Zhongyuan Shen

Sequence and Phylogenetic Analysis of SSU rRNA Gene of Five Microsporidia

The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosemaheliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia.

Phylogenetic analysis of complete rRNA gene sequence of Nosema philosamiae isolated from the lepidopteran Philosamia cynthia ricini.

ABSTRACT. The microsporidian Nosema philosamiae is a pathogen that infects the eri‐silkworm Philosamia cynthia ricini. The complete sequence of rRNA gene (4,314 bp) was obtained by polymerase chain reaction amplification with specific primers and sequencing. The sequence analysis showed that the organization of the rRNA of N. philosamiae was similar to the pattern of Nosema bombycis. Phylogenetic analysis of rRNA gene sequences revealed that N. philosamiae had a close relationship with other Nosema species, confirming that N. philosamiae is correctly assigned to the genus Nosema.

Identification of a protein interacting with the spore wall protein SWP26 of Nosema bombycis in a cultured BmN cell line of silkworm.

Nosema bombycis is a silkworm parasite that causes severe economic damage to sericulture worldwide. It is the first microsporidia to be described in the literature, and to date, very little molecular information is available regarding microsporidian physiology and their relationships with their hosts. Therefore, the interaction between the microsporidia N. bombycis and its host silkworm, Bombyx mori, was analyzed in this study. The microsporidian spore wall proteins (SWPs) play a specific role in spore adherence to host cells and recognition by the host during invasion. In this study, SWP26 fused with enhanced green fluorescence protein (EGFP) was expressed in BmN cells by using a Bac-to-Bac expression system. Subsequently, the turtle-like protein of B. mori (BmTLP) was determined to interact with SWP26 via the use of anti-EGFP microbeads. This interaction was then confirmed by yeast two-hybrid analysis. The BmTLP cDNA encodes a polypeptide of 447 amino acids that includes a putative signal peptide of 27 amino acid residues. In addition, the BmTLP protein contains 2 immunoglobulin (IG) domains and 2 IGc2-type domains, which is the typical domain structure of IG proteins. The results of this study indicated that SWP26 interacts with the IG-like protein BmTLP, which contributes to the infectivity of N. bombycis to its host silkworm.

Early responses of silkworm midgut to microsporidium infection – A Digital Gene Expression analysis

Host-pathogen interactions are complex processes, which have been studied extensively in recent years. In insects, the midgut is a vital organ of digestion and nutrient absorption, and also serves as the first physiological and immune barrier against invading pathogenic microorganisms. Our focus is on Nosema bombycis, which is a pathogen of silkworm pebrine and causes great economic losses to the silk industry. A complete understanding of the host response to infection by N. bombycis and the interaction between them is necessary to prevent this disease. Silkworm midgut infected with N. bombycis is a good model to investigate the early host responses to microsporidia infection and the interaction between the silkworm and the microsporidium. Using Digital Gene Expression analysis, we investigated the midgut transcriptome profile of P50 silkworm larvae orally inoculated with N. bombycis. At 6, 12, 18, 24, 48, 72, and 96 h post-infection (hpi), 247, 95, 168, 450, 89, 80, and 773 DEGs were identified, respectively. KEGG pathway analysis showed the influence of N. bombycis infection on many biological processes including folate biosynthesis, spliceosome, nicotinate and nicotinamide metabolism, protein export, protein processing in endoplasmic reticulum, lysosome, biosynthesis of amino acids, ribosome, and RNA degradation. In addition, a number of differentially expressed genes involved in the immune response were identified. Overall, the results of this study provide an understanding of the strategy used by silkworm as a defense against the invasion by N. bombycis. Similar interactions between hosts and pathogens infection may exist in other species.

Identification of sumoylated proteins in the silkworm Bombyx mori.

Small ubiquitin-like modifier (SUMO) modification (SUMOylation) is an important and widely used reversible modification system in eukaryotic cells. It regulates various cell processes, including protein targeting, transcriptional regulation, signal transduction, and cell division. To understand its role in the model lepidoptera insect Bombyx mori, a recombinant baculovirus was constructed to express an enhanced green fluorescent protein (eGFP)-SUMO fusion protein along with ubiquitin carrier protein 9 of Bombyx mori (BmUBC9). SUMOylation substrates from Bombyx mori cells infected with this baculovirus were isolated by immunoprecipitation and identified by LC–ESI-MS/MS. A total of 68 candidate SUMOylated proteins were identified, of which 59 proteins were functionally categorized to gene ontology (GO) terms. Analysis of kyoto encyclopedia of genes and genomes (KEGG) pathways showed that 46 of the identified proteins were involved in 76 pathways that mainly play a role in metabolism, spliceosome and ribosome functions, and in RNA transport. Furthermore, SUMOylation of four candidates (polyubiquitin-C-like isoform X1, 3-hydroxyacyl-CoA dehydrogenase, cyclin-related protein FAM58A-like and GTP-binding nuclear protein Ran) were verified by co-immunoprecipitation in Drosophila schneide 2 cells. In addition, 74% of the identified proteins were predicted to have at least one SUMOylation site. The data presented here shed light on the crucial process of protein sumoylation in Bombyx mori.

Functional characterization of an aquaporin from a microsporidium, Nosema bombycis

Microsporidia are a diverse group of eukaryotic organisms, capable of causing parasitic infections in both vertebrates and invertebrates. During the germination process, there is an increase in the osmotic pressure of microsporidian spores. As part of this study, we cloned a homologous aquaporin gene in Nosema bombycis, and named it Nosema bombycis aquaporin (NbAQP). Sequence analysis revealed that the NbAQP contains an open reading frame with a length of 750 bp and encodes a polypeptide of 249 amino acids. Amino acid sequence homology was greater than 50% that of five aquaporins from other microsporidian species. Indirect immunofluorescence (IFA) and immunogold electron microscopy showed NbAQP to be located predominantly in the spore wall of N. bombycis spores. The results of qRT-PCR analysis revealed that NbAQP expression remained high 0 h after inoculation and decreased sharply to 24 h, increased gradually from 2 days and peaked at 6 days. After expression of NbAQP in Xenopus laevis oocytes, it was observed that NbAQP can promote rapid penetration of water into oocytes. The associated permeation rate was 2–3 times that of the water-injected and uninjected oocytes. Antibody blocking experiments showed that the inhibition rate of spore germination was approximately 28% after antibody blocking. The difference in germination rate between the control group and the NbAQP group was significant (P < 0.05). This study shows for the first time that N. bombycis contains functional water channel proteins and provides a platform suitable for further research into the mechanisms underlying the regulation of NbAQP protein expression. Further study of NbAQP and their inhibitors may have significance for prevention of microsporidiosis.

Differentially expressed genes in the cuticle and hemolymph of the silkworm, Bombyx mori, injected with the fungus Beauveria bassiana

Abstract The most important pathogenic fungus of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is Beauveria bassiana (Balsamo-Crivelli ) Vuillemin (Hypocreales: Clavicipitaceae), which causes significant damage to sericulture production. Therefore, diagnosing fungal disease and developing new control measures are crucial to silk production. To better understand the responsive and interactive mechanisms between the host silkworm and this fungus, variations in silkworm gene expression were investigated using the suppression subtractive hybridization method following the injection of B. bassiana conidia. Two cDNA libraries were constructed, and 140 cDNA clones were isolated. Of the 50 differentially expressed genes identified, 45 (112 clones) were identified in the forward library, and 5 (28 clones) were identified in the reverse library. Expression profiling of six of these genes by quantitative polymerase chain reaction (qPCR) verified that they were induced by the fungal challenge. The present study provides insight into the interaction between lepidopteran insects and pathogenic fungi.

Complete sequence and gene organization of the Nosema heliothidis ribosomal RNA gene region.

ABSTRACT. By sequencing the entire ribosomal RNA (rRNA) gene region of Nosema heliothidis isolated from cotton bollworm (Helicoverpa armigera), we showed that its gene organization is similar to the type species, Nosema bombycis: the 5′‐large subunit rRNA (2,490 bp)‐internal transcribed spacer (192 bp)‐small subunit rRNA (1,232 bp)‐intergenic spacer (274 bp)‐5S rRNA (115 bp)‐3′. We constructed two phylogenetic trees, analyzed phylogenetic relationships, examined rRNA organization of microsporidia, and compared the secondary structure of small subunit rRNA with closely related microsporidia. The latter two features may provide important information for the classification and phylogenetic analysis of microsporidia.

In vitro expression and functional characterization of NPA motifs in aquaporins of Nosema bombycis

Nosema bombycis contains functional aquaporins (NbAQPs), which are key targets for exploring the mechanism of N. bombycis infection; however, the regulation of these NbAQPs remains unknown. The two highly conserved asparagine-proline-alanine sequences (NPA motifs) play important roles in AQP biogenesis. As part of this study, we constructed a series of NbAQP mutants (NbAQP_NPA1, NbAQP_NPA2, and NbAQP_NPA1,2) and expressed them in BmN cells. The results showed that mutations in either NPA motif or in both NPA motifs did not affect NbAQP expression in vitro. After expression in Xenopus laevis oocytes, those injected with wild-type NbAQP rapidly expanded, whereas oocytes injected with NbAQP_NPAs did not significantly change in size. The associated water permeability (pf) of NbAQP_NPAs was significantly reduced five–six times compared to that of wild-type NbAQP. These results indicated that NPA motifs are necessary for the water channel function of AQPs in N. bombycis. The present study shows for the first time that the NbAQP NPA motif has an impact on the water permeability of aquaporin in N. bombycis, thereby providing a platform for further research into the mechanisms underlying the regulation of NbAQP expression.

Identification and localization of SAS-6 in the microsporidium Nosema bombycis

The centriole in eukaryotes functions as the cells microtubule-organizing center (MTOC) to nucleate spindle assembly. The evolutionarily conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. Microsporidia possess the spindle plaque instead of centriole as their MTOC to nucleate spindle assembly. However, little is known about the components of spindle plaques in microsporidia. In our present study, we identified a SAS-6 protein in the microsporidium Nosema bombycis and named it as NSAS-6. The NSAS-6 gene contains a complete ORF of 1104 bp in length that encodes a 367-amino acid polypeptide. NSAS-6 consists of a conserved N-terminal domain and a coiled-coil domain. The high identity of SAS-6 homologous sequences from microsporidia indicates that SAS-6 is a conserved protein in microsporidia. Immunolocalization in sporoplasms, intracellular stages and mature spores showed that NSAS-6 probably localizes to the nucleus of N. bombycis and exists throughout the life cycle of N. bombycis. These results suggest that NSAS-6 is required in cell morphogenesis and division in N. bombycis. The function and structure of NSAS-6 should be the focus for further studies, which is essential to elucidate the role of SAS-6 in spindle plaque assembly.