Functional annotation of a divergent genome using sequence and structure-based similarity.

BACKGROUND: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes. RESULTS: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes. CONCLUSION: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

Intraspecific polymorphism of rDNA among five Nosema bombycis isolates from different geographic regions in China.

The microsporidian Nosema bombycis is the causative agent of pébrine, a highly infectious disease of the silkworm Bombyx mori. Three regions of the multicopy rDNA gene were examined in order to investigate the relationships among five Nosema isolates from various regions of China. Ribosomal DNA alleles are present on each of the 18 chromosomes of N. bombycis and show a high degree of variation. In this study the small subunit (SSU) rDNA, internal transcribed spacer (ITS) and intergenic spacer (IGS) regions for up to 10 different rDNA copies from each N. bombycis isolate were cloned and sequenced. As expected we see greater polymorphism in the ITS region (88 variable sites in 179 nucleotides) and IGS (200 variable sites in 279 nucleotides) than in the SSU rDNA (24 variable sites in 1232 nucleotides). Phylogenetic analysis shows greater differences between alleles within an isolate than between the same alleles from different isolates. The data reveal two very different groups, one from the Sichuan province and the other with a broad distribution including four provinces in southeast China and Japan. The Sichuan isolate does not have any rDNA alleles with sequences identical to those in the other isolates, implying that it is a separate, non-intermixing, population or perhaps a separate species from the other isolates. In light of the polymorphic nature of the rDNA alleles in N. bombycis and their presence on every chromosome, the rDNA gene may be useful for understanding the movement and ultimately the source of pébrine infections.

SilkPathDB: a comprehensive resource for the study of silkworm pathogens.

Silkworm pathogens have been heavily impeding the development of sericultural industry and play important roles in lepidopteran ecology, and some of which are used as biological insecticides. Rapid advances in studies on the omics of silkworm pathogens have produced a large amount of data, which need to be brought together centrally in a coherent and systematic manner. This will facilitate the reuse of these data for further analysis. We have collected genomic data for 86 silkworm pathogens from 4 taxa (fungi, microsporidia, bacteria and viruses) and from 4 lepidopteran hosts, and developed the open-access Silkworm Pathogen Database (SilkPathDB) to make this information readily available. The implementation of SilkPathDB involves integrating Drupal and GBrowse as a graphic interface for a Chado relational database which houses all of the datasets involved. The genomes have been assembled and annotated for comparative purposes and allow the search and analysis of homologous sequences, transposable elements, protein subcellular locations, including secreted proteins, and gene ontology. We believe that the SilkPathDB will aid researchers in the identification of silkworm parasites, understanding the mechanisms of silkworm infections, and the developmental ecology of silkworm parasites (gene expression) and their hosts. Database URL: http://silkpathdb.swu.edu.cn.

The Genome of Nosema sp. Isolate YNPr: A Comparative Analysis of Genome Evolution within the Nosema/Vairimorpha Clade.

The microsporidian parasite designated here as Nosema sp. Isolate YNPr was isolated from the cabbage butterfly Pieris rapae collected in Honghe Prefecture, Yunnan Province, China. The genome was sequenced by Illumina sequencing and compared to those of two related members of the Nosema/Vairimorpha clade, Nosema ceranae and Nosema apis. Based upon assembly statistics, the Nosema sp. YNPr genome is 3.36 x 106bp with a G+C content of 23.18% and 2,075 protein coding sequences. An "ACCCTT" motif is present approximately 50-bp upstream of the start codon, as reported from other members of the clade and from Encephalitozoon cuniculi, a sister taxon. Comparative small subunit ribosomal DNA (SSU rDNA) analysis as well as genome-wide phylogenetic analysis confirms a closer relationship between N. ceranae and Nosema sp. YNPr than between the two honeybee parasites N. ceranae and N. apis. The more closely related N. ceranae and Nosema sp. YNPr show similarities in a number of structural characteristics such as gene synteny, gene length, gene number, transposon composition and gene reduction. Based on transposable element content of the assemblies, the transposon content of Nosema sp. YNPr is 4.8%, that of N. ceranae is 3.7%, and that of N. apis is 2.5%, with large differences in the types of transposons present among these 3 species. Gene function annotation indicates that the number of genes participating in most metabolic activities is similar in all three species. However, the number of genes in the transcription, general function, and cysteine protease categories is greater in N. apis than in the other two species. Our studies further characterize the evolution of the Nosema/Vairimorpha clade of microsporidia. These organisms maintain variable but very reduced genomes. We are interested in understanding the effects of genetic drift versus natural selection on genome size in the microsporidia and in developing a testable hypothesis for further studies on the genomic ecology of this group.

Molecular phylogeny of the Microsporidia: ecological, ultrastructural and taxonomic considerations.

The Microsporidia are a group of obligate intracellular parasites, now thought to be derived fungi. Presented here is a comparative small subunit rDNA (ssrDNA) analysis of 125 species of Microsporidia (sequences obtained from GenBank). This analysis shows that groups or clades are formed based largely on habitat and host. This result is supported by comparative molecular analyses of the past decade, and indicates that structural and ultrastructural characters are unreliable for distinguishing among higher-level microsporidian taxa. Our findings indicate the presence of five major clades of Microsporidia which group according to habitat. We present three new classes of Microsporidia based on natural phylogenetic groupings as illustrated by the ssrDNA analysis: Aquasporidia, Marinosporidia and Terresporidia. The names of the proposed classes reflect the habitat of each group. The class Aquasporidia, found primarily in freshwater habitats, is a paraphyletic group consisting of three clades. The Marinosporidia are found in hosts of marine origin and the Terresporidia are primarily from terrestrial environments.