| Microsporidia,a kind of obligate intracellular and unicellular eukaryotic parasite,which are composed of at least 200 genera and 1,500 species,can infect nearly all invertebrate and vertebrate,including humans.Several species of microsporidia have been listed as biological defense class B agents by the centers for disease control and prevention(CDC).Nosema bombycis,the first reported microsporidium,infects silkworm and transmits a highly fatal disease referred to as pébrine through horizontal and vertical transmissions,which causes huge economic losses in the silk-producing industry,but the molecular mechanism of its infection remains to be clarified.With the development of high-throughput sequencing technologies,the analysis of the microsporidia genome is greatly advanced.However,due to microsporidia cannot being cultured in cell-free medium and the lack of an effective genetic operating system,the biology and pathogenesis of microsporidia is seriously hindered.The infection of microsporidia starts from sporoplasm ejected from spores through the polar tube with the inside and outside the host cell,which is an important morphological and developmental stage of microsporidia.However,the characteristics,infectivity,and invasion mechanism of sporoplasm are still unclear.In order to study the sporoplasm,N.bombycis,which can germinate and eject the tube in vitro,is selected to isolate the sporoplasm in this study,analyzing the morphological and structural characteristics of the sporoplasm,and studying the cell-free culture and genetic transformation of sporoplasm.The main research results of this paper are as follows:1.Isolation and characteristic analysis of N.bombycis sporoplasmSeparation system of sporoplasm with KOH as germinating liquid is established by using the trait that N.bombycis can germinate and eject the polar tube in vitro.Light microscopic observation revealed that the sporoplasm was pear-shaped immediately after extrusion through the polar tube and was non-refractive.The sporoplasm diameter ranged from 2.7 to 5.2 ?m.After several minutes,all sporoplasm were spherical and averaged 3.64 ± 0.41 ?m in diameter(n = 80).CCK-8 assay result showed that sporoplasm were still metabolically active.TEM results show that sporoplasm was surrounded by smooth single cell membrane with the typical two nuclei located close to the cell membrane.Three nuclei were observed in a sporoplasm.The mean length of the nuclei of the mature spore(0.78 ± 0.07 ?m)and sporoplasm(0.59 ± 0.05 ?m)were measured by TEM(n = 50).These results suggested that the sporoplasm appeared to be in a flexible state,being conducive extruded from the polar tube.Meanwhile,the sporoplasm membrane can not be stained by Wheat Germ Agglutinin(WGA)and the nucleus can be labeled by Trypan Blue and Propidium Iodide(PI),indicating the sporoplasm membrane lacked lectin-binding sites and was strongly permeable.2.Gene expression characteristics of N.bombycis sporoplasmTranscriptome sequencing analysis of the mature spore(MS)and sporoplasm(SP)were performed by RNA-seq.After sequencing,146,865,590 and 135,344,316high-quality reads were obtained in the MS group and SP group,respectively,with matching rate of 82.54% and 83.18% to the reference genome.By calculating the FPKM value of expressed genes in the SP group and MS group,a total of 541 significantly different expressed genes were screened(padj < 0.05),among which 302 were up-regulated and 239 were down-regulated in sporoplasm.We found that the down-regulated expression gene is mainly key enzymes involved in trehalose synthesis metabolism,glycolysis,pentose phosphate and chitin synthesis.Then,ten transporter genes were up-regulated expression,including energy substance related transporters such as ADP/ATP carrier protein,mechanical sensitive ion channel proteins(MscS)and amino acid transporter.This result revealed that sporoplasm may inhibit their own metabolic activity and obtain the substances needed for proliferation through transporter proteins located on the surface of the plasma membrane.KEGG pathways enrichment analysis suggested that there is an obvious difference between the number of DEGs participated in the Ubiquitin system and Protein phosphatases.Then,we found that the ubiquitination level of total spore protein increased while the phosphorylation level of total spore protein decreased by Western blotting,suggesting that protein modification played an important role in the transformation of microsporidia from mature spores to sporoplasm.3.Interaction between N.bombycis sporoplasm and host cellsBm E cells are infected by sporoplasm staining the nucleus and plasma membrane.The result showed that after 6 h of infection,the sporoplasm could stick to the surface of host cells and is enveloped by protruding projections from the host cell,indicating that sporoplasm,an infectious state,may have the ability to infect host cells.After 18 h of infection,the fluorescent-labeled sporoplasm has invaded the host cell,thus confirming the infectivity of the sporoplasm.After 48 h of infection,early piriform spores with red fluorescence were observed in the host cells,suggesting that sporoplasm possesses the ability of infection and proliferation.The results of SEM and TEM showed that the sporoplasm is closely bound to the surface of the host cell,and the host cell membrane produces a distinct invagination and extends a pseudopod to enclose the sporoplasm.Then,biotin-streptomyces affinity system is used to isolate and identify the interaction molecules between sporoplasm and BmE cells.Through sequence analysis,32 candidate proteins in sporoplasm and 188 candidate proteins in BmE cell were obtained finally.KEGG analysis is performed to use these candidate proteins,and many genes are involved in the Membrane trafficking pathway in the silkworm data,which may be involved in the invasion of sporoplasm.After that,the function and subcellular localization of the candidate proteins was predicted and 6 genes of sporoplasm and 10 genes of Bm E cell was selected as candidate interaction molecules.Therefore,the final screening of Nb1 BD,Nb4BD and Nb6 BD proteins may interact with Bm4 AD and Bm6 AD,respectively,through yeast two-hybrid assays.Nb1 BD,Nb4BD and Nb6 BD have no clear functional annotation,Bm4 AD is ABCC family protein,and Bm6 AD is amino acid transporter protein4.In vitro culture and genetic transformation of N.bombycis sporoplasmIn order to establish the genetic operation system of microsporidia based on sporoplasm,cell-free culture of sporoplasm was carried out first.Based on the culture medium of silkworm cell line,the medium for sporoplasm was prepared.We found that after 6-day culture,many granular substances appear in the sporoplasm cytoplasm,and granulated substances gradually increase.However,there was no significant change in size of the sporoplasm at different time points,and no meront was observed.Meanwhile,after 3-day culture,we found that the nuclei of some sporoplasm were separated and moved toward the both ends,and multiple nuclei were found in the sporoplasm.Therefore,EdU was used to detect DNA replication of sporoplasm in culture,suggesting that DNA replication occurs in the sporoplasm at the 3rd and 6th day and the nucleus tended to separate.qPCR results proved that the genome copy number of sporoplasm begins to rise on the 6th day of culture,while the content of ATP in the culture solution decrease significantly within 1-5 day culture,but the content of glucose does not change significantly,thus indicating that the sporoplasm would absorb the ATP supporting for metabolism.Based on the established sporoplasm maintenance system in vitro,a genetic manipulation method for sporoplasma was established.Then,using RNAi technology to down-regulate the expression of Nobo ABCG1.1 gene.RT-qPCR and Western Blotting analysis revealed that the expression of Nobo ABCG1.1 gene is significantly decreased on the third day,indicating that sporoplasm could be used for RNAi analysis of genes.In order to realize the transgenic operation method of microsporidia,we constructed five vectors with different promoters successfully,including IE,A3,NB76,NB64 and NB451,to express EGFP and transfect the sporoplasm.Western Blotting detection result demonstrated that EGFP expression is detected in sporoplasm transfected with A3::EGFP vector,suggesting that the transient expression of exogenous genes was realized in the sporoplasm.To sum up,the present study represents the successful isolation of sporoplasm in microsporidia and will contribute to a comprehensive and in-depth understanding of sporoplasm at the morphological and molecular levels.The infectivity of sporoplasm was proved;the survival of sporoplasm in vitro and the transient expression of exogenous genes were achieved.This study provides important information for understanding the infection mechanism and establishing genetic manipulation system of microsporidia. |
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