Impact of Genome Reduction in Microsporidia

Abstract

Microsporidia represent an evolutionary outlier in the tree of life and occupy the extreme edge of the eukaryotic domain with some of their biological features. Many of these unicellular fungi-like organisms have reduced their genomic content to potentially the lowest limit. With some of the most compacted eukaryotic genomes, microsporidia are excellent model organisms to study reductive evolution and its functional consequences. While the growing number of sequenced microsporidian genomes have elucidated genome composition and organization, a recent increase in complementary post-genomic studies has started to shed light on the impacts of genome reduction in these unique pathogens. This chapter will discuss the biological framework enabling genome minimization and will use one of the most ancient and essential macromolecular complexes, the ribosome, to illustrate the effects of extreme genome reduction on a structural, molecular, and cellular level. We outline how reductive evolution in microsporidia has shaped DNA organization, the composition and function of the ribosome, and the complexity of the ribosome biogenesis process. Studying compacted mechanisms, processes, or macromolecular machines in microsporidia illuminates their unique lifestyle and provides valuable insights for comparative eukaryotic structural biology.

1.1 Causes and Effects of Reductive Evolution

There is a common bias to describe evolution as a march toward decreased entropy and increased complexity. After all, the regular ordering of atoms to create larger and more complex systems is an intrinsic feature of life on earth. While this suggests that a gradual increase in organism complexity is inevitable, complexity is balanced in many environmental niches by selective pressures that favor rapid and efficient reproduction, leading to the elimination of superfluous traits. This process of ablation is known as “reductive evolution” and can result in simpler organisms deriving from more complex ancestors.

Reductive evolution typically results from efficient nutrient usage. Macroscopic examples of this include the loss or atrophy of eyes in a wide variety of cave fish or the somewhat ironic loss of a digestive tract in tapeworms (Castro 1996; Morris et al. 2012). At the microscopic level, reductive evolution usually involves eliminating extraneous metabolic processes. Many freshwater chrysophytes, for example, have switched from autotrophy/mixotrophy to heterotrophy in order to combat limited carbon availability and have lost photosynthetic pathways and large swaths of their genomes along the way (Olefeld et al. 2018; Majda et al. 2021).

Gene loss is the most common example of reductive evolution and is often made feasible by co-occurring organisms. One interesting case of this is the loss of the catalase-peroxidase protein, KatG, in the marine cyanobacteria Prochlorococcus spp. (Scanlan et al. 2009). KatG serves an important role in protecting many cyanobacteria from hydrogen peroxide (Perelman et al. 2003), which builds up in oceans due to photooxidation of dissolved organic carbon (Cooper et al. 1988). In a few hours of direct sunlight, enough hydrogen peroxide can be produced to kill off Prochlorococcus cultures (Morris et al. 2011), indicating that they have a very high sensitivity for the molecule. It is surprising then that Prochlorococcus spp. would have lost katG. Instead, co-occurring cyanobacteria have retained katG and scavenge surrounding hydrogen peroxide from marine environments (Petasne and Zika 1997). The loss of katG is therefore only possible because organisms in the natural community provide a protective function.

Interactions between Prochlorococcus spp. and other marine cyanobacteria inspired the “Black Queen Hypothesis,” which posits that natural selection for genomic streamlining breeds dependencies on co-occurring organisms (Morris et al. 2012). This contrasts with the “Red Queen Hypothesis,” inspired by Lewis Carroll’s Through the Looking-Glass, which postulates that competition breeds coevolution (Van Valen 1973). As a corollary to the Black Queen Hypothesis, the more dependent an organism is on other organisms, the more thoroughly a genome will be streamlined. We might therefore hypothesize that genome reduction scales with metabolic dependence on other organisms, i.e., the average genome size of evolutionarily related phototrophs > heterotrophs, and facultative parasites > obligate parasites, which seems to be the case (de Castro et al. 2009; Merhej et al. 2009; Clark et al. 2010; Majda et al. 2021) (Fig. 1.1).

Fig. 1.1

Microsporidia have the smallest known eukaryotic genomes. Logarithmic plot of the number of annotated protein coding genes as a function of the respective organism’s genome size. All entries present in NCBI (https://www.ncbi.nlm.nih.gov/) were included, but the data were broadly filtered to remove untenable outliers, partial sequences, and nucleomorphs. Because of the broad filtering, some partially sequenced or annotated entries are still present. The plot was generated using source code from https://github.com/smsaladi/genome_size_vs_protein_count. Eukaryotes are colored in different shades of red, with microsporidia in black. Prokaryotes and viruses are represented in shades of green

Parasites are some of the greatest beneficiaries of reductive evolution, and nowhere is this more conspicuous than in microsporidia. As obligate intracellular parasites, microsporidia have dramatically reduced many elements of their genomes. In the sections below, we describe the various factors facilitating genome reduction and outline elements of the genome that are absent in microsporidia. We then delve more deeply into the effects of genome ablation at the protein and RNA level by comparing aspects of ribosome structure, function, and maturation in microsporidia to other eukaryotes.

1.2 The Price of a Large Genome

The cost of genome replication is threefold and requires payment in time, nutrients, and space. All three costs increase with genome size, although there is some variation between prokaryotes and eukaryotes. In this section, we discuss the impact of each of these factors on genome replication and describe how they contribute to reduction in microsporidian genome sizes.

1.2.1 Time

Time is required both to collect materials for DNA synthesis and to physically duplicate the genome. The amount of time required for genome replication depends largely on the catalytic rate of the DNA polymerase. In E. coli, DNA polymerase III copies around 1000 nucleotides/second (Kelman and O’Donnell 1995; Naufer et al. 2017). On the other hand, the equivalent yeast polymerase, Pol ϵ, has a maximal catalytic rate of only 350 nt/s (Ganai et al. 2015). Yeast replication is further decreased to 50 nt/s by proofreading, lagging strand synthesis, etc. Fortunately, eukaryotes are able to offset slower catalytic rates and considerably larger genomes by segregating genetic material into different chromosomes and amplifying from multiple origins of replication. Consequently, yeast and E. coli grown in ideal conditions have replication times commensurate to their genome sizes: 90–120 min for 12 Mbp in yeast (Salari and Salari 2017), versus 40 min for 4.6 Mbp in E. coli (Fossum et al. 2007). Interestingly, replication rates for many cancerous human cells are on the order of only 20 h (Pereira et al. 2017), despite having genomes 250 times larger than yeast. This shows that various factors contribute to dramatically decrease the necessary time for eukaryotic replication, but that total replication time typically increases with increasing genome sizes. Thus, it is beneficial for intracellular parasites like microsporidia to reduce their genome size in order to decrease doubling times. Unfortunately, very little is currently known about microsporidian polymerases, or even whether their chromosomes harbor multiple origins of replication. One study on the microsporidia Nematocida parisii determined that their population doubles in around 140 min (Balla et al. 2016). Although there are a variety of confounding factors, such as growth occurring in infected nematodes rather than in an optimized broth, the replication rate of the 4.3 Mbp N. parisii genome is considerably slower than in yeast (about 1/4 the rate). This suggests that the catalytic rate of the polymerase is slower and/or that N. parisii has fewer chromosomes and origins of replication per Mbp than yeast.

1.2.2 Nutrients

Nitrogen and phosphorous are key elements in DNA and are considered the limiting nutrients for growth in most ecosystems (Ågren et al. 2012; Elser 2012). The biosynthesis of DNA is thus an extremely resource-intensive investment. In fact, comprehensive estimates for the ATP requirements for DNA replication suggest that it costs as much as 500 high-energy bonds/bp in diploid eukaryotes (Lynch and Marinov 2015). While this estimate includes indirect costs such as the production of nucleosomes to stabilize the DNA, most expenses scale linearly with genome size. The larger the genome, the more NTPs are required, and the less high-energy bonds are available for alternative functions like protein production or cell defense. Many organisms therefore pass through a cell-cycle checkpoint, called START, which acts as a nutrient-sensing step to assess available resources prior to replication (Foster et al. 2010). Cells lacking requisite nutrients enter a quiescent state until conditions are more favorable for DNA biosynthesis.

Nutrient limitations are even more restrictive for obligate intracellular parasites. Indeed, microsporidia are almost completely reliant on their hosts and are metabolically inactive in nutrient-poor, extracellular environments (Weiss and Becnel 2014). The hijacking of host systems allows them to bypass much of the innate cost of DNA replication, and simply importing nucleotides instead of synthesizing their own reduces the ATP requirements per base pair by nearly 50% (Lynch and Marinov 2015). Intriguingly, microsporidia have opted to eliminate the majority of enzymes required for nucleotide biosynthesis (Dean et al. 2016) and have instead expanded families dedicated to nucleotide import (Cuomo et al. 2012). This indicates that microsporidia have increased import proteins but greatly decreased biosynthetic pathways, facilitating a net decrease in overall genome size (Dean et al. 2016). Similar trends are identifiable in microsporidia for many other central eukaryotic pathways, such as glycolysis or fatty acid metabolism (Wiredu Boakye et al. 2017).

1.2.3 Space

Although space is perhaps the least conspicuous cost for DNA, many studies have noted and discussed the intricate relationship between genome size and cell size in eukaryotes (Gregory 2001; Cavalier-Smith 2005). The crux of this argument lies in the relatively invariant karyoplasmic ratio, i.e., the ratio of the nuclear volume to cytoplasm is important for cell function, and is generally conserved (Huxley 1925; Trombetta 1942; Cavalier-Smith 2005). The nuclear size is in turn proportional to the total volume of the chromatin (Cavalier-Smith 2005). Although the underlying causes of this effect are still being determined (Cantwell and Nurse 2019; Blommaert 2020), a decrease in genome size will generally lead to a decreased nuclear size, catalyzing a decrease in cell size. The reverse also holds true, where a decrease in cell size will herald a decrease in genome size. This relationship was cleanly demonstrated in a eukaryotic phytoplankton by Malerba et al. (2020). In this study, 72 different Dunaliella tertiolecta lineages with cell volumes spanning two orders of magnitude were placed under selective pressures favoring smaller cells. After 100 generations, lineages that were initially much larger displayed an up to 11% decrease in genome size, while smaller lineages were unaffected. This suggests that (1) selective pressures favoring smaller cells indirectly select for smaller genomes and (2) lineages with larger genomes contain a set of superfluous genes that can be lost, while smaller lineages are already operating at closer to the minimal genome (Malerba et al. 2020).

For intracellular parasites like microsporidia, the space available within their hosts directly restricts the number of spores produced. Cells infected with microsporidia are often saturated with spores (Weiss and Becnel 2014; Grigsby et al. 2020), suggesting the host cell walls limit the number of spores created per infection. In fact, the spatial costs of DNA are twofold, as not only does DNA indirectly determine the size of the spores or meronts, but it also takes up valuable real estate within the cell. It is therefore extremely beneficial for microsporidia to minimize genome size, and it is unsurprising that they are some of the physically smallest eukaryotes. As a consequence of cell-wall limitations to genome size, microsporidian species that exit via exocytosis may have less stringent spatial costs than lytic species. Mature spores are constantly being shed in exocytosed species, increasing the effective available space compared to lytic species. Currently, only one pair of species can be used as an example: Nematocida displodere is primarily released via cell lysis, while N. parisii can be exocytosed in vesicles (Luallen et al. 2016). Although the genome of N. displodere is, in fact, smaller than the genome of N. parisii (Luallen et al. 2016), more data are required to determine whether the “spore release method” contributes to genome size variation between related microsporidians.

1.3 Paths of Reductive Evolution in Microsporidia

Microsporidia are characterized by many unique and interesting features, such as a lack of innate mobility (Weiss and Becnel 2014) and a fishing-line like infection apparatus (Han et al. 2017). Despite their innovations, microsporidia are perhaps most frequently referenced for their exquisitely small genomes (Keeling and Slamovits 2004; Corradi et al. 2010; Corradi and Slamovits 2011) and minimized macromolecular complexes (Melnikov et al. 2018a; Barandun et al. 2019; Ehrenbolger et al. 2020). Microsporidian genomes are indeed very small and have the honors of claiming both the smallest known eukaryotic genome (Corradi et al. 2010) and one of the highest known eukaryotic gene densities (Fig. 1.2) (Keeling and Slamovits 2004; Keeling 2007). The genome of Encephalitozoon intestinalis, for example, is only 2.3 Mbp (Corradi et al. 2010). That is only half the size of the E. coli genome (4.6 Mbp) and 1/65,000 the size of Paris japonica (150 Gbp), a flowering perennial with the largest confirmed eukaryotic genome (Pellicer et al. 2010).

Fig. 1.2

Microsporidia have one of the most gene-dense eukaryotic genomes. Gene density across different kingdoms was calculated by dividing the number of annotated protein coding genes by the genome size of the respective organism in kilobase pairs. All entries present in NCBI (https://www.ncbi.nlm.nih.gov/) were included, but the data were broadly filtered to remove untenable outliers, partial sequences, and nucleomorphs. Because of the broad filtering, some partially sequenced or annotated entries are still present. Eukaryotes are colored in different shades of red, with microsporidia in black. Prokaryotes and viruses are represented in shades of green

Early studies on microsporidia noted the absence or modification of several cellular structures characteristic of eukaryotes. For example, microsporidia lack peroxisomes, have unstacked Golgi bodies, and have highly reduced mitochondria called mitosomes (Corradi and Keeling 2009; Vávra and Ronny Larsson 2014). These observations led to speculation that microsporidia represent an ancient and unsophisticated eukaryotic lineage. They were therefore classified as Archezoa, with the prevailing hypothesis stating that they diverged prior to endosymbiosis of the mitochondrial ancestor (Cavalier-Smith 1983). This theory was disproven when further genetic analyses demonstrated that a subset of genes found in eukaryotic mitochondria have been transferred to microsporidian chromosomes (Germot et al. 1996; Katinka et al. 2001), indicating that microsporidia diverged after endosymbiosis and are therefore simplified organisms derived from more complex ancestors. Likewise, the small genomes of microsporidia are not a representation of a primitive ancestral state but are instead the result of minimization of multifarious genomic features. In this section, we describe several features affecting genome size, such as gene loss, intron minimization/removal, reductions in gene length, deletions of redundant genes, and the shortening of intergenic regions (IGRs) (Fig. 1.3a).

Fig. 1.3

Mechanisms of genome compaction in microsporidia. (a) Schematic representation of a relatively expanded (top) and compacted genome (bottom). Different genomic elements are colored, and the processes leading to their compaction (lower panel) are labeled on top. (b) The size of intergenic sequence (IGS) regions correlates with the directionality of adjacent genes, likely due to the presence of transcriptional control elements upstream of the transcriptional start site (e.g., enhancers, promotors). Gene directionality is indicated with arrows and 5′ or 3′ labels. Transcriptional control elements and their binding partners (e.g., transcription factors, RNA polymerases etc.) are shown as symbolic cartoons and colored in shades of green

1.3.1 Non-coding Regions

Microsporidian genomes are similar to those of other eukaryotes in structure and organization. Multiple linear chromosomes can be segregated into telomeres, subtelomeres containing ribosomal DNA (rDNA) and repetitive elements, and gene-rich cores (Dia et al. 2016). Variation is more localized to individual elements of the genome, like coding sequences and intergenic regions. The regions between genes are essential for efficient transcription and contain binding sites for various promoters and enhancers, which are often thousands of nucleotides away from the gene they enhance. It is intriguing then that many microsporidia have tiny IGRs, with E. intestinalis averaging only 115 bp between genes (Corradi et al. 2010). The genes themselves are an average of 1.04 kbp (Corradi et al. 2010). By taking into account the gene density (1.16 kbp/gene) (Corradi et al. 2010), we can determine that coding regions account for as much as 90% of the E. intestinalis genome. To put this in perspective, around 70% of the yeast genome codes for proteins (Dujon 1996) and only 2% of the human genome is protein coding (Piovesan et al. 2019). The low ratio of non-coding to coding sequences suggests that microsporidia have extremely streamlined IGRs. In fact, contrary to most eukaryotes, non-coding regions in microsporidia have higher sequence conservation than coding regions (Corradi et al. 2010; Corradi and Slamovits 2011; Whelan et al. 2019), indicating that the remaining bases form important molecular recognition motifs.

Most regulatory elements are found upstream of the 5′ end of a gene. Tellingly, the length of microsporidian IGRs appears to correlate with the directionality of adjacent genes (Fig. 1.3b) (Keeling and Slamovits 2004). For Encephalitozoon cuniculi, regions wedged between the termini of two genes (the 3′ ends) are about 20% shorter than regions between parallel genes (one 3′, and one 5′ end), while regions abutting divergent 5′ ends are a further 20% longer on average. This pattern is indicative of severe reductive selection operating on IGRs (Keeling and Slamovits 2004), as zero, one, or two sets of upstream transcription factors need to bind between convergent, parallel, and divergent genes, respectively.

Several other factors suggest that Encephalitozoon spp. are operating at the limit of IGR reduction. Firstly, the length of IGRs sometimes dips into negative values, i.e., genes overlap one another (Katinka et al. 2001; Akiyoshi et al. 2009; Corradi et al. 2010). Secondly, multiple studies have noted that transcripts initiate in upstream genes and read through into downstream genes (Williams et al. 2005; Corradi et al. 2008; Gill et al. 2010), suggesting that transcriptional start sites and termination sequences are often located within adjacent genes. Finally, microsporidia produce many multigene transcripts, which surprisingly encode both sense and antisense genes (Peyretaillade et al. 2009; Corradi and Slamovits 2011; Watson et al. 2015). These transcripts, known as “noncontiguous operons,” are thought to regulate protein expression levels and result from evolutionary pressure to minimize genome size (Sáenz-Lahoya et al. 2019). These three examples provide evidence that microsporidia trim and eliminate IGRs wherever possible and have adapted more spatially efficient mechanisms to regulate protein expression levels.

Microsporidian parsimony is not only directed toward IGRs but also impacts other non-coding regions like introns. Splicing machinery and introns appear to have been convergently eliminated in at least three microsporidian genera: Edhazardia, Nematocida, and Enterocytozoon (Keeling et al. 2010; Desjardins et al. 2015). Even when introns are retained, they are reduced in both number and length (Lee et al. 2010; Campbell et al. 2013). In E. cuniculi, for example, a total of 36 introns have been identified, ranging from only 23 to 76 bases in length (Lee et al. 2010). The splicing efficiency for many of these introns is very low, often around 10–25%, and many putative introns display no active splicing (Grisdale et al. 2013; Campbell et al. 2013; Desjardins et al. 2015). For comparison, the yeast genome contains at least 300 introns ranging from around 100 to 1000 bases (Spingola et al. 1999; Xia 2020), which are frequently spliced with 100% efficiency (Xia 2020). The convergent loss of introns and splicing machinery in several microsporidian clades implies there is little functional utility for most introns. Microsporidian species retaining spliceosomal machinery may therefore represent an earlier evolutionary snapshot in the decay of intron usage.

Interestingly, a large proportion of the retained introns are found in ribosomal proteins (Lee et al. 2010). Splice sites for ribosomal proteins are heavily biased toward the 5′ end of the coding sequence, and Exon-1 often encodes only a few amino acids (Fig. 1.4a). Ribosome structures are available for Vairimorpha necatrix and Paranosema locustae, two microsporidia that retain functional splicing machinery. The protein product of a spliced mRNA can be visualized by inspecting the cryo-EM density for proteins encoded by genes with introns. For example, the N-terminal region of eS6 in P. locustae displays apparent density for Met1-Lys2 amino acids (Exon-1), followed by amino acids from Exon-2, providing visual evidence for the successful splicing of a 23-nucleotide intron (Fig. 1.4b).

Fig. 1.4

Visualizing the protein product of eS6 mRNA splicing in Paranosema locustae. (a) Schematic representation of the 5′ end of the gene encoding eS6 in P. locustae, with DNA and corresponding amino acid sequence indicated. The splice sites are underlined. An in-frame stop codon in the intron is shown in red. (b) The corresponding cryo-electron microscopy density of eS6 is shown in isolation. The right side displays the entire ribosomal protein density in light blue with its N-terminus indicated in purple. The left side zoom in depicts the superposition of the model (PDB 6ZU5) and cryo-EM density (EMDB 11437) for the protein eS6 (Ehrenbolger et al. 2020). Density and cartoon model are colored according to their coding exons, analogous to (a) (Exon 1, purple; Exon 2, light-blue)

1.3.2 Gene Deletion and Minimization

Constant selective pressure favoring reductive evolution has led to widespread gene deletions, resulting in a core of only 800 conserved microsporidian proteins (Nakjang et al. 2013). These proteins are generally involved in essential processes, such as replication, DNA repair, and protein synthesis or recycling (Galindo et al. 2018). Categories of deleted proteins span the gamut; however, metabolic and regulatory pathways are particularly depleted (Nakjang et al. 2013; Dean et al. 2016; Wiredu Boakye et al. 2017; Galindo et al. 2018). Encephalitozoon spp. have lost almost all proteins involved in glycolysis, oxidative phosphorylation, fatty acid metabolism, and amino acid/nucleotide biosynthesis (Dean et al. 2016; Wiredu Boakye et al. 2017). Recent work identified one mechanism by which microsporidia survive without these important anabolic and catabolic pathways (Kurze et al. 2016; Luo et al. 2021). During infections, microsporidia secrete enzymatic proteins like hexokinase and trehalase into host cells (Senderskiy et al. 2014). These secreted proteins are incorporated into host metabolic pathways, leading to the upregulation of genes important for the biosynthesis of amino acids, nucleotides, and fatty acids (Kurze et al. 2016; Luo et al. 2021). Interestingly, transporters are one of the few classes of proteins that have experienced expansion rather than reduction in microsporidia (Nakjang et al. 2013; Dean et al. 2016). The slight radiation in transport genes has facilitated a much more substantial elimination of metabolic genes, allowing for a large net decrease in genome size.

Not only have many proteins been lost in microsporidia, the remaining proteins are also shorter. In fact, E. cuniculi proteins are on average 15% shorter than the yeast orthologs (Katinka et al. 2001). The impetus for this gene shortening remains to be proven, but Katinka et al. (2001) speculate that the loss of proteins led to more simplified interaction networks, which has facilitated the removal of protein-protein interaction domains from remaining proteins. One potential example of this can be seen in Taf5, a subunit of the transcription initiation factor TFIID. In most eukaryotes, Taf5 contains a conserved, N-terminal Lis1 Homology motif (LisH) (Romier et al. 2007; Wang et al. 2020). LisH domains are short, ~33 amino acid motifs that assist in protein dimerization and subcellular targeting (Gerlitz et al. 2005). In Taf5, the LisH domain is known to both facilitate dimerization (Bhattacharya et al. 2007) and help mediate interactions between Taf5 and the Spt20 subunit of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex (Wang et al. 2020). Interestingly, the LisH domain is absent in microsporidia (Romier et al. 2007). Although the absence of spt20 is yet to be verified, a BLAST (Altschul et al. 1990) search against all available microsporidian genomes produced no reliable hits. Additionally, other core components of the SAGA complex are absent in microsporidia (Miranda-Saavedra et al. 2007). As the LisH domain is primarily a structural domain that promotes protein-protein interactions, the loss of its binding partner would render its function moot. These data therefore support the hypothesis that simplified protein-protein interaction networks lead to the ablation of superfluous domains and the minimization of microsporidian gene length.

Although we have thus far described the loss of a gene as the loss of a function, functional redundancy is common in most eukaryotes (Dean et al. 2008). Organisms operating under strong reductive selection frequently minimize genomes by eliminating redundancies (Luo et al. 2011). In highly reduced picoplanktonic eukaryotic organisms, for example, the total number of gene families is conserved despite extensive gene loss (Derilus et al. 2020). Instead, the average size of each gene family is decreased as a result of deletions of paralogous genes. A similar trend can be identified in the most minimal microsporidia, like Encephalitozoon spp., which are largely devoid of duplications and repetitive elements (Katinka et al. 2001; Keeling and Slamovits 2004; Cormier et al. 2021). These findings only hold true, however, for the most reduced microsporidia. Other species, like Nematocida spp., have markedly expanded a small number of gene families (Reinke et al. 2017), while the comparatively large Edhazardia aedis and Hamiltosporidium tvaerminnensis are quite rich in duplications and repetitive elements (Williams et al. 2008; Cormier et al. 2021).

1.3.3 Gene Retention and Expansion

Gene families that are retained or expanded despite reductive selection provide an abundance of valuable information. By considering which proteins are retained, it is possible to identify biologically essential systems. As described above, gene families retained in microsporidia are often involved in core cellular processes, and their removal is typically lethal (Nakjang et al. 2013). Additionally, yeast orthologs of these conserved proteins are significantly more likely to be highly expressed and have a large number of interaction partners. These traits persist in microsporidia (Nakjang et al. 2013), revealing the importance of connectivity and expression levels in gene retention. Unexpectedly, conserved core proteins only account for around 800 of the 1750 (Enterocytozoon bieneusi) to 4500 (Nosema bombycis) predicted genes (Peyretaillade et al. 2009; Nakjang et al. 2013; Pan et al. 2013). The remaining genes serve species-specific functions and are often members of novel expanded gene families (Reinke et al. 2017).

One group that has undergone expansion is the Small Conductance Mechanosensitive Ion Channel (MscS) family. These membrane proteins are found in both prokaryotes and eukaryotes and are involved in the regulation of intracellular pressure in response to extracellular stimuli. Most frequently, this stimulus takes the form of mechanical stress on membranes resulting from hypo- or hyperosmotic conditions (Kung et al. 2010). These proteins function by forming a channel, which allows for the influx or efflux of water and small molecules to relieve the stress by reducing pressure. Microsporidia encode at least five copies of MscS proteins, derived from a combination of horizontal gene transfers and lineage-specific expansions (Nakjang et al. 2013). Based on other MscS functions, it has been proposed that they play a role in the regulation of osmotic stress during microsporidian germination (Nakjang et al. 2013). Previous studies have demonstrated that the rapid degradation of metabolites like trehalose, followed by a subsequent increase in turgor pressure, provides the impetus for release of the microsporidian polar tube (Undeen and Vander Meer 1999). Therefore, it is unsurprising that proteins involved in the regulation of turgor pressure would be both enriched and conserved in microsporidian species.

The most expanded microsporidian gene families are novel and have no known function (Heinz et al. 2012; Peyretaillade et al. 2012). Examination of these proteins in Nematocida spp. demonstrated that they are recently generated and rapidly evolving, as many members are either species- or clade-specific (Reinke et al. 2017). Tellingly, the genes are typically located within the subtelomeric regions of the chromosomes, an area often associated with rapid evolution and immune evasion (Fischer et al. 2003; Brown et al. 2010; Pombert et al. 2012; Reinke et al. 2017). As such, it is likely that these families are involved in direct interactions with hosts and are expanding and evolving in a species-specific way in response to preferred hosts. In support of this idea, Reinke et al. (2017) identified host-exposed proteins in N. parisii using spatially restricted enzymatic tagging and found that 49% of the experimentally identified proteins belonged to a large gene family and that 88% of all host-exposed proteins lacked orthologs outside of closely related Nematocida spp. Although further work is required to understand the role these families play in host-parasite interactions, their expansion against a background of reductive evolution suggests a unique and important function.

1.3.4 Variation Between Species

The genome sizes of microsporidia differ considerably between species, from 2.3 Mbp (E. intestinalis) to 51.3 Mbp (E. aedis). This variation is not mirrored in the number of protein coding genes, which fluctuates within a much narrower window (1750–4500) (Peyretaillade et al. 2009; Pan et al. 2013). The larger genome size variation instead reflects the accumulation of non-coding regions in larger microsporidia. In fact, non-coding regions sometimes accrete to such a degree that the microsporidian clade contains both one of the most gene dense and one of the least gene dense fungal species (Fig. 1.2) (Muszewska et al. 2019). This surprising finding is evidence that not all microsporidia are undergoing aggressive reductive evolution.

Repetitive sequences make up a large proportion of the non-coding regions of gene-sparse species (Parisot et al. 2014). These repetitions, largely transposable elements, are associated with mildly deleterious effects in eukaryotes (Hua-Van et al. 2011). This begs the questions, what differs between gene-dense and gene-sparse species, and why are gene-sparse species experiencing less stringent reductive selection? To address these questions, recent work compared and contrasted the life cycles of various microsporidia and discovered a correlation between genome size and mode of transmission. Microsporidia that are transmitted through purely horizontal means have small and compact genomes, while microsporidia with mixed-mode (vertical and horizontal) transmission have larger genomes with a higher concentration of transposable elements (Haag et al. 2020; De Albuquerque et al. 2020). These studies suggest that population bottlenecks resulting from vertical transmission lead to reduced selective pressure and facilitate the expansion of repetitive sequences. Although more work is required, it is clear that the mode of transmission contributes to the significant variation in microsporidian genome sizes.

1.4 The Ribosome as a Molecular Fossil Record

As seen above, reductive evolution operates on all facets of the microsporidian genome. Although we have thus far focused on the factors leading to genome reduction, it is just as important to understand the structural and functional adaptations resulting from that reduction, i.e., which pathways are lost or minimized, and how does the loss or minimization of these pathways fit in with what we know about the microsporidian life cycle? For the remainder of this chapter, we will describe the results of genome compaction on cellular systems, using ribosome structure, function, and biosynthesis as our case study. There are a variety of reasons to choose the ribosome for this analysis. Firstly, microsporidia suffer from a dearth of structural data. There are only 43 microsporidian structures available on the PDB (using keyword “microsporidia,” www.rcsb.org; June 2021), as opposed to over 5000 for Saccharomyces cerevisiae. Two of the microsporidian structures encompass the whole ribosome, making it one of the most studied microsporidian structures (Barandun et al. 2019; Ehrenbolger et al. 2020). Secondly, the ribosome is an essential macromolecular complex responsible for protein synthesis in all known “living” organisms. Thirdly, despite a relatively conserved core, ribosomes differ significantly between clades (Fig. 1.5). They are dramatically expanded in most eukaryotes compared to prokaryotes, but highly reduced in intracellular parasites like microsporidia or apicomplexans. Finally, because of both its variation and ubiquity, the ribosome has long been used to build gene-based phylogenetic trees, promoting a function for ribosomes as evolutionary timekeepers.

Fig. 1.5

The evolution of the microsporidian ribosome is shaped by an unusual reversal of eukaryotic expansions. A simplified schematic phylogenetic tree (James et al. 2006, 2013; Haag et al. 2014), depicting the expansive evolution of the ribosome from prokaryotes to eukaryotes and the reduction in microsporidia. Ribosomes are displayed for representative organisms in four views, related by 90-degree rotations [bacteria, E. coli PDB 4YBB (Noeske et al. 2015); archaea, Pyrococcus furiosus PDB 4V6U (Armache et al. 2013); plants, Triticum aestivum PDB 4V7E (Gogala et al. 2014); animals, Homo sapiens PDB 6EK0 (Natchiar et al. 2017); fungi, S. cerevisiae PDB 4 V88 (Ben-Shem et al. 2011); and microsporidia, V. necatrix PDB 6RM3 (Barandun et al. 2019)]. The shared rRNA core and all ribosomal proteins are shown in white, 5S rRNAs are in red, and rRNA elements absent in microsporidia are colored in blue (LSU rRNA) or gold (SSU rRNA). The organism’s name, number and size of rRNAs, and number of ribosomal proteins (rps) are indicated on the right side

In 1977 Carl Woese and George Fox recognized the ribosomal genes’ potential to serve as a molecular fossil record of life and revolutionized biology by establishing ribosomal RNA (rRNA) sequencing as a tool in molecular phylogenetics (Woese and Fox 1977). Their work led to the discovery of the Archaea and a fundamental re-drawing of the tree of life. When analyzing the ribosomal RNA of the microsporidia V. necatrix in 1987, Vossbrinck and Woese found a highly reduced ribosomal RNA sequence, more akin to a prokaryote than a eukaryote. It was concluded with the data available at the time, that microsporidia might be early branching, primitive eukaryotes (Vossbrinck et al. 1987). However, with the advent of genome sequencing and the increasing availability of protein and ribosomal RNA sequences from different species in the decades since, mounting evidence has shown that microsporidia are closely related to fungi (James et al. 2006; Haag et al. 2014), rather than being primitive eukaryotes. Hence, the “prokaryote-like” ribosomal gene arrangement, reduced size of the ribosomal RNA, and minimized proteins in microsporidia are the result of genome compaction and represent an unusual reversal of the drastic expansion that occurred in eukaryotes (Fig. 1.5).

1.5 The Microsporidian Ribosome: An Outlier in Ribosome Evolution

The ribosome is a complex macromolecular machine responsible for the production of all proteins. To perform this vital function, many ribosomes are produced, accounting for nearly 30% of the dry mass of a rapidly dividing bacterial cell (Bremer and Dennis 2008; Piir et al. 2011). Ribosomes are composed of both proteins and RNA (i.e., they are ribonucleoproteins) and are typically segregated into two sections: the small subunit (SSU, 40S in eukaryotes) and the large subunit (LSU, 60S in eukaryotes). As a result of the ribosome’s large size and essential role, as much as 80% of a cell’s resources are dedicated to its biosynthesis and functional upkeep in nutrient-rich conditions (Tempest and Neijssel 1984; Maitra and Dill 2015). Although all known living organisms produce ribosomes, the ribosome composition varies significantly between clades (Fig. 1.5).

Most prokaryotes have simple genomes smaller than 10 Mbp (Fig. 1.1) and are tightly packed with protein-coding genes (Fig. 1.2). In extreme cases, genomes can be as small as 0.112 Mbp (Nasuia deltocephalinicola) (Bennett and Moran 2013), or as large as 13 Mbp (Sorangium cellulosum) (Han et al. 2013). Despite the tiny genome of N. deltocephalinicola containing only 137 coding genes, it still manages to produce ribosomes, which are composed of 3 rRNAs (4445 nucleotides total) and ~ 50 proteins (Moran and Bennett 2014). In more typical bacteria like Escherichia coli, ribosomes still consist of 3 ribosomal RNAs (rRNAs) with a combined size of 4567 nt. The surface of the rRNA is coated with 51 ribosomal proteins (Fig. 1.5), and the biogenesis process requires dozens of additional proteins (Shajani et al. 2011). The eukaryotic ribosome, on the other hand, is significantly expanded in size and number of components. For example, the cytoplasmic ribosome from S. cerevisiae contains 4 rRNAs of a combined 5475 nt, and a total of 79 proteins. The eukaryotic assembly process also differs drastically from prokaryotic ribosome assembly and utilizes over 300 trans-acting factors for ribosomal maturation (Woolford and Baserga 2013; Klinge and Woolford 2019).

While translational machinery is expanded in most eukaryotes, it has been significantly affected by genomic erosion in microsporidia. Recently, the ribosome structures have been solved for two microsporidian species: V. necatrix and P. locustae (Figs. 1.5 and 1.6) (Barandun et al. 2019; Ehrenbolger et al. 2020). These structures provide one of the first glimpses of the effects of reductive evolution on macromolecular complexes in microsporidia. Interestingly, microsporidian ribosomes have been reduced to such an extent that their rRNAs are smaller than many bacterial ribosomes (3 rRNAs totaling ~3850 nt), including those from N. deltocephalinicola, and are significantly smaller than the yeast ribosome. Many parts of the ribosome have been lost or reduced in microsporidia; in this section, we will describe some regions that have been altered, analyze the functional significance of those regions, and postulate on the implications of those changes for microsporidia.

Fig. 1.6

Extensive rRNA expansion segment loss in microsporidia. (a, b) Schematic secondary structure diagram of the small (a) and the large subunit ribosomal rRNA (b), based on the S. cerevisiae structure, with expansion segments that have been lost in V. necatrix colored (SSU, shades of orange and yellow; LSU, shades of blue and green). (c–e) Related views of the ribosome from S. cerevisiae PDB 4 V88 (c), P. locustae PDB 6ZU5 (d), and V. necatrix PDB 6RM3 (e). The middle section displays the full ribosome, while two 90-degree-related views of the SSU and the LSU solvent-exposed sides are shown in isolation on the left and right. Ribosomes are colored in white, while locations of expansion segments or other elements that have been lost in V. necatrix are colored as in (a, b). The microsporidian ribosomal protein (msL1) is shown in light-green, while MDF1 and MDF2 are colored purple and red

1.5.1 Minimization of Expansion Segments

Eukaryotic ribosomes are characterized by approximately 30 additional eukaryote-specific proteins and 50 additional rRNA elements known as expansion segments (ES). These segments are aptly named, as they are regions of the rRNA that have been expanded in eukaryotes compared to prokaryotes. The functions of most ESs have not been thoroughly studied; however, many ESs seem to stabilize the additional layer of proteins present in eukaryotic ribosomes (Ben-Shem et al. 2011). Others aid in recruiting and organizing components of the much more complex eukaryotic ribosome biogenesis process (see Sect. 1.6) (Ramesh and Woolford 2016). More targeted studies have suggested that specific expansion segments are involved in recruiting regulatory factors (Fujii et al. 2018), or serving auxiliary roles by engaging and stabilizing mRNA during translation (Parker et al. 2018). Regardless of function, ESs are a hallmark of typical eukaryotic ribosomes.

Microsporidia have reversed the evolutionary trend to expand rRNA elements and have removed the vast majority of eukaryotic ESs (Fig. 1.6). Those that do remain are significantly reduced in size. A comparison of the SSU rRNAs indicates that microsporidia are indeed evolving toward the loss of ESs (Fig. 1.7), rather than simply not expanding in the first place. Step-wise deletions lead to early branching species like Rozella allomycis and Mitosporidium daphinae encoding partial versions of ESs, while more recently diverged species have removed many ESs altogether (see, e.g., es9 or es3; Fig. 1.7). If ESs were instead convergently evolving from a minimal core in a last common ancestor, we would not see ES sequence homology between early diverging microsporidia and other eukaryotes. Although most ESs do not have defined roles or specific known interaction partners, several segments have been studied in more detail, allowing us to draw conclusions on the causes and effects of ES loss in microsporidia.

Fig. 1.7

Expansion segment loss and sequence divergence in the microsporidian ribosomal RNA. Small subunit rRNA sequence alignment of selected microsporidian and eukaryotic organisms, created with Clustal Omega using RNA settings (Sievers et al. 2011). A structurally impossible sequence insertion and potential sequencing issue in the N. ceraneae rRNA was manually removed (nt 700–728). Not included are sequences from E. romaleae, P. neurophilia, and N. displodere, which are only partially available. Organisms are labeled on the top left with microsporidia in the dark box and other eukaryotes in light-gray. Conservation is indicated with shades of gray, from white (variable) to dark gray (conserved). Elements which are not present in the V. necatrix rRNA are indicated with colored boxes and labeled on top using the same coloring scheme as in Fig. 1.6. The SILVA ribosomal RNA gene database (Quast et al. 2013) was used to obtain sequences

N-terminal acetylation of proteins is extremely common, with 60% of the yeast proteome and 85% of the human proteome containing this modification (Arnesen et al. 2009). Acetylation plays a role in protein half-life, most commonly by protecting proteins from ubiquitination of N-terminal residues, thereby preventing their proteasomal degradation (Ree et al. 2018). Nearly 40% of all acetylation in humans happens co-translationally and is mediated by the NatA acetylation complex (Ree et al. 2018). Co-translational acetylation is achieved via direct interactions between NatA and multiple ribosome ESs, including H24, ES7, ES27, and ES39 (Knorr et al. 2019). In microsporidia, these ESs are extremely reduced (as in the case of P. locustae; Figs. 1.6 and 1.7), or completely absent (V. necatrix), indicating N-terminal acetylation is either not performed co-translationally or is mediated by a different complex. Consistently, an analysis of microsporidian proteomes demonstrated that, while some subunits of the NatA complex are present, there is a significant depletion in NatA substrate motifs in microsporidia (Rathore et al. 2016), suggesting a greatly diminished role for the acetylation complex. Intriguingly, N-terminal acetylation has also been associated with cellular targeting, where cytoplasmic proteins are enriched and secreted proteins are depleted in the modification (Forte et al. 2011). As intracellular parasites, microsporidia interact extensively with hosts and are known to have a large number of secreted proteins (Reinke et al. 2017). It is therefore possible that the loss of NatA-interacting ESs serves to both conserve nutrients by minimizing the ribosome and facilitate protein secretion into host cells.

In addition to recruiting the acetylation complex, ES27 has a purported role in translational fidelity (Fujii et al. 2018). The deletion of segments of ES27 in yeast leads to the misincorporation of amino acids during translation. Microsporidia lack ES27 (Barandun et al. 2019; Ehrenbolger et al. 2020), indicating that they ensure translational fidelity via alternative mechanisms or have higher error rates. A recent study on the microsporidia Vavraia culicis supports the latter possibility and shows that nearly 6% of leucine residues are erroneously translated (Melnikov et al. 2018b). For comparison, E. coli has a mistranslation rate of only 0.2% (Zaher and Green 2009). A low translational fidelity is utilized by many organisms as an adaptive strategy, facilitating immune evasion by increasing proteomic diversity (Miranda et al. 2013; Ling et al. 2015). For organisms like microsporidia that have both high numbers of host-exposed proteins and extremely restricted proteomes, the additional flexibility garnered by mistranslation may be particularly beneficial. Therefore, the loss of ES27, and the potential decrease in translational fidelity, would be both economically and functionally advantageous.

The P. locustae ribosome structure provides an extremely economical example of ES ablation. In most eukaryotic ribosomes, ES39 contains a highly conserved nucleotide that appears to stabilize the interface between two ribosomal proteins (Ehrenbolger et al. 2020). The microsporidian ribosome has eliminated the vast majority of ES39; however, extra density consistent with a single nucleotide is present at the same location. These data indicate that the free nucleotide is a relic of ES39 and serves an important role as an architectural cofactor to stabilize the protein-protein interface. The near-complete reduction of ES39 to a single nucleotide is an exceedingly economical solution and lends credence to the idea that microsporidia are under high levels of reductive selection.

The minimization of this ES to a single-nucleotide relic, in conjunction with the previous examples of ES loss, demonstrates that ES deletion is a common mechanism by which microsporidia reduce genome size and cut nutrient costs in the biosynthesis of ribosomes. Interestingly, the localization of rRNA elements within the subtelomeric regions of the genome may facilitate rRNA minimization. Subtelomeres are often repetitive, associated with higher evolutionary rates, and have increased frequencies of double-strand breaks (Brown et al. 2010; Muraki and Murnane 2017). The high repetition may accelerate the deletion of ESs during double-strand break repair. Regardless of the underlying mechanism, it is clear that microsporidia have removed the vast majority of ESs, resulting in the smallest known cytoplasmic ribosome in eukaryotes.

1.5.2 Changes to the Proteinaceous Composition of Microsporidian Ribosomes

Ribosomal proteins are some of the most widely conserved across the tree of life. Approximately half of the protein subunits are present in both prokaryotic and eukaryotic ribosomes and are thus called the “universal” or “u” ribosomal proteins (Ban et al. 2014). However, as seen with ESs, eukaryotic ribosomes have greatly expanded their proteinaceous repertoire, developing the “eukaryotic” or “e” proteins of the ribosome. It is somewhat surprising then that the drastic microsporidian reduction in ESs is not accompanied by a concomitant loss in the number of ribosomal proteins (Fig. 1.5).

To better understand the proteinaceous changes to microsporidian ribosomes, we have collected sequences from genomes available on MicrosporidiaDB (Aurrecoechea et al. 2011) and compared their conservation in Fig. 1.8. Caution is advised while drawing conclusion from these data, as many microsporidian genomes are derived from incomplete assemblies and microsporidian proteins are rapidly evolving. It is therefore very likely that some of the proteins marked as absent were simply not identified via our methods. That said, microsporidia have retained most of the ribosomal proteins found in yeast, and only a few of the 80 yeast proteins are potentially absent in many microsporidian species. Remaining proteins have a 38% average sequence identity to yeast homologs and are often considerably shorter (Fig. 1.8a and b). Some proteins have lost loops or linkers, while others have been truncated at the N- or C-terminus. Additionally, low levels of sequence identity can be used to demarcate proteins that have structurally diverged from yeast (Fig. 1.8c).

Fig. 1.8

Microsporidian ribosomal protein phylogeny, identity, and structure relative to their yeast homologs. (a) Ribosomal protein phylogeny generated by using protein sequences conserved in all listed microsporidian species. Connected to the microsporidian phylogeny (black) is a simplified tree for other non-microsporidian species, based on (James et al. 2006, 2013; Haag et al. 2014). For the microsporidian phylogenetic tree, the protein sequences were obtained by performing translated nucleotide blast (tblastn) searches with an E-value cutoff of 0.05, using the S. cerevisiae sequences or verified microsporidian hits as query and MicrosporidiaDB (Aurrecoechea et al. 2011) as database. For P. locustae and V. necatrix, protein sequences were obtained from (Barandun et al. 2019; Ehrenbolger et al. 2020) or local genome databases. For the non-microsporidian species, sequences were obtained from https://www.ncbi.nlm.nih.gov/. Proteins were aligned using MUSCLE 3.8.31 (Edgar 2004) and trimmed using trimAl (Capella-Gutiérrez et al. 2009) with the –gappyout option. The trimmed alignments were then concatenated using FASconcat 1.11 (Kück and Meusemann 2010). The phylogenetic tree was constructed with RAxML 8.2.12 using the model PROTGAMMAILGF, determined with ProtTest 3.4.2, and 1000 bootstrap replicates. The sequence identity heatmap was constructed using MUSCLE 3.8.31 (Edgar 2004) and Clustal-Omega (Sievers et al. 2011). The S. cerevisiae sequences were set as reference, except for eL28 and msL1, where H. sapiens and V. necatrix were used. The different shades of blue describe the percentage identity of the protein sequence compared to the reference. The row for S. cerevisiae contains viability data, color coded for lethal (dark yellow), slow-growing (yellow), and normal-growing (cream) ribosomal gene knockouts (Giaever et al. 2002; Gao et al. 2015). A black dot is used to mark genes that are duplicated in the yeast genome. Only single gene knockouts were performed in the referenced study. The sequence of eS31* was modified by removing the ubiquitin moiety to create the mature protein. (b) Difference in length between the V. necatrix or P. locustae and S. cerevisiae ribosomal proteins. (c) Comparison of the region around ES4 between the S. cerevisiae (left, PDB 4 V88), V. necatrix (middle, PDB 6RM3), and P. locusate ribosome (PDB 6ZU5). Selected ribosomal proteins are colored and labeled with name and N- and C-termini in shades of red. The lost eL38 and the gained msL1 are shown in shades of green. (d) The same view is shown as in (c) with selected proteins colored solid and the ribosome structure transparent

Genome-wide knockout screens have been performed in yeast, which allows us to identify essential ribosomal proteins (Giaever et al. 2002; Gao et al. 2015) (Fig. 1.8a). These studies further noted knockouts that led to slow-growth defects. It is important to mention, however, that yeast have duplicated the majority of ribosomal genes. Some deleterious effects may have therefore been ameliorated by the presence of paralogs during single-gene deletion studies. Nevertheless, comparisons between gene conservation and essentiality reveal several interesting results. Firstly, as might be expected, many of the essential genes in yeast were not duplicated. Secondly, essential genes are still extant in almost all microsporidia. Instances of their loss, such as uL16 in Enterocytozoon hepatopenaei, are more likely a result of incomplete genome assemblies or low sequence conservation. This is evinced by the isolation of purported losses. Only in the case of uL23 are essential genes unidentifiable in a related cluster of microsporidia (Trachipleistophora hominis and Pseudoloma neurophilia). Numerous studies have demonstrated the essentiality of uL23 for the formation of the polypeptide exit tunnel (Kaur and Stuart 2011; Polymenis 2020). We therefore find it more probable that its absence is a matter of incomplete genome assemblies; however, a genuine absence would undoubtedly provide useful insights into evolutionary strategies developed by microsporidia to minimize the ribosome exit tunnel. Thirdly, all of the yeast proteins unidentifiable in most microsporidia are nonessential (see eL28, eL38, eL41, P1, and P2), as are some of the frequently missing proteins (eS12, eS25, and eL29). The nonessential eL38 is present in all earlier branching eukaryotes and is absent in all but two microsporidian species (M. daphinae, Amphiamblys sp.), suggesting a relatively recent loss of this ribosomal protein (Barandun et al. 2019). These findings demonstrate that microsporidia have typically retained essential proteins and eliminated nonessential ones.

The nonessential protein eL41 is the only yeast subunit absent in all sequenced microsporidia (Fig. 1.8a) (Barandun et al. 2019; Ehrenbolger et al. 2020). It is remarkably short in other eukaryotes, only ~25 amino acids, and forms a small bridge between the LSU and the SSU (Tamm et al. 2019). Deletions of eL41 are easily tolerated, with knockout yeast strains displaying growth rates similar to wild-type strains (Giaever et al. 2002). More in-depth analyses have revealed that eL41 plays a role in translational efficiency (Dresios et al. 2003; Meskauskas et al. 2003). Ribosomes lacking eL41 had both lower translational fidelity and slower rates of peptidyltransferase activity. This suggests that the removal of eL41 in microsporidia may be another factor contributing to their markedly high rate of missense mutations (Melnikov et al. 2018b). The deletion of eL41 may also result in a slower translation rate, although no information is currently available on the kinetics of microsporidian ribosomes.

The ribosomal stalk proteins, which also have a purported role in translational efficiency (Wawiórka et al. 2017), are reduced in most microsporidia. A typical eukaryotic ribosomal stalk is composed of uL10, two subunits of P1, and two subunits of P2. All five protomers contain a highly conserved, C-terminal SDDDMGFGLFD motif, preceded by a long and flexible linker (Choi et al. 2015). This organization and motif is found in organisms as diverged as humans and the archaeon Pyrococcus horikoshii (Ito et al. 2014). During active translation, the C-termini of the pentamer bind to and recruit the essential elongation factor EF1α, which delivers charged aminoacyl-tRNA to the ribosome. It is proposed that the five redundant motifs aid in the rapid and efficient recruitment of the correct aminoacyl-tRNA, by greatly increasing the local concentrations of EF1α (Wawiórka et al. 2017). Additionally, this kinetic model of decoding suggests that ribosomal pausing leads to the acceptance of near-cognate anticodons, resulting in missense mutations. It is therefore interesting that the majority of microsporidia do not to encode P1, and some may have lost P2 (Fig. 1.8), implying a single EF1α-binding motif is present. Previous work has demonstrated that P1 and P2 are nonessential in eukaryotes only because uL10 retains an EF1α binding domain (Santos and Ballesta 1995; Remacha et al. 1995). On the other hand, the prokaryotic equivalents to P1/P2 are required for translation (Huang et al. 2010), as prokaryotic L10 lacks the binding motif. Remarkably, the uL10 homologs for microsporidian clades have lost the linker and the SDDDMGFGLFD motif (data not shown). Some microsporidia therefore have no identified proteins that can recruit EF1α to ribosomes. This finding may indicate that the translation rate and fidelity are much lower in microsporidia. Alternatively, microsporidia might have developed novel proteins or binding motifs to recruit EF1α. This possibility is of particular interest, as the C-terminal motif utilized by eukaryotes and archaea is a common target for potent toxins like ricin (Choi et al. 2015; Fan et al. 2016). A unique motif would represent an attractive target for therapeutics or pesticides.

1.5.3 Retained and Gained Ribosomal Proteins

Most microsporidian proteins have relatively low sequence identity to yeast proteins (Fig. 1.8). This is not entirely unexpected, as even proteins from two closely related Nematocida species share only ~70% of their amino acid sequence (Balla and Troemel 2013). A noticeable outlier in this divergence is eS31, an essential protein located in the beak of the SSU. Interestingly, eS31 is always produced as a fusion with a ubiquitin moiety. The ubiquitin acts as a chaperone protein to assist in the production and folding of eS31 and is cleaved off before eS31 is incorporated into ribosomes (Martín-Villanueva et al. 2019). The high sequence identity for eS31 derives from this ubiquitin moiety, as a realignment without ubiquitin results in much lower values (see eS31 vs eS31* in Fig. 1.8). Another highly conserved protein is eL15, which is present in all sequenced microsporidia. Little is known about eL15’s function other than that it is essential; however, it is a structural protein that is mostly buried and is therefore likely to have many conserved intermolecular interactions. Additionally, eL15 seems to mediate concentrations of other core ribosomal proteins, and its dysregulation leads to various cancers and diseases (Wlodarski et al. 2018; Ebright et al. 2020). Despite the lack of focused studies, the high conservation of eL15 in microsporidia evinces a high level of functional significance, which is not amenable to mutations in sequence or structure.

In addition to retaining most ribosomal proteins, microsporidia have also gained at least one novel subunit. The microsporidia-specific ribosomal protein (msL1) binds to V. necatrix ribosomes in a gap left by the loss of four ESs (Fig. 1.5) (Barandun et al. 2019). Although the specific role of this protein is unknown, it may be required to stabilize the ribosome in the absence of ESs. Genomic erosion in organelles, such as mitochondria, has resulted in a similarly minimized rRNA. In response, many mitochondria have acquired unique proteins used to patch unstable ribosomes (Petrov et al. 2019). It is likely that msL1 serves a similar patching function in microsporidia where rRNA reduction led to structural instability.

1.5.4 Conserving Energy by Utilizing Ribosome Hibernation Factors

Translational costs are high, and an estimated 30 ATPs are required for the biosynthesis and attachment of each amino acid (Wagner 2007). Such costs are unsustainable in nutrient-poor conditions. Organisms therefore express proteins known as hibernation factors, which bind to and inhibit ribosomes when nutrients are scarce (Prossliner et al. 2018). These factors allow cells to sequester intact ribosomes instead of degrading them (Brown et al. 2018; Trösch and Willmund 2019). The ability to inactivate ribosomes and recover them post-quiescence is of vital importance to microsporidia, as they spend a significant portion of their lifecycle as metabolically inactive spores (Weiss and Becnel 2014).

Microsporidia encode multiple hibernation factors, including the late-annotated short open reading frame 2 (Lso2), and microsporidian dormancy factors (MDF) 1 and 2 (Barandun et al. 2019; Ehrenbolger et al. 2020). All three of these proteins block active sites of the ribosome (Fig. 1.6) and are incompatible with active translation. In yeast, Lso2 is important for recovery of ribosomes post-starvation (Wang et al. 2018), and roughly 10% of ribosomes isolated from starved yeast are bound by Lso2 (Wells et al. 2020). Microsporidian ribosomes isolated from spores, on the other hand, displayed an approximately 92% occupancy rate, indicating that the vast majority of ribosomes in spores are in an inactivated state (Ehrenbolger et al. 2020). MDF1 and MDF2 have not been biochemically characterized; however, their high occupancy in spores and mechanisms of binding indicate that they are likely hibernation factors (Barandun et al. 2019). While MDF1 is broadly conserved in eukaryotes, MDF2 may be species-specific. Orthologs have thus far only been identified in V. necatrix, Nosema ceranae, and Nosema apis. The high occupancy of these factors bound to spore-stage ribosomes, and the fact that microsporidia have potentially evolved species-specific hibernation factors, demonstrates that sequestration of ribosomes during the spore stage is crucial. Although hibernation factors are not specifically associated with reductive evolution, they provide an additional example of the mechanisms by which microsporidia conserve energy.

1.6 Microsporidian Ribosome Assembly

In eukaryotes, ribosome biogenesis is a multidimensional process requiring the action of all three RNA polymerases (Pol) and a complex repertoire of over 300 assembly factors and snoRNAs (Woolford and Baserga 2013; Ebersberger et al. 2014; Klinge and Woolford 2019). The pathway starts in the nucleolus, a subcompartment of the nucleus, where the transcription of a precursor ribosomal RNA (pre-rRNA) initiates a co-transcriptional maturation pathway. In yeast, the precursor contains the rRNAs of both the small subunit (18S) and the large subunit (5.8S, 25S). These rRNAs are flanked by four transcribed spacer regions, two external and two internal (ETS, ITS; Fig. 1.9a). The third rRNA of the large subunit (5S) is transcribed from a different locus and is not part of this long precursor RNA. Assembly factors associate in a co-transcriptional manner with the rRNA precursor, including the transcribed spacers, to assist in the folding and enzymatic processing of the pre-rRNA and to incorporate ribosomal proteins. Several co-transcriptional endonucleolytic cleavage events are required to process the spacers and release the partially matured pre-ribosomal particles. Maturation then continues in the nucleus, where the pre-mature rRNA ends (e.g., 5′ ETS or ITS2) are further processed and degraded. After a controlled export through the nuclear pore complex, the last ribosomal maturation steps and quality control events occur in the cytoplasm.

Fig. 1.9

Compaction of the microsporidian rDNA locus to a prokaryotic-like organization. Schematic representation of a single rDNA locus, below a diagram indicating the genomic distribution of all rDNA loci, from (a) S. cerevisiae, (b) microsporidia (nucleotide sizes from V. necatrix), and (c) E. coli. The genes and known spacer sizes are indicated and drawn to scale for comparative purposes

The transcribed spacers are not present in the mature ribosome, but are essential elements required to recruit ribosome assembly factors. The level of spacer processing is also used to demarcate the maturation stage of this complex particle (Klinge and Woolford 2019). In addition, eukaryotic ribosomal expansion segments, which are part of the mature ribosome, are also involved in recruiting specific assembly factors. Genome compaction in microsporidia has not only removed rRNA elements, such as eukaryotic ESs, but also drastically affected the transcribed spacers of the ribosomal precursor (e.g., removal of ITS2; Fig. 1.9b). While the pre-ribosomal and ribosomal RNA have been minimized, the number of ribosomal proteins associated with the mature microsporidian ribosome has been less affected (see Sect. 1.5.2) (Barandun et al. 2019; Ehrenbolger et al. 2020). This raises the question of whether ribosome assembly factors and the maturation pathway have been similarly reduced overall, or if specific assembly factor categories have been more impacted by genome reduction than others. Have microsporidia lost ribosome assembly factors with a role in maturing eukaryotic-specific RNA or protein elements? The following section discusses the impact of reductive evolution on the organization of rDNA loci and the maturation of pre-rRNA in microsporidia.

1.6.1 Impact of Genome Compaction on Number and Localization of the rDNA Loci

In most organisms, the ribosomal RNAs are transcribed from one or more polycistronic ribosomal DNA loci. The number of rDNA loci increases considerably from prokaryotes to eukaryotes: from a single rDNA locus in slow-growing bacteria (e.g., Mycobacterium tuberculosis) to ~150–200 copies in yeast (Petes 1979) to more than 10,000 in some plants (Kobayashi 2014). In S. cerevisiae, the primary model organism to study eukaryotic ribosome assembly, all rDNA loci are clustered head to tail on a single chromosome (Petes 1979) (Fig. 1.9a). Within eukaryotes, the size of one pre-rRNA coding locus varies substantially. These size variations are mostly due to differences in the lengths of external and internal spacer elements or eukaryotic-specific ribosomal expansion segments. Eukaryotic rDNA sizes range from the minimal microsporidian version with approximately 4.5 kbp (calculated from the V. necatrix sequences), which has lost many regulatory spacers and ESs, to ~9.1 kbp in yeast or ~ 43 kbp in humans, which contain long ETSs and extensive intergenic spacer regions.

In microsporidia, the rDNA organization and localization within the genome differ between species. While other eukaryotes contain large numbers of clustered rDNA repeats, microsporidia are left with fewer and often not clustered rDNA genes. Twenty-two rDNA copies have been reported for E. cuniculi, located on both telomeric ends of its 11 chromosomes (Brugère et al. 2000; Katinka et al. 2001; Dia et al. 2016). Forty-six partial and polymorph rDNA loci have been found in N. ceraneae (Cornman et al. 2009), and similar to the rDNA loci in N. bombycis, they appear to be distributed over all chromosomes (Liu et al. 2008). While the individual loci are scattered throughout different chromosomes in many microsporidian species, in N. apis, the rDNA genes cluster as repeats head to tail (Gatehouse and Malone 1998), which is more similar to the classical arrangement observed in other eukaryotic organisms.

In most eukaryotes, the 5S encoding gene is dispersed throughout the genome and is not adjacent to the other three rRNAs. One exception to this observation is S. cerevisiae, where the 5S rRNA gene clusters in the intergenic spaces between rDNA repeats (Fig. 1.9a). Both arrangements have been observed in microsporidia. Similar to yeast, in N. bombycis, the 5S gene is located next to the rDNA locus (Huang et al. 2004). Other species, such as E. cuniculi and E. intestinalis, have dispersed the 5S throughout the genome. In these two microsporidia, three copies for the 5S have been detected (Katinka et al. 2001; Corradi et al. 2010), in contrast to the 22 rDNA loci. While the rDNA locus is transcribed by RNA Pol I, the 5S rRNA is transcribed by RNA Pol III (Ciganda and Williams 2011). The microsporidian transcription machinery includes elements for RNA pol I, II, and III (Katinka et al. 2001), indicating that the use of separate polymerases for 5S and rDNA transcription may be retained in microsporidia.

The comparatively small number of rDNA repeats in microsporidia may be a result of their diminutive cell size, simple genomes, and low proteomic complexity. Fewer and shorter genes might require a reduced number of ribosomes, which in turn can be synthesized from fewer rDNA repeats. Indeed, a strong positive correlation between genome size and the number of rDNA repeats in eukaryotes has been noted (Prokopowich et al. 2003). Although this correlation exists, in general, only a fraction of all rDNA repeats are transcriptionally active. The actual rRNA synthesis rate is more so determined by the rate of RNA polymerase recruitment. A yeast strain with only 42 rDNA repeats, compared to the original 142 repeats, grows as well as wild type because two times more RNA polymerases are recruited to the rDNA locus (French et al. 2003). In addition to a potentially reduced need for ribosomes and increased RNA polymerase recruitment to a single locus, the simplified microsporidian rDNA gene organization might allow for a more streamlined ribosome maturation. Fewer pre-rRNA processing steps might be required than in other eukaryotes, due to missing pre-rRNA elements such as internal transcribed spacer 2 (ITS2).

1.6.2 Loss and Minimization of Transcribed Spacers

In many microsporidian species, genome compaction and gene fusion led to a reduction in the total number of ribosomal RNAs from four to three, which represents a reversal of the evolutionary trend seen in eukaryotic ribosomes. The eukaryotic 5.8S rRNA sequence and the 5′ end of the prokaryotic large subunit gene are homologous (Jacq 1981). In typical eukaryotes, ITS2 separates the 5.8S from the remainder of the large subunit rRNA gene (Fig. 1.9b). Early branching microsporidia like M. daphnia and Chytridiopsis typographi still contain highly reduced versions of ITS2 and thereby preserve the traditional eukaryote-specific separation of the 5.8S from the LSU gene (Corsaro et al. 2019). In all later-branching microsporidia, ITS2 has been removed (Vossbrinck and Woese 1986). The reductive evolution in these organisms led to a complete loss of ITS2 and fusion of the 5.8S rRNA with the LSU rRNA (23S), which has created a unique eukaryotic rDNA locus (Fig. 1.9b) with prokaryotic features (Fig. 1.9c). The remaining ITS has been reduced to a surprisingly short sequence in some microsporidia. While N. bombycis (Huang et al. 2004) contains an ITS of ~179 nt, other microsporidians, such as V. necatrix or N. apis (Gatehouse and Malone 1998), compacted this element to only ~33/34 nt. The intergenic spacer regions are important signal sequences for co-transcriptional endonucleolytic processing of the pre-rRNA fragment. Together with an apparent reduction of the 5′ and 3′ ETS regions and the removal of ITS2, the shortening of the ITS has significant implications for the ribosome maturation process, which is tightly controlled by ribosome assembly factors binding to these regions.

1.6.3 Impact of rDNA Compaction on Ribosome Biogenesis Factors

In 2014, Ebersberger et al. performed an evolutionary analysis of 255 yeast protein factors involved in ribosome biogenesis and included four microsporidian species in their analyses (Ebersberger et al. 2014). From these initial factors, 244 were proposed to be present in the last common ancestor shared with the microsporidia. Remarkably, only about half of them could be identified in microsporidia, which was highlighted as “the most remarkable gene loss” observed among the eukaryotic supertaxa (Ebersberger et al. 2014). Although extensive lists of factors involved in yeast ribosome biogenesis existed at the time, the precise functions or binding sites of most of these factors were unknown due to a lack of structural and biochemical data. During the decade since, our knowledge of fungal ribosome biogenesis has advanced to a detailed structural and functional description of the individual factors. This is mainly due to the technical progress made in cryo-EM, which provided high-resolution information and enabled the study of previously inaccessible pre-ribosomal particles from the fungi S. cerevisiae or Chaetomium thermophilum. These structures now provide an updated and comprehensive picture of fungal ribosome maturation and depict the intricate interaction network of assembly factors and ribosomal proteins bound to pre-ribosomal rRNA elements (Barandun et al. 2018; Klinge and Woolford 2019). They show how ribosome maturation proceeds in a hierarchical manner through several different conformational states to produce the final mature eukaryotic ribosome (Klinge and Woolford 2019). The emerging structural data on the fungal biogenesis process, together with recent studies on the microsporidian ribosomes (Barandun et al. 2019; Ehrenbolger et al. 2020), allows us to give a few selected examples of why expansion segment and transcribed spacer removal or shortening might have enabled assembly factor loss (or vice versa).

In yeast, the 5′ ETS is 700 nt long and is involved in the co-transcriptional recruitment of up to 27 ribosome biogenesis factors and the formation of an assembly platform for the SSU. In microsporidia, the exact size and structure of the 5′ ETS pre-rRNA fragment are not known. However, several factors that typically bind to this region have not been identified in microsporidia (Fig. 1.10). One of the first and largest multi-subunit complexes bound to the newly synthesized 5′ ETS is UtpA (Fig. 1.10b) (Hunziker et al. 2016). UtpA is a 7-subunit complex in yeast but appears to be absent or drastically reduced in microsporidia. The UtpA binding site on the 5′ ETS is shared with Utp18, a subunit of another early binding biogenesis complex, UtpB. The potential absence of Utp18 and the entire UtpA complex (Fig. 1.10a) suggests microsporidia may contain a shorter 5′ ETS sequence, which recruits a minimal small subunit assembly platform. Alternatively, assembly factors may be too divergent to be identified.

Fig. 1.10

A reduced set of ribosome biogenesis factors and selected examples of assembly factor and expansion segment loss in microsporidia. (a) Presence and conservation of ribosome assembly factors in selected eukaryotes and microsporidia. The protein sequences were obtained by performing translated nucleotide blast (tblastn) or protein blast (blastp) searches with an E-value cutoff of 0.05, using the S. cerevisiae sequences and MicrosporidiaDB (Aurrecoechea et al. 2011) as database. For P. locustae and V. necatrix, protein sequences were obtained from local genome databases. For the non-microsporidian species, sequences were obtained from https://www.ncbi.nlm.nih.gov/. For phylogenetic tree calculation, see legend of Fig. 1.8. Many biogenesis factors display significant sequence similarity (e.g., WD40 domain proteins). It was therefore common for the same open reading frame to be identified as homologous to multiple different biogenesis factors. In such cases, we selected hits with the lowest E-value. It should be noted that the figure thus serves as only a guide to general trends for absent and present proteins, since annotations may be inaccurate. Proteins that were not identifiable (NI) are shown in cream. Biogenesis factors are clustered based on known or predicted binding regions within the 5’ ETS, SSU, ITS2, or LSU of the pre-rRNA. Conservation correlates between members of the same complex. Example complexes are labelled below (a) in shades of gray. (b-d) Structures of S. cerevisiae pre-ribosomal particles denoting selected maturation factors that are often absent in microsporidia, as highlighted in (a). Pre-SSU structures from PDB 5WLC (Barandun et al. 2017) (b), PDB 7AJU (Lau et al. 2021) (c), and a pre-LSU-structure from PDB 6C0F (Sanghai et al. 2018) (d) are displayed with expansion segments missing in V. necatrix, colored in shades of orange and yellow (SSU) or shades of blue and green (LSU), and selected biogenesis factors colored as in (a). These examples demonstrate a correlation between ES reduction and the loss of biogenesis factors that typically bind to those ESs

During pre-rRNA maturation, several eukaryotic expansion segments of the ribosomal RNA are bound and remodeled by assembly factors. In general, the absence of an assembly factor correlates with removal of its binding site in other organisms (Fig. 1.10). One striking example includes the SSU segments es3 and es6, which are bound and stabilized by the large HEAT repeat protein Utp20 (Fig. 1.10c). Es3 and es6 are the two largest small subunit expansion segments and have been completely lost or strongly reduced in microsporidia (Barandun et al. 2019; Ehrenbolger et al. 2020). Similarly, Utp20 appears to be absent in all microsporidian species. This suggests the primary role of Utp20 in chaperoning the maturation of these two expansion segments is no longer required in microsporidia. Similarly, the loss of h41 correlates with the loss of Utp30, an assembly factor binding to this rRNA element in pre-ribosomal particles (Fig. 1.10b). In the large subunit, ES7 is bound by two assembly factors, Rrp1 and Nsa1. Again, both the ES7 and the two assembly factors seem to be eliminated from microsporidian genomes.

A key step in large subunit maturation in S. cerevisiae involves processing of the ITS2 prior to nuclear export. Absence of ITS2, the spacer separating the two LSU rRNAs, explains the absence of many ribosome assembly factors binding this RNA region, such as Cic1, Rlp7, or the Las1 complex (Woolford and Baserga 2013). ITS1 processing in yeast is catalyzed by the essential ribozyme-protein complex RNAse MRP. While microsporidia still contain a highly reduced version of RNAse MRP (Zhu et al. 2006), ITS1 has been ablated to only 33 nt. It is unclear if this short ITS region can fold into a structure recognized by the minimized RNAse MRP, or if a simpler mechanism is used.

Apart from the mature ribosome structure, genomic data, and bioinformatics, very little is known about ribosome assembly in microsporidia. By studying the process in these minimal organisms, we can learn more about the still relatively unknown role of expansion segments during the assembly process in other eukaryotic organisms. The compaction of rRNAs together with the removal of transcribed spacer regions appears to have significantly affected the assembly process in microsporidia. A more thorough analysis of how expansion segment removal correlates with assembly factor loss will be required to understand the process in microsporidia and relate loss and compaction to a potential functional role in other eukaryotes.

1.7 Conclusion and Future Perspectives

Genome reduction and size appear to correlate with the degree of metabolic dependence on other organisms. Consequently, an obligate intracellular lifestyle provides a plausible explanation for the loss of redundant metabolic pathways and the invention of novel and more energetically efficient mechanisms of host exploitation. The drastic impact of genome compaction in microsporidia, however, has not only reduced the complexity of metabolic pathways but also affected intergenic regions, minimized gene sizes, and removed regulatory elements and features considered to be essential in eukaryotic organisms.

Genome erosion has significantly altered the microsporidian ribosomal DNA locus. By removing eukaryote-specific elements, such as ITS2 and nearly all expansion segments, the rDNA gene arrangement regressed to a prokaryote-like organization. The recent structural characterization of the microsporidian ribosome has illustrated the impact of genome reduction on the composition and assembly of this essential and ancient particle. It provided the surprising information that despite the loss of their rRNA binding site, almost all eukaryote-specific ribosomal proteins, albeit shortened, are still retained in the structure. Could limited access to primary metabolites precipitate a more compact ribosome? Are nucleotides more “rare” than amino acids, and could this be one reason why the rRNA is much more compacted than ribosomal proteins? Does the extensive rRNA loss affect the fidelity of the ribosome? Further studies are required to delineate the functional implications of ribosome compaction on protein synthesis and to reveal the suitability of ribosome-targeting antibiotics as translation inhibitors in microsporidia.

Microsporidia are of great interest in the fields of infection biology and comparative structural biology. They act as a reservoir for many unique and peculiar structures and have developed the most minimized versions of eukaryotic macromolecular complexes. Additional biochemical and structural studies in microsporidia not only will illuminate their own lifecycle but will also shed light on optional elements in many highly conserved cellular processes.