org/10. 1086/693564 44. Huang Y, Zheng S, Mei X, Yu B, Sun B, Li B, et al. A secretory hexokinase plays an active role in the proliferation of Nosema bombycis. PeerJ. 2018; 6: e5658. https://doi.org/10.7717/peerj.5658 PMID: 45. Ferguson S, Lucocq J. The invasive cell coat at the microsporidian Trachipleistophora hominis-host cell interface contains secreted hexokinases. MicrobiologyOpen. 2019; 8: e00696. https://doi.org/10.1002/ mbo3.696 PMID: 30051624 46. Senderskiy IV, Timofeev SA, Seliverstova EV, Pavlova OA, Dolgikh VV. Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes. PLoS ONE. 2014; 9: e93585. https://doi.org/10.1371/ journal.pone.0093585 PMID: 24705470 47. Major P, Sendra KM, Dean P, Williams TA, Watson AK, Thwaites DT, et al. A new family of cell surface located purine transporters in Microsporidia and related fungal endoparasites. eLife. 2019; 8. https:// doi.org/10.7554/eLife.47037 PMID: 31355745 48. Han B, Ma Y, Tu V, Tomita T, Mayoral J, Williams T, et al. Microsporidia Interact with Host Cell Mito- chondria via Voltage-Dependent Anion Channels Using Sporoplasm Surface Protein 1. mBio. 2019;10. https://doi.org/10.1128/mBio.01944-19 PMID: 31431557 49. Reinke AW, Troemel ER. The Development of Genetic Modification Techniques in Intracellular Para- sites and Potential Applications to Microsporidia. PLoS Pathog. 2015; 11: e1005283. https://doi.org/10. 1371/journal.ppat.1005283 PMID: 26720003 50. Franchet A, Niehus S, Caravello G, Ferrandon D. Phosphatidic acid as a limiting host metabolite for the proliferation of the microsporidium Tubulinosema ratisbonensis in Drosophila flies. Nat Microbiol. 2019; 4: 645–655. https://doi.org/10.1038/s41564-018-0344-y PMID: 30692666 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 8 / 8 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS Pathogens Public Library of Science (PLoS) Journal

Evolution of microsporidia: An extremely successful group of eukaryotic intracellular parasites

PLoS Pathogens, Volume 16 (2) – Feb 13, 2020

Loading next page...
 
/lp/public-library-of-science-plos-journal/evolution-of-microsporidia-an-extremely-successful-group-of-eukaryotic-Nm5sxJ0L1T
Publisher
Public Library of Science (PLoS) Journal
Copyright
Copyright: © 2020 Wadi, Reinke. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was funded by the Canadian Institutes of Health Research grant PJT-159479 and the Alfred P. Sloan Research Fellowship FG-2019-12040. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
ISSN
1553-7366
eISSN
1553-7374
DOI
10.1371/journal.ppat.1008276
Publisher site
See Article on Publisher Site

Abstract

PEARLS Evolution of microsporidia: An extremely successful group of eukaryotic intracellular parasites Lina Wadi , Aaron W. Reinke * ID ID Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada * aaron.reinke@utoronto.ca Overview Microsporidia are obligate intracellular parasites that can infect a wide range of hosts. They cause death and disease in both humans and agriculturally important animals. They have also become a powerful model for understanding the evolution of intracellular parasites. Recent work has explored how microsporidia genomes have evolved, the evolutionary origins of a1111111111 microsporidia, and how microsporidia adapt to interact with their hosts. a1111111111 a1111111111 What do microsporidia have in common? a1111111111 Microsporidia can only exist outside of their host as environmentally resistant, chitin-contain- a1111111111 ing spores. These spores contain a unique infectious apparatus known as the polar tube. Dur- ing infection, the polar tube is rapidly discharged and can pierce a host cell, depositing the parasite’s sporoplasm within the host cell [1]. The sporoplasm then proliferates, eventually producing spores that then exit the host to cause subsequent infections. Microsporidia are OPENACCESS thought to reproduce mostly asexually, although most are likely diploid based on genomic het- Citation: Wadi L, Reinke AW (2020) Evolution of erozygosity and conservation of meiotic genes [2–5]. microsporidia: An extremely successful group of Because of the intimate relationship between microsporidia and their hosts, they are heavily eukaryotic intracellular parasites. PLoS Pathog 16 dependent on host resources and have undergone extensive genomic reduction [6,7]. Analysis (2): e1008276. https://doi.org/10.1371/journal. of sequenced microsporidia genomes (Fig 1A) has revealed a wide loss of protein families that ppat.1008276 are present in other eukaryotes, leaving only approximately 800 conserved microsporidia pro- Editor: Laura J. Knoll, University of Wisconsin teins [6]. The proteins that have been retained function in essential core cellular processes Medical School, UNITED STATES such as DNA replication, transcription, and translation [6,8,9]. Proteins that have been lost Published: February 13, 2020 include many metabolic enzymes, regulatory pathways, and proteins involved in vesicular transport such as TOR (Target of Rapamycin) and clathrin [8–10]. In addition, the proteins Copyright:© 2020 Wadi, Reinke. This is an open access article distributed under the terms of the that are retained are shorter than their fungal orthologs, with Encephalitozoon cuniculi proteins Creative Commons Attribution License, which being on average only 85% as long as their yeast counterparts [3,11]. One such instance of pro- permits unrestricted use, distribution, and teins that have decreased in size are the aminoacyl-tRNA synthetases, which have lost many reproduction in any medium, provided the original regulatory regions, including a domain in leucyl-tRNA synthetase (LeuRS) that edits mis- author and source are credited. charged tRNAs. The microsporidia Vavraia culicis was shown to have mistranslation error Funding: This project was funded by the Canadian rates of 5.9% at positions that coded for leucine—although valine, which has a corresponding Institutes of Health Research grant PJT-159479 tRNA synthetases with an intact editing domain, has a mistranslation rate of 7.5%, suggesting and the Alfred P. Sloan Research Fellowship FG- that there are other factors responsible for the high levels of mistranslated proteins [12]. 2019-12040. The funders had no role in study design, data collection and analysis, decision to Microsporidia also have reduced noncoding RNAs, including the 18s RNA, which is only publish, or preparation of the manuscript. approximately 2/3 as large as in other eukaryotes and results in the smallest eukaryotic ribo- somes [13]. Although this reduction has led to several binding sites being eliminated, struc- Competing interests: The authors have declared that no competing interests exist. tural and mass spectrometry analysis of ribosomes from Vairimorpha necatrix demonstrated PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 1 / 8 Fig 1. Phylogeny, hosts, and characteristics of microsporidia species. (A) Phylogenetic tree of whole-genome sequenced microsporidia and related species. Tree was generated as described previously using single-copy orthologs present in all species [19,35]. Predicted proteins for each genome were obtained from https://www.ncbi. nlm.nih.gov, except for Metchnikovella incurvata and Enterospora canceri, which were predicted from genome assemblies using Prodigal 2.6.3. Orthologous gene −5 families were identified using OrthoMCL 2.0.9 using an inflation index of 1.5 and a BLAST E-value cutoff of 10 . Phylogeny was constructed from 43 single-copy orthologs present in all 28 species. Proteins from each orthogroup were aligned using MUSCLE 3.8.31. These alignments were trimmed using trimAl 1.4 with the option-gappyout. Each orthogroup alignment was then concatenated into a single alignment using FASconCAT 1.11. Using ProtTest 3.4.2, it was determined that PROTGAMMAIWAGF was the best fitting model for the data. Phylogeny was then inferred using RAxML 8.2.12 with the best fitting model and 1,000 bootstrap replicates. Scale bar indicates changes per site. To the right of each species are illustrations of the host(s) that each species has been reported to infect. This phylogenetic tree is largely in agreement with recent previously published phylogenies [3,4,8,9,19,23,27], with the exception of Anncaliia algerae or Edhazardia aedis, which have different placements in several reports [3,8,23,27]. (B) Comparision of protein content for each species. (C) Comparision of genome size for each species. (D) Morphological and molecular characteristics of each species. E-value, expect value; TOR, target of rapamycin. https://doi.org/10.1371/journal.ppat.1008276.g001 that most conserved ribosomal proteins can still bind [14]. Microsporidia also have reduced organelles, including mitochondrial remnants known as mitosomes [15]. These mitosomes do not encode a mitochondrial genome and are incapable of carrying out oxidative phosphoryla- tion; microsporidia must instead import ATP from their host [16]. Interestingly, there are 32 conserved protein families in microsporidia that are not found in any other eukaryote [6]. Sev- eral of these proteins function as part of the polar tube or spore wall, and the function of most of these proteins is unknown [6]. How do microsporidia genomes differ from one another? Microsporidia genome sizes vary significantly. The largest is Edhazardia aedis at 51.3 Mb (encoding approximately 4,200 proteins), and Encephalitozoon genomes are the smallest, with PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 2 / 8 some being only 2.3 MB (encoding approximately 1,800 proteins) [17] (Fig 1B and 1C). Much of this size disparity results from noncoding DNA, with only 9% of the E. aedis genome coding for proteins, compared to approximately 90% for E. cuniculi. The regions between genes can become very compact, with the intergenic regions averaging only 115 base pairs in Encephali- tozoon intestinalis [17,18]. Though microsporidia have much fewer proteins overall compared to other eukaryotes, genomic reduction was followed by a large expansion in lineage-specific protein families [6]. For example, Nematocida parisii comprises 2,661 proteins, of which only 1,074 are conserved with other eukaryotes or with other microsporidia species outside of its genus. This means that over half of the proteins in N. parisii are unique to the Nematocida genus, with 318 not even present in N. ironsii, a closely related sister species [19]. Many of these unique genes are found in subtelomeric regions and likely arose through local duplica- tion events [19]. Additional mechanisms that have changed the protein content of microspori- dia species include whole-genome duplications and genes arising from noncoding regions [20]. Several key eukaryotic processes have been lost in some, but not all, microsporidia. The ability to splice mRNA transcripts has been lost multiple times in microsporidia evolution, including in E. aedis, Nematocida species, and Enterocytozoon species, which no longer contain functional spliceosome machinery [21] (Fig 1D). This has resulted in some genomes having no detectable introns and the loss of most conserved splicing proteins [3]. Although many micro- sporidia genomes have retained Dicer and Argonaute, the two key effectors of the RNA inter- ference (RNAi) pathway, both of these proteins have been lost several times, including in all of the species in Nematocida and Encephalitozoon [4,22]. Although all microsporidia species have lost many metabolic enzymes, there are important distinctions such as differential losses in lipid biosynthesis genes [3]. Additionally, Enterocytozoon species have lost genes necessary for glycolysis, leaving them completely dependent on their host for energy production [23]. What did the last common ancestor of microsporidia look like? Microsporidia were once considered the earliest diverging eukaryotes, but sequencing of a large number of microsporidia genomes in the last several decades has made it clear that they belong to a group of the earliest diverging fungi [2,24]. More recently, the discovery and subse- quent genome sequencing of related basal species has shed light on the evolutionary origin of microsporidia. Microsporidia are closely related to a group of obligate intracellular parasites called the Cryptomycota or Rozellomycota [25] (Fig 1A). The only one of these species to have its genome sequenced so far, Rozella allomycis, infects water mold using motile flagellated spores [26]. Different than microsporidia, R. allomycis possesses mitochondria and encodes a much larger set of proteins that is more conserved with other eukaryotes. To date, the two most early diverging microsporidian species that have been sequenced are Paramicrospori- dium saccamoebae (classified as a Rozellomycota), an intranuclear parasite of amoeba, and Mitosporidium daphniae (classified as a microsporidia), which infects daphnia [27,28]. Although there has been debate about whether these species should be classified as microspori- dia, recent analysis of environmental samples supports the existence of an expanded micro- sporidia that includes a diversity of early diverging species [29]. P. Saccamoebae and M. daphniae differ from R. allomycis in that their spores have lost flagella and contain a polar fila- ment, although invasion by P. saccamoebae is carried out through host phagocytosis [30]. While P. saccamoebae and M. daphnia have a reduced gene set compared to other fungi, they have undergone less genome reduction than canonical microsporidia. Both species also have mitochondrial genomes, although P. saccamoebae has a conserved electron-transport chain whereas both R. allomycis and M. daphnia have lost Complex I and have different nuclear- PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 3 / 8 encoded genes that are thought to facilitate ATP generation [26–28]. Additionally, P. sacca- moebae has more genes in common with distantly related fungi than with R. allomycis or M. daphnia, suggesting that these species have undergone independent gene loss during coevolu- tion with their hosts [27]. Another early diverging group of microsporidia are the metchnikovellids, which parasitize apicomplexan gregarines. Recent genome sequencing of two of these species, Metchnikovella incurvata and Amphiamblys sp., has revealed that this group is most similar to the canonical microsporidia, as these species have undergone dramatic gene loss and do not contain mito- chondrial genomes [8,9] (Fig 1). One of the most striking differences in metchnikovellids is the presence of clathrin, which has been lost in the canonical microsporidia [8,9]. Additionally, the 32 conserved protein families that were found to be specific to the canonical microsporidia are not present in Amphiamblys sp. [9], indicating that the canonical microsporidia have a number of proteins that differentiate themselves from the metchnikovellids. Taken together, these genomic analyses suggest that the earliest steps in microsporidia evo- lution were the development of the defining polar filament and the loss of flagellum. This was followed by genomic reduction, including loss of the mitochondrial genome and subsequent expansion of microsporidia-specific and genus-specific gene families. Recent environmental sampling has revealed a great diversity of early diverging microsporidia species, and it is expected that additional whole-genome sequencing of these species will provide even greater clarity into the evolution of microsporidia [29]. Are there species that have evolved polar fila- ments while maintaining a flagella apparatus? Was the mitochondria genome lost in indepen- dent lineages? In species that have retained the mitochondrial genome, are there proteins besides Complex I that are commonly lost? Have other branches of early diverging microspori- dia undergone as dramatic of gene reduction as in canonical microsporidia? Are there features associated with the transition to infecting animals? Was the last common ancestor of Rozella and microsporidia a parasite, or did this ancestor have a free-living lifestyle? Additional com- parative studies of microsporidia and related species will help in answering these and other questions of how microsporidia evolved. What is the host specificity of microsporidia? Microsporidia infections are extremely common in animal species. Fig 1A shows a variety of invertebrate and vertebrate species that microsporidia infect. Microsporidia also infect protists such as free-living ciliates and other parasites, including paramyxids, which infect bivalve mol- lusks [29]. Although most microsporidia have a narrow host range and infect only one or sev- eral closely related host species, several microsporidia species are generalists and have broad host ranges [3,31–33]. Examples of generalists include Enterocytozoon bieneusi and the Ence- phalitozoon species, which can infect a wide variety of birds and mammals [34]. Many genera of microsporidia show specialization to a certain group of animals, such as the nine known Nematocida species that parasitize nematodes [19,31,35]. Species within a clade have also been observed to have different host specificity. For example, Encephalitozoon romaleae infects grasshoppers, unlike the other vertebrate-infecting members of the genus. This suggests that host specificity can rapidly switch between vertebrates and insects [36]. There are also several examples in which microsporidia have independently evolved to infect the same species, including humans, mosquitos, and nematodes [31]. Interestingly, host ranges can be extended when microsporidia encounter immunocompromised hosts [37]. This is thought to be the case for several species that infect immunocompromised humans, such as Trachipleistophora hominis, whose natural host is thought to be an insect, and Anncaliia algerae, whose natural host is a mosquito [38,39]. Tissue specificities can also differ within a host [2]. Of two different PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 4 / 8 species of Nematocida that infect Caenorhabditis elegans, N. parisii only infects the intestine, whereas N. displodere proliferates in several tissues including the muscle and epidermis. Differ- ences in subcellular localization have also been observed, such as with the related crab-infect- ing species Hepatospora eriocheir and Enterospora canceri, which infect the cytoplasm and nuclei, respectively [23]. Although approximately 1,400 microsporidia species have been described, there is likely a large amount of undiscovered species diversity [7,40]. For example, a study sampling terres- trial nematodes identified 12 distinct microsporidia species, and C. elegans alone has been reported to be infected by seven different species [19,31,35]. Additionally, environmental sam- pling has identified dozens of novel species [41,42]. Based on the wide diversity of known ani- mal hosts and the specificity of microsporidia species to infect one or several closely related hosts, it has been suggested that the number of microsporidia species equals the number of animal species. This has led to estimates of over 100 million microsporidia species [43]. How have microsporidia adapted to their hosts? Microsporidia use many proteins to directly interact with and manipulate their hosts. These host-exposed proteins contain targeting signals that direct them for secretion into the host or to the membrane of the parasite. Microsporidia species are predicted to have approximately 100 to 1,300 host-exposed proteins, which includes lineage-specific, expanded gene families [6,19]. These large gene families are particularly interesting as they can make up over 10% of a single microsporidia genome [35]. Although the function of these families is unknown, many family members contain protein–protein interaction domains, suggesting that they may inter- act with host proteins [19]. Because canonical microsporidia cannot undergo oxidative phosphorylation, they rely on their hosts for ATP and other metabolites. The most extensively studied microsporidia protein that directly interacts with hosts is hexokinase. Microsporidia hexokinases have gained a signal peptide and were experimentally shown to be secreted in several species [19,44–46]. This enzyme potentially increases host metabolism to provide nutrients for the developing para- sites, and consistent with this idea, knockdown of the enzyme reduces microsporidia prolifera- tion [44]. To obtain ATP from their hosts, microsporidia encode a nucleotide transport protein that was likely acquired via horizontal gene transfer from bacteria. These proteins allow for the transport of ATP into the parasite and have been diversified to acquire other sub- strates [16]. A second family of nucleotide transporters, the major facilitator superfamily, was recently shown to transport ATP and was found in many eukaryotes and in Rozella and all of the sequenced microsporidia [47]. Microsporidia can interact with host mitochondria, which is thought to increase the transport of ATP into the parasite. Recent work identified a protein from Encephalitozoon hellem, sporoplasm surface protein 1 (SSP1), that is on the sporoplasm that is involved in both invasion of host cells and association with the host mitochondria [48]. Future perspective Microsporidia were discovered over 160 years ago, but our understanding of these organisms has lagged compared to many other eukaryotic parasites of humans [24]. The use of genomic sequencing technologies has allowed for rapid advances in our understanding of how these cryptic parasites function and how they have evolved. Many other technologies are now being successfully applied to microsporidia. The application of whole-genome amplification to single infected host cells has allowed for the sequencing of species that are challenging to culture [8]. The use of ancestral gene reconstruction has revealed how nucleotide transporter genes evolved [16]. Proteomic technologies such as mass spectrometry have been used to study the PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 5 / 8 localization of microsporidia proteins as well as their translational fidelity [12,14,19]. Cryo- electron microscopy has been used to determine the structures of microsporidia ribosomes. This work allowed the identification of a conserved, microsporidia-specific ribosomal protein (msL1) and also identified two conserved microsporidia dormancy factors, one that is con- served throughout eukaryotes (MDF1) and the other that was only found in several species of microsporidia (MDF2) [14]. Although it is currently not possible to genetically modify micro- sporidia, using RNAi to knockdown genes provides a powerful approach to directly study microsporidia protein function in the context of infection [44,49]. Finally, the discovery of microsporidia that infect model organisms such as C. elegans and Drosophila melanogaster provides easily cultured, genetically tractable hosts for studying how microsporidia function [2,19,35,50]. Continued use of the technological advances highlighted here is likely to provide additional insight into the function and evolution of these fascinating pathogens. Acknowledgments We thank Alexandra R. Willis for the illustrations in Fig 1 and Calvin A. Mok, Hala Tamim El Jarkass, and Alexandra R. Willis for helpful comments on the manuscript. References 1. Han B, Weiss LM. Microsporidia: Obligate Intracellular Pathogens Within the Fungal Kingdom. Microbiol Spectr. 2017; 5. https://doi.org/10.1128/microbiolspec.FUNK-0018-2016 PMID: 28944750 2. Cuomo CA, Desjardins CA, Bakowski MA, Goldberg J, Ma AT, Becnel JJ, et al. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome Res. 2012; 22: 2478– 2488. https://doi.org/10.1101/gr.142802.112 PMID: 22813931 3. Desjardins CA, Sanscrainte ND, Goldberg JM, Heiman D, Young S, Zeng Q, et al. Contrasting host- pathogen interactions and genome evolution in two generalist and specialist microsporidian pathogens of mosquitoes. Nat Commun. 2015; 6: 7121. https://doi.org/10.1038/ncomms8121 PMID: 25968466 4. Ndikumana S, Pelin A, Williot A, Sanders JL, Kent M, Corradi N. Genome Analysis of Pseudoloma neu- rophilia: A Microsporidian Parasite of Zebrafish (Danio rerio). J Eukaryot Microbiol. 2017; 64: 18–30. https://doi.org/10.1111/jeu.12331 PMID: 27230544 5. Selman M, Sak B, Kvac M, Farinelli L, Weiss LM, Corradi N. Extremely reduced levels of heterozygosity in the vertebrate pathogen Encephalitozoon cuniculi. Eukaryot Cell. 2013; 12: 496–502. https://doi.org/ 10.1128/EC.00307-12 PMID: 23376943 6. Nakjang S, Williams TA, Heinz E, Watson AK, Foster PG, Sendra KM, et al. Reduction and expansion in microsporidian genome evolution: new insights from comparative genomics. Genome Biol Evol. 2013; 5: 2285–2303. https://doi.org/10.1093/gbe/evt184 PMID: 24259309 7. Keeling PJ, Fast NM. Microsporidia: biology and evolution of highly reduced intracellular parasites. Annu Rev Microbiol. 2002; 56: 93–116. https://doi.org/10.1146/annurev.micro.56.012302.160854 PMID: 12142484 8. Galindo LJ, Torruella G, Moreira D, Timpano H, Paskerova G, Smirnov A, et al. Evolutionary Genomics of Metchnikovella incurvata (Metchnikovellidae): An Early Branching Microsporidium. Genome Biol Evol. 2018; 10: 2736–2748. https://doi.org/10.1093/gbe/evy205 PMID: 30239727 9. Mikhailov KV, Simdyanov TG, Aleoshin VV. Genomic Survey of a Hyperparasitic Microsporidian Amphiamblys sp. (Metchnikovellidae). Genome Biol Evol. 2017; 9: 454–467. https://doi.org/10.1093/ gbe/evw235 PMID: 27694476 10. Shertz CA, Bastidas RJ, Li W, Heitman J, Cardenas ME. Conservation, duplication, and loss of the Tor signaling pathway in the fungal kingdom. BMC Genomics. 2010; 11: 510. https://doi.org/10.1186/1471- 2164-11-510 PMID: 20863387 11. Katinka MD, Duprat S, Cornillot E, Metenier G, Thomarat F, Prensier G, et al. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature. 2001; 414: 450–453. https://doi.org/10.1038/35106579 PMID: 11719806 12. Melnikov SV, Rivera KD, Ostapenko D, Makarenko A, Sanscrainte ND, Becnel JJ, et al. Error-prone protein synthesis in parasites with the smallest eukaryotic genome. Proc Natl Acad Sci U S A. 2018; 115: E6245–E6253. https://doi.org/10.1073/pnas.1803208115 PMID: 29915081 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 6 / 8 13. Melnikov SV, Manakongtreecheep K, Rivera KD, Makarenko A, Pappin DJ, Soll D. Muller’s Ratchet and Ribosome Degeneration in the Obligate Intracellular Parasites Microsporidia. Int J Mol Sci. 2018;19. https://doi.org/10.3390/ijms19124125 PMID: 30572624 14. Barandun J, Hunziker M, Vossbrinck CR, Klinge S. Evolutionary compaction and adaptation visualized by the structure of the dormant microsporidian ribosome. Nat Microbiol. 2019. https://doi.org/10.1038/ s41564-019-0514-6 PMID: 31332387 15. Williams BAP, Hirt RP, Lucocq JM, Embley TM. A mitochondrial remnant in the microsporidian Trachi- pleistophora hominis. Nature. 2002; 418: 865–869. https://doi.org/10.1038/nature00949 PMID: 16. Dean P, Sendra KM, Williams TA, Watson AK, Major P, Nakjang S, et al. Transporter gene acquisition and innovation in the evolution of Microsporidia intracellular parasites. Nat Commun. 2018; 9: 1709. https://doi.org/10.1038/s41467-018-03923-4 PMID: 29703975 17. Corradi N, Pombert J-F, Farinelli L, Didier ES, Keeling PJ. The complete sequence of the smallest known nuclear genome from the microsporidian Encephalitozoon intestinalis. Nat Commun. 2010; 1: 77. https://doi.org/10.1038/ncomms1082 PMID: 20865802 18. Pombert J-F, Xu J, Smith DR, Heiman D, Young S, Cuomo CA, et al. Complete genome sequences from three genetically distinct strains reveal high intraspecies genetic diversity in the microsporidian Encephalitozoon cuniculi. Eukaryot Cell. 2013; 12: 503–511. https://doi.org/10.1128/EC.00312-12 PMID: 23291622 19. Reinke AW, Balla KM, Bennett EJ, Troemel ER. Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins. Nat Commun. 2017; 8: 14023. https://doi.org/10.1038/ ncomms14023 PMID: 28067236 20. Williams TA, Nakjang S, Campbell SE, Freeman MA, Eydal M, Moore K, et al. A Recent Whole- Genome Duplication Divides Populations of a Globally Distributed Microsporidian. Mol Biol Evol. 2016; 33: 2002–2015. https://doi.org/10.1093/molbev/msw083 PMID: 27189558 21. Whelan TA, Lee NT, Lee RCH, Fast NM. Microsporidian Introns Retained against a Background of Genome Reduction: Characterization of an Unusual Set of Introns. Genome Biol Evol. 2019; 11: 263– 269. https://doi.org/10.1093/gbe/evy260 PMID: 30496512 22. Huang Q. Evolution of Dicer and Argonaute orthologs in microsporidian parasites. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2018; 65: 329–332. https://doi.org/10.1016/j.meegid.2018.08.011 PMID: 30142384 23. Wiredu Boakye D, Jaroenlak P, Prachumwat A, Williams TA, Bateman KS, Itsathitphaisarn O, et al. Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia. Environ Microbiol. 2017; 19: 2077–2089. https://doi.org/10.1111/1462-2920.13734 PMID: 28345194 24. Keeling P. Five questions about microsporidia. PLoS Pathog. 2009; 5: e1000489. https://doi.org/10. 1371/journal.ppat.1000489 PMID: 19779558 25. Jones MDM, Forn I, Gadelha C, Egan MJ, Bass D, Massana R, et al. Discovery of novel intermediate forms redefines the fungal tree of life. Nature. 2011; 474: 200–203. https://doi.org/10.1038/nature09984 PMID: 21562490 26. James TY, Pelin A, Bonen L, Ahrendt S, Sain D, Corradi N, et al. Shared signatures of parasitism and phylogenomics unite Cryptomycota and microsporidia. Curr Biol CB. 2013; 23: 1548–1553. https://doi. org/10.1016/j.cub.2013.06.057 PMID: 23932404 27. Quandt CA, Beaudet D, Corsaro D, Walochnik J, Michel R, Corradi N, et al. The genome of an intranuc- lear parasite, Paramicrosporidium saccamoebae, reveals alternative adaptations to obligate intracellu- lar parasitism. eLife. 2017; 6. https://doi.org/10.7554/eLife.29594 PMID: 29171834 28. Haag KL, James TY, Pombert J-F, Larsson R, Schaer TMM, Refardt D, et al. Evolution of a morphologi- cal novelty occurred before genome compaction in a lineage of extreme parasites. Proc Natl Acad Sci U S A. 2014; 111: 15480–15485. https://doi.org/10.1073/pnas.1410442111 PMID: 25313038 29. Bass D, Czech L, Williams BAP, Berney C, Dunthorn M, Mahe F, et al. Clarifying the Relationships between Microsporidia and Cryptomycota. J Eukaryot Microbiol. 2018; 65: 773–782. https://doi.org/10. 1111/jeu.12519 PMID: 29603494 30. Corsaro D, Walochnik J, Venditti D, Steinmann J, Muller K-D, Michel R. Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota. Parasitol Res. 2014; 113: 1909–1918. https://doi.org/10.1007/s00436-014-3838-4 PMID: 24652444 31. Zhang G, Sachse M, Prevost M-C, Luallen RJ, Troemel ER, Felix M-A. A Large Collection of Novel Nematode-Infecting Microsporidia and Their Diverse Interactions with Caenorhabditis elegans and Other Related Nematodes. PLoS Pathog. 2016; 12: e1006093. https://doi.org/10.1371/journal.ppat. 1006093 PMID: 27942022 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 7 / 8 32. Shaw RW, Kent ML, Brown AM, Whipps CM, Adamson ML. Experimental and natural host specificity of Loma salmonae (Microsporidia). Dis Aquat Organ. 2000; 40: 131–136. https://doi.org/10.3354/ dao040131 PMID: 10782347 33. Solter LF, Pilarska DK, McManus ML, Zubrik M, Patocka J, Huang W-F, et al. Host specificity of micro- sporidia pathogenic to the gypsy moth, Lymantria dispar (L.): field studies in Slovakia. J Invertebr Pathol. 2010; 105: 1–10. https://doi.org/10.1016/j.jip.2010.04.009 PMID: 20435042 34. Hinney B, Sak B, Joachim A, Kvac M. More than a rabbit’s tale—Encephalitozoon spp. in wild mammals and birds. Int J Parasitol Parasites Wildl. 2016; 5: 76–87. https://doi.org/10.1016/j.ijppaw.2016.01.001 PMID: 28560162 35. Luallen RJ, Reinke AW, Tong L, Botts MR, Felix M-A, Troemel ER. Discovery of a Natural Microspori- dian Pathogen with a Broad Tissue Tropism in Caenorhabditis elegans. PLoS Pathog. 2016; 12: e1005724. https://doi.org/10.1371/journal.ppat.1005724 PMID: 27362540 36. Johny S, Larson TM, Solter LF, Edwards KA, Whitman DW. Phylogenetic characterization of Encephali- tozoon romaleae (Microsporidia) from a grasshopper host: relationship to Encephalitozoon spp. infect- ing humans. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2009; 9: 189–195. https://doi.org/ 10.1016/j.meegid.2008.10.010 PMID: 19027883 37. Stentiford GD, Bass D, Williams BAP. Ultimate opportunists-The emergent Enterocytozoon group Microsporidia. PLoS Pathog. 2019; 15: e1007668. https://doi.org/10.1371/journal.ppat.1007668 PMID: 38. Watson AK, Williams TA, Williams BAP, Moore KA, Hirt RP, Embley TM. Transcriptomic profiling of host-parasite interactions in the microsporidian Trachipleistophora hominis. BMC Genomics. 2015; 16: 983. https://doi.org/10.1186/s12864-015-1989-z PMID: 26589282 39. Watts MR, Chan RCF, Cheong EYL, Brammah S, Clezy KR, Tong C, et al. Anncaliia algerae microspor- idial myositis. Emerg Infect Dis. 2014; 20: 185–191. https://doi.org/10.3201/eid2002.131126 PMID: 40. Vavra J, Lukes J. Microsporidia and “the art of living together”. Adv Parasitol. 2013; 82: 253–319. https://doi.org/10.1016/B978-0-12-407706-5.00004-6 PMID: 23548087 41. Ardila-Garcia AM, Raghuram N, Sihota P, Fast NM. Microsporidian diversity in soil, sand, and compost of the Pacific Northwest. J Eukaryot Microbiol. 2013; 60: 601–608. https://doi.org/10.1111/jeu.12066 PMID: 23869987 42. Williams BAP, Hamilton KM, Jones MD, Bass D. Group-specific environmental sequencing reveals high levels of ecological heterogeneity across the microsporidian radiation. Environ Microbiol Rep. 2018; 10: 328–336. https://doi.org/10.1111/1758-2229.12642 PMID: 29578312 43. Larsen BB, Miller EC, Rhodes MK, Wiens JJ. Inordinate Fondness Multiplied and Redistributed: the Number of Species on Earth and the New Pie of Life. Q Rev Biol. 2017; 92: 229–265. https://doi.org/10. 1086/693564 44. Huang Y, Zheng S, Mei X, Yu B, Sun B, Li B, et al. A secretory hexokinase plays an active role in the proliferation of Nosema bombycis. PeerJ. 2018; 6: e5658. https://doi.org/10.7717/peerj.5658 PMID: 45. Ferguson S, Lucocq J. The invasive cell coat at the microsporidian Trachipleistophora hominis-host cell interface contains secreted hexokinases. MicrobiologyOpen. 2019; 8: e00696. https://doi.org/10.1002/ mbo3.696 PMID: 30051624 46. Senderskiy IV, Timofeev SA, Seliverstova EV, Pavlova OA, Dolgikh VV. Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes. PLoS ONE. 2014; 9: e93585. https://doi.org/10.1371/ journal.pone.0093585 PMID: 24705470 47. Major P, Sendra KM, Dean P, Williams TA, Watson AK, Thwaites DT, et al. A new family of cell surface located purine transporters in Microsporidia and related fungal endoparasites. eLife. 2019; 8. https:// doi.org/10.7554/eLife.47037 PMID: 31355745 48. Han B, Ma Y, Tu V, Tomita T, Mayoral J, Williams T, et al. Microsporidia Interact with Host Cell Mito- chondria via Voltage-Dependent Anion Channels Using Sporoplasm Surface Protein 1. mBio. 2019;10. https://doi.org/10.1128/mBio.01944-19 PMID: 31431557 49. Reinke AW, Troemel ER. The Development of Genetic Modification Techniques in Intracellular Para- sites and Potential Applications to Microsporidia. PLoS Pathog. 2015; 11: e1005283. https://doi.org/10. 1371/journal.ppat.1005283 PMID: 26720003 50. Franchet A, Niehus S, Caravello G, Ferrandon D. Phosphatidic acid as a limiting host metabolite for the proliferation of the microsporidium Tubulinosema ratisbonensis in Drosophila flies. Nat Microbiol. 2019; 4: 645–655. https://doi.org/10.1038/s41564-018-0344-y PMID: 30692666 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008276 February 13, 2020 8 / 8

Journal

PLoS PathogensPublic Library of Science (PLoS) Journal

Published: Feb 13, 2020

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create folders to
organize your research

Export folders, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off