The varying microsporidian genome: Existence of long-terminal repeat retrotransposon in domesticated silkworm parasite Nosema bombycis

International Journal for Parasitology 36 (2006) 1049–1056 www.elsevier.com/locate/ijpara

The varying microsporidian genome: Existence of long-terminal repeat retrotransposon in domesticated silkworm parasite Nosema bombycis q Jinshan Xu a, Guoqing Pan a, Lin Fang b, Jun Li b, Xiangjun Tian b, Tian Li a, Zeyang Zhou a,c,*, Zhonghuai Xiang a a

The Key Sericultural Laboratory of Agricultural Ministry of China, Southwest University, Beibei, Chongqing 400716, China b Beijing Institute of Genomics, Chinese Academy of Sciences, Peking 101300, China c Laboratory of Animal Biology, Chongqing Normal University, Chongqing 400047, China Received 9 January 2006; received in revised form 17 April 2006; accepted 25 April 2006

Abstract Microsporidia are a group of intracellular parasites with an extremely compact genome and there is no confirmed evidence that retroelements are parasitised in these organisms. Using the dataset of 200,000 genomic shotgun reads of the silkworm pebrine Nosema bombycis, we have identified the eight complete N. bombycis long-terminal repeat retrotransposon (Nbr) elements. All of the Nbr elements are Ty3/gypsy members and have close relationships to Saccharomycetes long-terminal repeat retrotransposons identified previously, providing further evidence of their relationship to fungi. To explore the effect of retrotransposons in microsporidian genome evolution, their distribution was characterised by comparisons between two N. bombycis contigs containing the Nbr elements with the completed genome of the human parasite Encephalitozoon cuniculi, which is closely related to N. bombycis. The Nbr elements locate between or beside syntenic blocks, which are often clustered with other transposable-like sequences, indicating that they are associated with genome size variation and syntenic discontinuities. The ratios of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site of the open reading frames among members of each of the eight Nbr families were estimated, which reveal the purifying selection acted on the N. bombycis long-terminal repeat retrotransposons. These results strongly suggest that retrotransposons play a major role in reorganization of the microsporidian genome and they might be active. The present study presents an initial characterization of some transposable elements in the N. bombycis genome and provides some insight into the evolutionary mechanism of microsporidian genomes.  2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: LTR retrotransposon; Evolution; Genome; Microsporidia

1. Introduction Microsporidia are obligate intracellular parasitic protozoa, which can infect a wide variety of organisms including vertebrate and invertebrate (particularly insects). Some q Note: Nucleotide sequences determined in this study have been deposited in GenBank, EMBL and DDBJ databases under the accession numbers: Nbr1–Nbr8, accession no. DQ444465–DQ444472; Nosema bombycis partial genomic DNA contig1–contig2, accession no. DQ445481–DQ445482. * Corresponding author. Address: The Key Sericultural Laboratory of Agricultural Ministry of China, Southwest University, Chongqing 400716, China. Tel.: +86 23 68251088; fax: +86 23 68251128. E-mail address: [email protected] (Z. Zhou).

species lead to several syndromes in immunocompetent hosts and cause opportunistic infections in acquired immunodeficiency syndrome (AIDS) patients (Desportes et al., 1985; Snowden, 2004). More than 1200 microsporidia species belonging to 150 genera have been reported (Wittner, 1999). However, the evolutionary origin of the microsporidia still puzzles biologists. Microsporidia were initially classified as an early branch from prokaryotes to eukaryotes (Vossbrinck et al., 1987). The rRNA of microsporidia is of prokaryotic type without a separate 5.8S rRNA (Vossbrinck and Woese, 1986). Microsporidia have a nucleus and an intracytoplasmatic membrane system, atypical golgi apparatus, while they lack peroxisomes and mitochondria.

0020-7519/$30.00  2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.04.010

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Recently, more and more evidence to support a fungal origin for microsporidia has emerged (Keeling et al., 2000; Hirt et al., 1999; Thomarat et al., 2004). In particular, it has been reported that several genes related to some mitochondrial functions, such as Fe–S cluster assembly, were found in microsporidia, suggesting that microsporidia have retained a mitochondrion-derived organelle (Williams et al., 2002). Even though a reverse transcriptase was reported in Vittaforma corneae and repeated sequences exist in Antonospora locustae (Mittleider et al., 2002; Fast et al., 2003), to our knowledge, no confirmed evidence of transposable elements have been identified in the microsporidian genome. Several types of retrotransposons have been identified in the genome of protozoan parasites and they are shown to be very similar to long interspersed nuclear element and short interspersed nuclear elements (Aksoy et al., 1987; Aksoy, 1991; Wickstead et al., 2003; Arkhipova and Morrison, 2001). More recently, it was found that Entamoeba histolytica has abundant retrotransposons dispersed in the genome, which accounts for about 6% of the genome (Bakre et al., 2005). For a fungal genome, however, the transposable elements most commonly isolated to date are retrotransposons and the number of distinct long-terminal repeat (LTR) retrotransposons families varies considerably among different genomes. For instance, the yeast Saccharomyces cerevisiae has five families of retrotransposons, which are defined as Ty elements (Boeke and Sandmeyer, 1991) and in Cryptococcus neoformans, 15 families of LTR retrotransposons were found with several elements transposed (Goodwin et al., 2001). As a member of the phylum microsporidia, Nosema bombycis is known as a pathogen of silkworm pebrine, which usually prevails in sericulture. Currently, the N. bombycis genome is being sequenced in our lab, which has approximately 15.3 Mb with 18 chromosomes (Kawakami, 1994). At the time of writing, the N. bombycis genomic database consists of 200,000 shotgun reads, totalling sixfold coverage of the 15.3 Mb haploid genome. It is of value to identify retrotransposons in this genome using the genomic data available. By bioinformatic approaches, we identified eight LTR retrotransposons and designated them as Nbr1–Nbr8 (N. bombycis retrotransposons), respectively. Genome size of microsporidia varies from 2.3 to 19.5 Mb (Street, 1994; Biderre et al., 1994; 1995; Peyretaillade et al., 1998). The Encephalitozoon cuniculi genome with 2.9 Mb was published and characterised as genome compaction (Katinka et al., 2001), and many biosynthetic pathways were lost, indicating a strong reliance on the host cell for energy and nutrients. The organization of the A. locustae genome with 685 Kb size also displays the same characteristic with genome compaction and stability (Slamovits et al., 2004). Genome evolution and size variation in eukaryotic organisms are profoundly influenced by the activity of retrotransposons. However, there is still no confirmed evidence of this in microsporidia. Here, we present the retroelements analyses in N. bombycis, which bring about a new view on variation of microsporidian genomes.

2. Materials and methods 2.1. Characterization of full-length Nbr LTR retrotransposons Two hundred thousand initial random shotgun reads were obtained by constructing the plasmid library of the N. bombycis genomic DNAs, in which the sequencing N. bombycis isolate CQ1 was isolated from infected silkworms in Chongqing, China, conserved in China Veterinary Culture Collection Center (CVCC number 102059). All reads were assembled to generate the dataset of contigs using the RePS program (Wang et al., 2002). By mining this N. bombycis genomic dataset, Nbr elements were identified in several steps. The elements were initially detected by performing TBLASTN (http://www.ncbi.nlm.nih.gov/ BLAST/) searches using the reverse transcriptase (RT) amino acid sequences of retrotransposons identified previously in other species. Nucleotide sequences with homology to the RTs were then analysed to reveal the presence of LTR using the bl2seq program (http://www.ncbi.nlm.nih.gov/BLAST/). In addition, several characteristics such as target-site duplications (TSD) and primer-binding sites (PBS) were singled out manually. Finally, other retrotransposon domains of each intact open reading frame (ORF) were determined. To estimate the abundance of those families, we use the local BLASTN programs run under the sensitive condition by using the eight complete LTR retrotransposons elements as repbase to compare the dataset of contigs. All high-scoring pairs (HSPs) with the length longer than 100 nucleotides and identities higher than 80% were used to define different copies in the same family. 2.2. Multiple sequence alignments and phylogenetic analyses The RT domains of the eight Nbr elements were identified according to criteria described previously (Xiong and Eickbush, 1990) and these RT domains and other protein domains were aligned with the previously reported sequence. ClustalX software was used to generate amino acid sequence alignments (Thompson et al., 1997). The neighbor-joining approach implemented in software MEGA 3.0 (Kumar, et al., 2004) was used for reconstructing the phylogenetic tree and 100 data replicates were generated to analyse bootstrap values. 2.3. Ka/Ks analysis Previous studies show that if elements display higher genomic replication rates, they can possess a selective advantage over less active elements. So, selective forces impact on the shape of the retrotransposon population at the genomic level between elements (Jordan and McDonald, 1999). To evaluate the effects of selection operating at the genomic level among Nbr elements, the ratio of the number of non-synonymous substitutions per non-synonymous site (Ka) to the number of synonymous substitutions

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per synonymous site (Ks) was calculated. When Ka/Ks1, it means that selection eliminates deleterious mutations, keeping the protein as it is (purifying selection), and when Ka/Ks1, it strongly indicates positive selection (Hurst, 2002). After having investigated the abundance of each of eight Nbr element families in the genome, the average pairwise values of Ka and Ks were calculated by the yn00 program in PAML (Yang and Nielsen, 2000) for Nbr element ORFs of each family.

Nbr3–Nbr7 all contain a CX2HX4HX4C motif, which is similar to the CX2CX4HX4C motif, however, a cysteine was substituted for a histidine. Further studies are needed to determine whether this common Cys motif is functional for the putative domain. Alignment of the protease and RNaseH domain shows a conserved D (T/S) G motif, a conserved DAS motif and conservation of several other amino acids residues compared to the retroelements of other species (Fig. 1).

2.4. Nucleotide sequence accession numbers

3.2. Phylogenetic characterization of Nbr elements in N. bombycis

Nucleotide sequences determined in this study have been deposited in the GenBank, EMBL and DDBJ databases. Genbank accession numbers are as follows: Nbr1–Nbr8, DQ444465–DQ444472; N. bombycis partial genomic DNA contig 1, DQ445481; N. bombycis partial genomic DNA contig 2, DQ445482. 3. Results 3.1. Isolation and characterization of Nbr elements in N. bombycis The present study was based on the dataset of N. bombycis genomic sequences which consists of 200,000 genomic shotgun reads and approximately 6· coverage of the genome. Eight complete LTR retrotransposon families of N. bombycis were found in the phylum Microsporidia (Table 1). The eight complete Nbr elements have lengths of 3–4 kb, consist of a large and intact ORF (1000 amino acids), short 100–200 bp LTRs and five or six base TSDs. Detailed analyses of predicted ORFs show that Nbr2– Nbr4 and Nbr6–Nbr8 have the putative Cys motifs, protease, RT, RNaseH and integrase (Fig. 1). For the ORF of Nbr5, only one domain, RT, was found. Multiple sequence alignments of Cys motifs, protease, RNase H and integrase show that Nbr2 and Nbr8 have a common Cys motif CX2CX4HX4C, which represents a structure similar to that in other species (Covey, 1986; Copeland et al., 2003), and Table 1 Characteristics of long-terminal repeat retrotransposons from the Nosema bombycis genome Family

LTR length (bp)

Inserted element length (bp)

LNI (%)

TSD

LTR end dinuceotides

Nbr1 Nbr2 Nbr3 Nbr4 Nbr5 Nbr6 Nbr7 Nbr8

227 194 181 120 65 123 142 136

4021 4060 4172 4153 3389 3930 3924 3568

98.5 99.0 98.1 100 100 98.8 97.2 100.0

AAATT/AAATT CCATTG/CCATTG AATGAA/AATGAG ND ND ATTAT/ATTAT ATAGT/ATAGT GTATC/GTATC

TG/CA TG/CA TG/CA ND TG/CA TG/CA TG/CA TG/CA

TSD, target site duplications; LNI, LTR nucleic acid identity; ND, not determined.

To determine the evolutionary origin of these eight novel retrotransposons, phylogenetic trees were reconstructed. Sequences from the RT domains of several members of the LTR group of retrotransposons and some retroviruses were aligned using the ClustalX software and a phylogenetic tree was reconstructed by the neighbor-joining method (Fig. 2). The result strongly suggests that all Nbr elements belong to Ty3/gypsy group and these elements always form several distinct clades, which is consistent with the analysis that the RT domain of elements among different clades shows high divergence at the amino acid level (data not shown). In order to determine which are closely related to N. bombycis among different species, a search of Genbank was done using the RT of these elements as queries. The results suggest that most elements are more closely related to that of fungi and these elements usually show about 50% similarity with that of Saccharomycetes at the amino acid level. Nbr6, Nbr7 and Nbr8 share 51, 48 and 52% similarity with Ty16 elements of Yarrowia lipolytica (GenBank accession no. CAG34127) at the amino acid level, respectively; Nbr2 and Nbr3 show 49 and 48% homology to the TyB3p element of Saccharomyces exiguous (GenBank accession no. AAO27306), respectively. Nbr1 shows 50% similarity with the element of Candida glabrata at the amino acid level (GenBank accession no. AAT76628). Meanwhile, most of these elements also share 42–48% identity with that of Schistosoma mansoni, such as Saci-2 and Saci-3(GenBank accession nos. BK004069, BK004070) and this similarity is lower than that of Saccharomyces. 3.3. Purifying selection We analysed the sequence variation by calculating the pairwise values of Ka and Ks for the ORFs among members of each of the eight Nbr families. Table 2 shows that all the Ka/Ks ratios are significantly less than 1, indicating that purifying selection has acted on the Nbr elements in the N. bombycis genome. 3.4. Locations of the Nbr elements in the N. bombycis genome To better understand the role of these elements in genome evolution, we investigated the locations of sever-

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Fig. 1. The presentation of structure and multiple alignments for several novel Nosema bombycis LTR retrotransposons. (A) Boxes with triangles represent the LTRs, and shaded boxes represent the ORFs. The probable locations of the various domains within the ORFs are indicated above each element. (B) ClustalX was used to align for conserved regions of gag, protease, RT, RNase H, and integrase. No apparent domain but RT was found for Nbr5. Arrows above the each amino acid point to the conserved motifs previously described. The multiple alignments were shaded by boxshade 3.2.

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genomes (Katinka et al., 2001), it was found that the Nbr elements inserted between two different syntenic blocks in contig 1 or inserted beside one particular syntenic block in contig 2, which were bounded with other transposable-like sequences or other unknown genes with long ORFs (Fig. 3). These results suggest that the Nbr elements are often associated with syntenic discontinuities, and they should be one driving factor of genome variation, including gene acquisition or gene rearrangement. 4. Discussion

Fig. 2. Neighbor-joining tree for the eight Nbr elements and homologues in other related species. The phylogenetic tree was reconstructed based on reverse transcriptase domains. The bootstrap value (100 replicates) is presented in bold at each branching node. Names in bold are N. bombycis elements involved in this study. The tree was generated with the MEGA3 software. The GenBank sequences and accession numbers used for construction of alignments and phylogenetic tree are as follows: TyB3p, AAO27306; Tse1, AAK56444; Tyl6, CAG34127; CgRT, AAT76628; Saci2, BK004069; Saci-3, BK004070; TPA, DAA04495; BEV, BAA89659; RSV, NP_056886; HIV, AAB50259; Ninja, AB042129; Pao, L09635; Moose, AF060859; Bel, U23420; Zam, CAA04050; Kabuki, AB032718; Gypsy, P10401; Ted, M32662; Mag, X17219; Cer1, U15406; Mdg3, CAA65152; 1731, X07656; Atcopia, BAB09923; Dmcopia, CAD27357. Table 2 Average non-synonymous (Ka) and synonymous (Ks) substitution rates for the eight Nbr families Element

Kaa

Ksa

Ka/Ks

Nbr1 Nbr2 Nbr3 Nbr4 Nbr5 Nbr6 Nbr7 Nbr8

0.036 0.085 0.082 0.074 0.030 0.043 0.055 0.111

0.189 0.292 0.331 0.663 0.917 1.589 0.319 0.625

0.581 0.272 0.279 0.434 0.139 0.191 0.226 0.181

a The values are averages of Ka’s and Ks’s for comparisons of each complete Nbr coding sequence with other member sequences of the same family.

al Nbr elements in N. bombycis contig 1 and contig 2, whose sizes are 25.8 and 20.4 kb, respectively. By the analysis of gene synteny between the N. bombycis and E. cuniculi

Few retroelements were previously found in microsporidia (Hinkle et al., 1997; Mittleider et al., 2002); however, we identified several complete LTR retrotransposons in N. bombycis and confirmed that the microsporidian genome still possesses the retrotransposon. Existence of the Nbr also suggests that the N. bombycis genome is more complex and the content is more abundant than the microsporidian E. cuniculi genome. All the elements belong to the Ty3/gypsy group through phylogenetic analysis and RTs of the Nbr elements share higher similarity to that of Saccharomycetes than that of other species, even though these identities are below 60%, respectively. Previous studies showed that microsporidia are related to fungi and now our results offer new proof of this. Transposable elements have mostly been regarded as parasitic DNA (Orgel and Crick, 1980; Doolittle and Sapienza, 1980) and what we are interested in is when these parasitic DNAs have invaded the Saccharomycetes or microsporidia genomes, and whether they inserted into these genomes at the same time or at different times. This should be associated with the evolutionary relationship between microsporidia and Saccharomycetes. It should be pointed out that microsporidia was considered an amitochondrial intracellular parasite (Peyretaillade et al., 1998b; Wang et al., 2002 ), but close relationships between N. bombycis and other amitochondrial parasites, such as Trichomonas vaginalis, E. histolytica and Trypanosoma cruzi (Tovar et al., 1999; Leon–Avila and Tovar, 2004), were not determined by analyzing the LTR retrotransposons. We found that elements of the Schistosoma mansoni parasite display some similarity to the Nbr elements, however, whose phylogenetic position is far from microsporidia. Perhaps, this is due to few elements identified previously in the related amitochondrial parasites. Thus, our results provide useful information for retroelement research of other amitochondrial organisms in the future. Purifying selection against insertions appears to limit the contribution of retrotransposon DNA to genome size expansion in some compact genomes (Pereira, 2004). The Ka/Ks analyses of the Nbr elements show that purifying selection acted on the Nbr elements in the N. bombycis genome, indicating that most of the N. bombycis LTR retrotransposons might be active. Genome elimination and compaction in microsporidia suggest a heavy reliance on the host, so these extrinsic inserts (retrotransposons), which are always subject to metabolism and biosynthesis, should undergo selective

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Fig. 3. The distribution of the N. bombycis retrotransposons in contig 1 and contig 2, which the sizes are 25.8 and 20.4 kb, respectively. (A) The Nbr elements inserted between the two gene syntenic blocks in the Nosema bombycis contig 1. (B) The Nbr elements inserted beside the syntenic block in contig 2. The synteny was evaluated by combining the N. bombycis and E. cuniculi chromosome VIII, chromosome XI, and chromosome VI. Genes were identified by similar search of NCBI database using the web-based BLAST interface (http://www.ncbi.nlm.nih.gov/blast). The E. cuniculi genes were retrieved from GenBank and labeled according to their designation in the E. cuniculi genome (for instance, ECU08_1520 represents chromosomeVIII, locus 150). The rectangles denote the gene distributed in genome, and transcriptional orientations are indicated by arrows. The scale is shown in thin lines, with one scale representing 1 kb.

pressure in the N. bombycis genome adapted to a parasitic way of life. Our result is consistent with this opinion. Some of the characteristics of microsporidian genomes were revealed by comparative genomic analyses, such as genome structure conservation (Keeling et al., 2005). Comparative genome analyses of E. cuniculi and A. locustae showed that these two microsporidian species had retained an unexpected degree of synteny and the phylogenetic analysis of microsporidia has suggested that the N. bombycis is more closely related to E. cuniculi than to A. locustae (Slamovits et al., 2004), so comparative genome analysis between the N. bombycis and E. cuniculi is suitable for studying microsporidian genomic variation and evolution. It seems that locations of Nbr elements in two contigs are just the breakpoint of syntenic block compared with the corresponding chromosomes of E. cuniculi. Did a retrotransposon event occurr in genomic expansion from E. cuniculi to the N. bombycis or were such elements lost in genome compaction from E. cuniculi to N. bombycis? More evidence is needed to confirm this genomic evolutionary process. We have not found Nbr elements inserted into the interior of one syntenic block at current genome depth. According to a recent study, there is strong selective pressure to maintain the core gene order and to keep directional gene clusters intact in several trypanosomatid parasitic protozoa

(E1-Sayed et al., 2005), so it was difficult for these retroelements to insert into the interior of gene clusters and this would cause less deleterious effects to the host genome. Some questions remain to be answered. The Encephalitozoon cuniculi genome is compact and stable and no transposable elements were found, however, we have identified the complete LTR retrotransposons in N. bombycis. It implies that in some stages of microsporidian evolution, some species was invaded by retroelements, while other species was not. However, what causes this diversity? Maybe it is associated with their living conditions, such as their host, and the pressure of immunity. A reasonable answer could be obtained after the fine map of the N. bombycis genome has been completed. It should be pointed out that the type and number of Nbr elements should certainly increase after global analyses of repetitive elements in the N. bombycis genome. Our findings provide the initial overview of retroelements at a genomic level but shed light on the evolution of microsporidian genome. Acknowledgements This work was supported by the National Basic Research Program of China under grant no. 2005CB121000

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