Eiji Nagayasu

Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads

Although many de novo genome assembly projects have recently been conducted using high-throughput sequencers, assembling highly heterozygous diploid genomes is a substantial challenge due to the increased complexity of the de Bruijn graph structure predominantly used. To address the increasing demand for sequencing of nonmodel and/or wild-type samples, in most cases inbred lines or fosmid-based hierarchical sequencing methods are used to overcome such problems. However, these methods are costly and time consuming, forfeiting the advantages of massive parallel sequencing. Here, we describe a novel de novo assembler, Platanus, that can effectively manage high-throughput data from heterozygous samples. Platanus assembles DNA fragments (reads) into contigs by constructing de Bruijn graphs with automatically optimized k-mer sizes followed by the scaffolding of contigs based on paired-end information. The complicated graph structures that result from the heterozygosity are simplified during not only the contig assembly step but also the scaffolding step. We evaluated the assembly results on eukaryotic samples with various levels of heterozygosity. Compared with other assemblers, Platanus yields assembly results that have a larger scaffold NG50 length without any accompanying loss of accuracy in both simulated and real data. In addition, Platanus recorded the largest scaffold NG50 values for two of the three low-heterozygosity species used in the de novo assembly contest, Assemblathon 2. Platanus therefore provides a novel and efficient approach for the assembly of gigabase-sized highly heterozygous genomes and is an attractive alternative to the existing assemblers designed for genomes of lower heterozygosity.

The genomic basis of parasitism in the Strongyloides clade of nematodes

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families—families encoding astacin-like and SCP/TAPS proteins—is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

Paragonimiasis in Japan: A Twelve-year Retrospective Case Review (2001-2012)

OBJECTIVE Paragonimiasis, or lung fluke infection, is a food-borne parasitic disease caused by infection with trematodes belonging to the genus Paragonimus. Although paragonimiasis was once considered successfully controlled in the 1970s, new cases began to emerge in the late 1980s. To apprehend the current-day situation of the re-emergent cases of paragonimiasis in Japan, we conducted a retrospective review of 443 patients who were referred to our laboratory and diagnosed as having paragonimiasis during 2001-2012. METHODS Patients were diagnosed as having paragonimiasis based primarily on immunodiagnostic methods in addition to clinical, laboratory, and radiographic findings. Patient data were extracted from consultation sheets from attending physicians and were analyzed. RESULTS Majority of the patients were residents of Kyushu Island. However, a substantial number of cases were also from other parts of Japan. Immigrants (mostly from China, Thailand, and Korea) accounted for a quarter of the cases. Native Japanese contracted paragonimiasis by consuming wild boar meat or freshwater crabs, whereas immigrants contracted the infection almost exclusively by consumption of freshwater crabs. Eosinophilia and elevated serum IgE levels were found in around 80% of the patients. Parasite egg detection was documented only in 11.7% of the cases, showing the reliance on serological tests for diagnosing paragonimiasis in current clinical practice. CONCLUSION Paragonimiasis remains a public health issue in Japan, and the situation should be closely monitored.

Novel Thioredoxin-Like Proteins Are Components of a Protein Complex Coating the Cortical Microtubules of Toxoplasma gondii

ABSTRACT Microtubules are versatile biopolymers that support numerous vital cellular functions in eukaryotes. The specific properties of microtubules are dependent on distinct microtubule-associated proteins, as the tubulin subunits and microtubule structure are exceptionally conserved. Highly specialized microtubule-containing assemblies are often found in protists, which are rich sources for novel microtubule-associated proteins. A protozoan parasite, Toxoplasma gondii, possesses several distinct tubulin-containing structures, including 22 microtubules closely associated with the cortical membrane. Early ultrastructural studies have shown that the cortical microtubules are heavily decorated with associating proteins. However, little is known about the identities of these proteins. Here, we report the discovery of a novel protein, TrxL1 (for Thioredoxin-Like protein 1), and an associating complex that coats the cortical microtubules. TrxL1 contains a thioredoxin-like fold. To visualize its localization in live parasites by fluorescence, we replaced the endogenous TrxL1 gene with an mEmeraldFP-TrxL1 fusion gene. Structured illumination-based superresolution imaging of this parasite line produced a detailed view of the microtubule cytoskeleton. Despite its stable association with the cortical microtubules in the parasite, TrxL1 does not seem to bind to microtubules directly. Coimmunoprecipitation experiments showed that TrxL1 associates with a protein complex containing SPM1, a previously reported microtubule-associated protein in T. gondii. We also found that SPM1 recruits TrxL1 to the cortical microtubules. Besides SPM1, several other novel proteins are found in the TrxL1-containing complex, including TrxL2, a close homolog of TrxL1. Thus, our results reveal for the first time a microtubule-associated complex in T. gondii.

A novel C-type lectin identified by EST analysis in tissue migratory larvae of Ascaris suum

C-type lectins (CTLs) are a group of proteins which bind to carbohydrate epitopes in the presence of Ca2+, which have been described in a wide range of species. In this study, a cDNA sequence coding a putative CTL has been identified from the cDNA library constructed from the pig round worm Ascaris suum lung L3 (LL3) larvae, which was designated as A. suum C-type lectin-1 (As-CTL-1). The 510 nucleotide open reading frame of As-CTL-1 cDNA encoded the predicted 169 amino acid protein including a putative signal peptide of 23 residues and C-type lectin/C-type lectin-like domain (CLECT) at residue 26 to 167. As-CTL-1 was most similar to Toxocara canis C-type lectin-1 and 4 (Tc-CTL-1 and 4), and highly homologous to namatode CTLs and mammalian CTLs as well, such as human C-type lectin domain family 4 member G (CLECG4). In addition, As-CTL-1 was strongly expressed in tissue migrating LL3 and the L4 larvae, which were developmental larvae stages within the mammalian host. These results suggest that A. suum larvae might utilize As-CTL-1 to avoid pathogen recognition mechanisms in mammalian hosts due to it is similarity to host immune cell receptors.

Transcripts analysis of infective larvae of an intestinal nematode, Strongyloides venezuelensis.

Free-living infective larvae of Strongyloides nematodes fulfill a number of requirements for the successful infection. They need to endure a long wait in harsh environmental conditions, like temperature, salinity, and pH, which might change drastically from time to time. Infective larvae also have to deal with pathogens and potentially hazardous free-living microbes in the environment. In addition, infective larvae must recognize the adequate host properly, and start skin penetration as quickly as possible. All these tasks are essentially important for the survival of Strongyloides nematodes, however, our knowledge is extremely limited in any one of these aspects. In order to understand how Strongyloides infective larvae meet these requirements, we examined transcripts of infective larvae by randomly sequencing cDNA clones constructed from S. venezuelensis infective larvae. After assembling successfully sequenced clones, we obtained 162 unique singletons and contigs, of which 84 had been significantly annotated. Annotated genes included those for respiratory enzymes, heat-shock proteins, neuromuscular proteins, proteases, and immunodominant antigens. Genes for lipase, small heat-shock protein, globin-like protein and cytochrome c oxidase were most abundantly transcribed, though genes of unknown functions were also abundantly transcribed. There were no hits found against NCBI or NEMABASE4 for 37 (22.3%) EST out of the total 162 EST. Although most of the transcripts were not infective larva-specific, the expression of respiration related proteins was most actively transcribed in the infective larva stage. The expression of astacin-like metalloprotease, small heat-shock protein, S. stercoralis L3Nie antigen homologue, and one unannotated and 2 novel genes was highly specific for the infective larva stage.

Loss of a doublecortin (DCX) domain protein causes structural defects in a tubulin-based organelle of Toxoplasma gondii and impairs host-cell invasion

The human pathogen Toxoplasma gondii builds a remarkable structure, the conoid, from the ubiquitous cytoskeletal protein tubulin but arranges it into a completely novel form. Deleting the T. gondii gene for a DCX-domain protein disrupts the structure of the conoid and cripples the ability of the parasite to invade host cells.

Genome-Wide Analyses of Individual Strongyloides stercoralis (Nematoda: Rhabditoidea) Provide Insights into Population Structure and Reproductive Life Cycles

The helminth Strongyloides stercoralis, which is transmitted through soil, infects 30–100 million people worldwide. S. stercoralis reproduces sexually outside the host as well as asexually within the host, which causes a life-long infection. To understand the population structure and transmission patterns of this parasite, we re-sequenced the genomes of 33 individual S. stercoralis nematodes collected in Myanmar (prevalent region) and Japan (non-prevalent region). We utilised a method combining whole genome amplification and next-generation sequencing techniques to detect 298,202 variant positions (0.6% of the genome) compared with the reference genome. Phylogenetic analyses of SNP data revealed an unambiguous geographical separation and sub-populations that correlated with the host geographical origin, particularly for the Myanmar samples. The relatively higher heterozygosity in the genomes of the Japanese samples can possibly be explained by the independent evolution of two haplotypes of diploid genomes through asexual reproduction during the auto-infection cycle, suggesting that analysing heterozygosity is useful and necessary to infer infection history and geographical prevalence.

A possible origin population of pathogenic intestinal nematodes, Strongyloides stercoralis , unveiled by molecular phylogeny

Humans and dogs are the two major hosts of Strongyloides stercoralis, an intestinal parasitic nematode. To better understand the phylogenetic relationships among S. stercoralis isolates infecting humans and dogs and to assess the zoonotic potential of this parasite, we analyzed mitochondrial Cox1, nuclear 18S rDNA, 28S rDNA, and a major sperm protein domain-containing protein genes. Overall, our analyses indicated the presence of two distinct lineages of S. stercoralis (referred to as type A and type B). While type A parasites were isolated both from humans and dogs in different countries, type B parasites were found exclusively in dogs, indicating that the type B has not adapted to infect humans. These epidemiological data, together with the close phylogenetic relationship of S. stercoralis with S. procyonis, a Strongyloides parasite of raccoons, possibly indicates that S. stercoralis originally evolved as a canid parasite, and later spread into humans. The inability to infect humans might be an ancestral character of this species and the type B might be surmised to be an origin population from which human-infecting strains are derived.

Venison, another source of Paragonimus westermani infection

Paragonimiasis is a typical food-borne parasitic disease, endemic in most parts of Asia, with sporadic case reports from American and African countries. The major source of infection is undercooked freshwater crab or crayfish, though consumption of wild boar meat is also responsible for the infection in Japan, because wild boar is a paratenic host for Paragonimus westermani. Recently, living juveniles of P. westermani were isolated from muscle of a sika deer, Cervus nippon, in Japan, raising the possibility that venison has been another source of infection. In order to clarify the potential contribution of venison consumption to the occurrence of paragonimiasis, we analysed dietary histories of those paragonimiasis patients in whose diagnoses we were involved between 2001 and 2015. Among 380 patients, freshwater crab had been consumed by 208 patients, wild boar meat by 190, and wild deer meat by 76 patients before the onset of the disease. Overall contribution of wild deer meat was estimated to be 6.8% to 20.0%, although in Oita and Gifu Prefectures, where a substantial proportion of patients had consumed raw venison, the contribution of venison consumption was much higher (27.5 to 62.1% and 42.1 to 78.9% in Oita and Gifu Prefectures, respectively). We demonstrated P. westermani-specific antibodies in the sera of 4 out of 160 sika deer from Gifu Prefecture, strongly suggesting that these deer were infected with P. westermani.