Microsporidian Nosema bombycis hijacks host vitellogenin and restructures ovariole cells for transovarial transmission.

Microsporidia are a group of obligate intracellular parasites that infect almost all animals, causing serious human diseases and major economic losses to the farming industry. Nosema bombycis is a typical microsporidium that infects multiple lepidopteran insects via fecal-oral and transovarial transmission (TOT); however, the underlying TOT processes and mechanisms remain unknown. Here, we characterized the TOT process and identified key factors enabling N. bombycis to invade the ovariole and oocyte of silkworm Bombyx mori. We found that the parasites commenced with TOT at the early pupal stage when ovarioles penetrated the ovary wall and were exposed to the hemolymph. Subsequently, the parasites in hemolymph and hemolymph cells firstly infiltrated the ovariole sheath, from where they invaded the oocyte via two routes: (I) infecting follicular cells, thereby penetrating oocytes after proliferation, and (II) infecting nurse cells, thus entering oocytes following replication. In follicle and nurse cells, the parasites restructured and built large vacuoles to deliver themselves into the oocyte. In the whole process, the parasites were coated with B. mori vitellogenin (BmVg) on their surfaces. To investigate the BmVg effects on TOT, we suppressed its expression and found a dramatic decrease of pathogen load in both ovarioles and eggs, suggesting that BmVg plays a crucial role in the TOT. Thereby, we identified the BmVg domains and parasite spore wall proteins (SWPs) mediating the interaction, and demonstrated that the von Willebrand domain (VWD) interacted with SWP12, SWP26 and SWP30, and the unknown function domain (DUF1943) bound with the SWP30. When disrupting these interactions, we found significant reductions of the pathogen load in both ovarioles and eggs, suggesting that the interplays between BmVg and SWPs were vital for the TOT. In conclusion, our study has elucidated key aspects about the microsporidian TOT and revealed the key factors for understanding the molecular mechanisms underlying this transmission.

New insights into Microsporidia polar tube function and invasion mechanism.

Microsporidia comprise a large phylum of single-cell and obligate intracellular parasites that can infect a wide range of invertebrate and vertebrate hosts including humans. These fungal-related parasites are characterized by a highly reduced genome, a strong energy dependence on their host, but also by their unique invasion organelle known as the polar tube which is coiled within the resistant spore. Upon appropriate environmental stimulation, the long hollow polar tube (ranging from 50 to 500 µm in length) is extruded at ultra-fast speeds (300 µm/s) from the spore acting as a harpoon-like organelle to transport and deliver the infectious material or sporoplasm into the host cell. To date, seven polar tube proteins (PTPs) with distinct localizations along the extruded polar tube have been described. For example, the specific location of PTP4 and PTP7 at the tip of the polar tube supports their role in interacting with cellular receptor(s). This chapter provides a brief overview on the current understanding of polar tube structure and dynamics of extrusion, primarily through recent advancements in cryo-tomography and 3D reconstruction. It also explores the various mechanisms used for host cell invasion. Finally, recent studies on the structure and maturation of sporoplasm and its moving through the tube are discussed.

Down-Regulation of Lipid Metabolism in the Hepatopancreas of Shrimp Litopenaeus vannamei upon Light and Heavy Infection of Enterocytozoon hepatopenaei: A Comparative Proteomic Study.

Enterocytozoon hepatopenaei (EHP) is the pathogen of hepatopancreatic microsporidiosis (HPM) in shrimp. The diseased shrimp Litopenaeus vannamei exhibits a slow growth syndrome, which causes severe economic losses. Herein, 4D label-free quantitative proteomics was employed to analyze the hepatopancreas of L. vannamei with a light (EHPptp2 < 103 copies/50 ng hpDNA, L group) and heavy (EHPptp2 > 104 copies/50 ng hpDNA, H group) load of EHP to better understand the pathogenesis of HPM. Exactly 786 (L group) and 1056 (H group) differentially expressed proteins (DEPs) versus the EHP-free (C group) control were mainly clustered to lipid metabolism, amino acid metabolism, and energy production processing. Compared with the L group, the H group exhibited down-regulation significantly in lipid metabolism, especially in the elongation and degradation of fatty acid, biosynthesis of unsaturated fatty acid, metabolism of α-linolenic acid, sphingolipid, and glycerolipid, as well as juvenile hormone (JH) degradation. Expression pattern analysis showed that the degree of infection was positively correlated with metabolic change. About 479 EHP proteins were detected in infected shrimps, including 95 predicted transporters. These findings suggest that EHP infection induced the consumption of storage lipids and the entire down-regulation of lipid metabolism and the coupling energy production, in addition to the hormone metabolism disorder. These were ultimately responsible for the stunted growth.

Septin homologs cooperating in the Proliferative Stage of Microsporidia Nosema bombycis.

The single-celled pathogen Nosema bombycis, that can infect silkworm Bombyx mori and other lepidoptera including Spodoptera, is the first identified Microsporidia which has diplokaryotic nuclei throughout the life cycle. Septin proteins can form highly ordered filaments, bundles or ring structures related to the cytokinesis in fungi. Here, three septin proteins (NbSeptin1, NbSeptin2 and NbSeptin3) from Nosema bombycis CQ I are described. These proteins, appear to be conserved within the phylum Microsporidia. NbSeptins transcripts were detected throughout the pathogen developmental cycle and were significantly enhanced from second days of infection, which lead to our hypothesis that NbSeptins play a role in merogony. Immunofluorescence assay (IFA) revealed a broad distribution of NbSeptins in meronts and partly co-localization of NbSeptins. Interestingly, in some of meronts, NbSeptin2 and NbSeptin3 showed localization between the nuclei of the diplokaryon. Yeast two-hybrid and co-immunoprecipitation analysis verified that NbSeptins can interact with each other. Our findings suggest that NbSeptins can cooperate in the proliferation stage of Nosema bombycis and contribute towards the understanding of the rols of septins in microsporidia development.

Nbseptin2 Expression Pattern and Its Interaction with NbPTP1 during Microsporidia Nosema bombycis Polar Tube Extrusion.

Nosema bombycis (Nb) is a deadly species of microsporidia capable of causing pébrine, leading to heavy losses in sericulture. Germination is an important biological event in the invasion process of microsporidia. Septins, a family of membrane-associated proteins, play a critical role in tissue invasion and have been recognized as a virulence factor in numerous pathogens. Previous work in our laboratory has shown that Nosema bombycis septin2 (Nbseptin2) interacts with subtilisin-like protease 2 (NbSLP2). Herein, we found that Nbseptin2 was mainly associated with the plasma membrane in spores. Following spore germination, Nbseptin2 was found to co-localize with polar tube protein 1 (NbPTP1) at the polar cap and proximal zone of the polar tube. Co-immunoprecipitation and yeast two-hybrid analysis further confirmed that Nbseptin2 interacted with NbPTP1. The translocation and interaction of Nbseptin2 in the spores suggest that Nbseptin2 may play a significant role in microsporidia polar tube extrusion process. Our findings improve understanding of the mechanisms underlying microsporidia germination.

Expression of Nosema bombycis polar tube protein 1 in lepidopteran Sf9 cells and its effect on microsporidian proliferation.

Nosema bombycis, the first identified microsporidium, causes heavy losses to the sericulture industry in China. During infection, microsporidia discharge a long and hollow polar tube, which delivers the sporoplasm into host cells. Polar tube protein 1 was the major component on the polar tube. Previously, we expressed the polar tube protein 1 from Nosema bombycis (NbPTP1) intercellularly in Drosophila S2 cells. Here, the microsporidian protein was expressed in Lepidopteran Sf9 cells. During heterologous expression, NbPTP1 protein was secreted and glycosylated. Microsporidian proliferation decreased in NbPTP1-expressing Sf9 cells. This confirms that NbPTP1 protein can interact with the host cell membrane receptor protein to facilitate microsporidian invasion.

Proliferation characteristics of the intracellular microsporidian pathogen Nosema bombycis in congenitally infected embryos.

Nosema bombycis is an obligate intracellular pathogen that can be transmitted vertically from infected females to eggs, resulting in congenital infections in embryos. Here we investigated the proliferation characteristics of N. bombycis in silkworm embryos using a histopathological approach and deep RNA sequencing. We found that N. bombycis proliferated mainly around yolk granules at the early stage of the embryonic development, 1-2 days post oviposition (dpo). At 4-6 dpo, a portion of N. bombycis in different stages adjacent to the embryo were packaged into the newly formed intestinal lumen, while the remaining parasites continued to proliferate around yolk granules. In the newly hatched larvae (9 dpo), the newly formed spores accumulated in the gut lumen and immediately were released into the environment via the faeces. Transcriptional profiling of N. bombycis further confirmed multiplication of N. bombycis throughout every stage of embryonic development. Additionally, the increased transcriptional level of spore wall proteins and polar tube proteins from 4 dpo indicated an active formation of mature spores. Taken together, our results have provided a characterization of the proliferation of this intracellular microsporidian pathogen in congenitally infected embryos leading to vertical transmission.

The role of microsporidian polar tube protein 4 (PTP4) in host cell infection.

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

Development of a nucleic acid lateral flow strip for rapid, visual detection of Nosema bombycis in silkworm eggs.

Nosema bombycis, the pathogen of silkworm pébrine, causes enormous economic losses to sericulture. As such, quarantine of commercial silkworm eggs represents an important safeguard to the silkworm industry. Here, we established a user-friendly detection system based on a nucleic acid lateral flow strip (NAFLS) that combines polymerase chain reaction (PCR) and a colloidal gold strip. PCR primers were designed based on the sequence of LSU rDNA of N. bombycis and has favourable specificity for common microsporidian isolates in silkworms. The forward and reverse primers were labeled on the 5' end with biotin and carboxyfluorescein (FAM), respectively. Genomic DNA was extracted from egg samples and was used as a template for PCR, followed by subsequent detection by NALFS. The detection limit of purified N. bombycis genomic DNA was 1 pg, 100× more sensitive than that of agarose gel electrophoresis (AGE). Furthermore, the sensitivity of detection of simulated "infected" silkworm eggs was 10-100× higher than that of AGE. NALFS detected infection in 27 of 29 samples of silkworm eggs oviposited by female moths infected in lab; ≥2% infected eggs per batch are detected as positive, while ≥40% infected eggs per batch are required for detection by AGE. Collectively, NALFS is easy to use and has great potential for widespread use in the detection of N. bombycis in silkworm egg production.

Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall.

All microsporidia possess a unique, highly specialized invasion mechanism that involves the polar tube and spore wall. The interaction between spore wall proteins (SWPs) and polar tube proteins (PTPs) in the formation, arrangement, orderly orientation, and function of the polar tube and spore wall remains to be determined. This study was undertaken to examine the protein interactions of Nosema bombycis SWP7 (NbSWP7), NbSWP9, and PTPs. Coimmunoprecipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and yeast two-hybrid data demonstrated that NbSWP9, but not NbSWP7, interacts with NbPTP1 and NbPTP2. Furthermore, immunoelectron microscopy (IEM) showed that NbSWP9 was localized mainly in the developing polar tube of sporoblasts, while NbSWP7 was found randomly in the cytoplasm. However, both NbSWP9 and NbSWP7 were located in the polar tube and spore wall of N. bombycis mature spores. The reason why NbSWP7 was localized to the polar tube may be due to the interaction between NbSWP9 and NbSWP7. Interestingly, the majority of NbSWP9, but not NbSWP7, accumulated in the beginning part of the extruded polar tube and the ruptured spore wall called the anchoring disk (AD) when the mature spores germinated under weak-alkaline environmental stimulation. Additionally, anti-NbSWP9 antibody reduced spore germination in a dose-dependent manner. In conclusion, our study further confirmed that NbSWP9 is a scaffolding protein that not only anchors and holds the polar tube but also tethers the polar tube to the spore wall.

Comparative proteomic analysis of differentially expressed proteins in the Bombyx mori fat body during the microsporidia Nosema bombycis infection.

Nosema bombycis is an obligate intracellular parasite, which can cause pébrine disease. To investigate the effects of N. bombycis infection, 5th-instar silkworms were challenged with N. bombycis isolate CQ1, and two-dimensional gel electrophoresis analysis was performed to analyze the differentially expressed proteins in infected and uninfected silkworm fat bodies 1, 2, 4, 6 and 8days post-infection (dpi). 46 differentially expressed proteins were identified at the 5 time points using MALDI-TOF/TOF MS. The changed proteins mainly involved in immune response, energy metabolism, and molecular synthesis. Overall, the identified proteins may provide important insights into the mechanisms of the silkworm response to N. bombycis infection.

Characterization of the First Fungal Glycosyl Hydrolase Family 19 Chitinase (NbchiA) from Nosema bombycis (Nb).

Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the ß-(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)(2-5) detected by high-performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)(3-5). On the basis of structure-based multiple-sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site-directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia.

Characterization of a novel otubain-like protease with deubiquitination activity from Nosema bombycis (Microsporidia).

Otubains are a recently identified family of deubiquitinating enzymes (DUBs). They are involved in diverse biological processes including protein degradation, signal transduction, and cell immune response. Several microsporidian genomes have been published in the last decade; however, little is known about the otubain-like protease in these widely-spread obligate intracellular parasites. Here, we characterized a 25 kDa otubain-like protease (NbOTU1) from the microsporidian Nosema bombycis, the pathogen causing pebrine disease in the economically important insect Bombyx mori. Sequence analysis showed that this protein contained a conserved catalytic triad of otubains composed of aspartate, cysteine, and histidine residues. The expression of Nbotu1 began on day 3 postinfection as determined by the RT-PCR method. Immunofluorescence analysis indicated that NbOTU1 is localized on the spore wall of N. bombycis. The subcellular localization of the NbOTU1 was further detected with immunoelectron microscopy, which showed that NbOTU1 is localized at the regions around endospore wall and plasma membrane. Deubiquitination analysis confirmed that the recombinant NbOTU1 possessed deubiquitination activity in vitro. Taken together, a novel microsporidian otubain-like protease NbOTU1 was partially characterized in N. bombycis, demonstrating its subcellular location and deubiquitination activity. This study provided a basic reference for further dissecting the function of otubains in microsporidia.

Development of an approach to analyze the interaction between Nosema bombycis (microsporidia) deproteinated chitin spore coats and spore wall proteins.

Nosema bombycis is an obligate intracellular parasite of the Bombyx mori insect. The spore wall of N. bombycis is composed of an electron-dense proteinaceous outer layer and an electron-transparent chitinous inner layer, and the spore wall is connected to the plasma membrane. In this study, the deproteinated chitin spore coats (DCSCs) were acquired by boiling N. bombycis in 1M NaOH. Under a transmission electron microscope, the chitin spore coat resembles a loosely curled ring with strong refractivity; organelles and nuclei were not observed inside the spore. The anti-SWP25, 26, 30 and 32 antibodies were used to detect whether spore wall proteins within the total soluble and mature spore proteins could bind to the DCSCs. Furthermore, a chitin binding assay showed that within the total soluble and mature spore proteins, the SWP26, SWP30 and SWP32 spore wall proteins, bound to the deproteinated chitin spore coats, although SWP25 was incapable of this interaction. Moreover, after the DCSCs were incubated with the alkali-soluble proteins, the latter were obtained by treating N. bombycis with 0.1M NaOH. Following this treatment, SWP32 was still capable of binding the DCSCs, while SWP26 and SWP30 were unable to bind. Collectively, the DCSCs are useful for investigating the arrangement of spore wall proteins, and they shed light on how the microsporidia spore wall is self-assembled.

Microsporidia dressing up: the spore polaroplast transport through the polar tube and transformation into the sporoplasm membrane.

Microsporidia are obligate intracellular parasites that infect a wide variety of hosts including humans. Microsporidian spores possess a unique, highly specialized invasion apparatus involving the polar filament, polaroplast, and posterior vacuole. During spore germination, the polar filament is discharged out of the spore forming a hollow polar tube that transports the sporoplasm components including the nucleus into the host cell. Due to the complicated topological changes occurring in this process, the details of sporoplasm formation are not clear. Our data suggest that the limiting membrane of the nascent sporoplasm is formed by the polaroplast after microsporidian germination. Using electron microscopy and 1,1'-dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine perchlorate staining, we describe that a large number of vesicles, nucleus, and other cytoplasm contents were transported out via the polar tube during spore germination, while the posterior vacuole and plasma membrane finally remained in the empty spore coat. Two Nosema bombycis sporoplasm surface proteins (NbTMP1 and NoboABCG1.1) were also found to localize in the region of the polaroplast and posterior vacuole in mature spores and in the discharged polar tube, which suggested that the polaroplast during transport through the polar tube became the limiting membrane of the sporoplasm. The analysis results of Golgi-tracker green and Golgi marker protein syntaxin 6 were also consistent with the model of the transported polaroplast derived from Golgi transformed into the nascent sporoplasm membrane.IMPORTANCEMicrosporidia, which are obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection by the microsporidia is their unique invasion apparatus which includes the polar filament, polaroplast, and posterior vacuole. When the mature spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that transports the sporoplasm components including the microsporidian nucleus into host cells. Details of the structural difference between the polar filament and polar tube, the process of cargo transport in extruded polar tube, and the formation of the sporoplasm membrane are still poorly understood. Herein, we verify that the polar filament evaginates to form the polar tube, which serves as a conduit for transporting the nucleus and other sporoplasm components. Furthermore, our results indicate that the transported polaroplast transforms into the sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of the polar tube and origin of the sporoplasm membrane, which provide important clarification of the microsporidian infection mechanism.

Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation.

BACKGROUND: Microsporidian Nosema bombycis has received much attention because the pébrine disease of domesticated silkworms results in great economic losses in the silkworm industry. So far, no effective treatment could be found for pébrine. Compared to other known Nosema parasites, N. bombycis can unusually parasitize a broad range of hosts. To gain some insights into the underlying genetic mechanism of pathological ability and host range expansion in this parasite, a comparative genomic approach is conducted. The genome of two Nosema parasites, N. bombycis and N. antheraeae (an obligatory parasite to undomesticated silkworms Antheraea pernyi), were sequenced and compared with their distantly related species, N. ceranae (an obligatory parasite to honey bees). RESULTS: Our comparative genomics analysis show that the N. bombycis genome has greatly expanded due to the following three molecular mechanisms: 1) the proliferation of host-derived transposable elements, 2) the acquisition of many horizontally transferred genes from bacteria, and 3) the production of abundnant gene duplications. To our knowledge, duplicated genes derived not only from small-scale events (e.g., tandem duplications) but also from large-scale events (e.g., segmental duplications) have never been seen so abundant in any reported microsporidia genomes. Our relative dating analysis further indicated that these duplication events have arisen recently over very short evolutionary time. Furthermore, several duplicated genes involving in the cytotoxic metabolic pathway were found to undergo positive selection, suggestive of the role of duplicated genes on the adaptive evolution of pathogenic ability. CONCLUSIONS: Genome expansion is rarely considered as the evolutionary outcome acting on those highly reduced and compact parasitic microsporidian genomes. This study, for the first time, demonstrates that the parasitic genomes can expand, instead of shrink, through several common molecular mechanisms such as gene duplication, horizontal gene transfer, and transposable element expansion. We also showed that the duplicated genes can serve as raw materials for evolutionary innovations possibly contributing to the increase of pathologenic ability. Based on our research, we propose that duplicated genes of N. bombycis should be treated as primary targets for treatment designs against pébrine. The genome data and annotation information of N. bombycis and N.antheraeae were submitted to GenBank (Accession numbers ACJZ01000001 -ACJZ01003558).

Identification of a novel chitin-binding spore wall protein (NbSWP12) with a BAR-2 domain from Nosema bombycis (microsporidia).

The spore wall of Nosema bombycis plays an important role in microsporidian pathogenesis. Protein fractions from germinated spore coats were analysed by two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF/TOF mass spectrometry. Three protein spots were identified as the hypothetical spore wall protein NbHSWP12. A BAR-2 domain (e-value: 1.35e-03) was identified in the protein, and an N-terminal protein-heparin interaction motif, a potential N-glycosylation site, and 16 phosphorylation sites primarily activated by protein kinase C were also predicted. The sequence analysis suggested that Nbhswp12 and its homologous genes are widely distributed among microsporidia. Additionally, Nbhswp12 gene homologues share similar sequence features. An indirect immunofluorescence analysis showed that NbHSWP12 localized to the spore wall, and thus we renamed it spore wall protein 12 (NbSWP12). Moreover, NbSWP12 could adhere to deproteinized N. bombycis chitin coats that were obtained by hot alkaline treatment. This novel N. bombycis spore wall protein may function in a structural capacity to facilitate microsporidial spore maintenance.

Germination of Microsporidian Spores: The Known and Unknown.

Microsporidia are a large group of mysterious obligate intracellular eukaryotic parasites. The microsporidian spore can survive in the absence of nutrients for years under harsh conditions and germinate within seconds under the stimulation of environmental changes like pH and ions. During germination, microsporidia experience an increase in intrasporal osmotic pressure, which leads to an influx of water into the spore, followed by swelling of the polaroplasts and posterior vacuole, which eventually fires the polar filament (PF). Infectious sporoplasm was transported through the extruded polar tube (PT) and delivered into the host cell. Despite much that has been learned about the germination of microsporidia, there are still several major questions that remain unanswered, including: (i) There is still a lack of knowledge about the signaling pathways involved in spore germination. (ii) The germination of spores is not well understood in terms of its specific energetics. (iii) Limited understanding of how spores germinate and how the nucleus and membranes are rearranged during germination. (iv) Only a few proteins in the invasion organelles have been identified; many more are likely undiscovered. This review summarizes the major resolved and unresolved issues concerning the process of microsporidian spore germination.

The microsporidian polar tube: origin, structure, composition, function, and application.

Microsporidia are a class of obligate intracellular parasitic unicellular eukaryotes that infect a variety of hosts, even including humans. Although different species of microsporidia differ in host range and specificity, they all share a similar infection organelle, the polar tube, which is also defined as the polar filament in mature spores. In response to the appropriate environmental stimulation, the spore germinates with the polar filament everted, forming a hollow polar tube, and then the infectious cargo is transported into host cells via the polar tube. Hence, the polar tube plays a key role in microsporidian infection. Here, we review the origin, structure, composition, function, and application of the microsporidian polar tube, focusing on the origin of the polar filament, the structural differences between the polar filament and polar tube, and the characteristics of polar tube proteins. Comparing the three-dimensional structure of PTP6 homologous proteins provides new insight for the screening of additional novel polar tube proteins with low sequence similarity in microsporidia. In addition, the interaction of the polar tube with the spore wall and the host are summarized to better understand the infection mechanism of microsporidia. Due to the specificity of polar tube proteins, they are also used as the target in the diagnosis and prevention of microsporidiosis. With the present findings, we propose a future study on the polar tube of microsporidia.

Proteomic profile of polar filament and polar tube from fungal pathogen microsporidium Nosema bombycis provides new insights into its unique invasion organelle.

Microsporidium is a kind of intracellular fungal pathogen that greatly threatens the human health, breeding industry, and food security. All members of microsporidia possess a unique, highly specialized invasion organelle, described as the polar filament. Like "reversing a finger of gloves", the polar filament discharges out of mature spores to transform as the polar tube, and pathogenic sporoplasm is transported to host cell through polar tube to complete infection. During the invasion process, the structure of polar filament and polar tube has changed, so does the protein composition on them? In this study, we firstly proposed a purification method for polar filament and polar tube from microsporidium Nosema bombycis which was infected silkworm Bombyx mori, and it was also found that the structure of polar filament and polar tube was obviously different. Therefore, the proteome of these two structures was comparatively analyzed. A total of 881 and 1216 proteins were respectively identified from the polar filament and polar tube. Ten potential novel polar tube proteins (PTPs) were screened, providing a reference for the novel PTPs identification. Compared with the polar filament, there were 35 upregulated and 41 downregulated proteins on the polar tube. GO and KEGG pathway analysis of all proteins from the polar filament and polar tube provided us with a profound understanding for the microsporidian germination process, which was of great significance for clarifying the infection mechanism of microsporidia. SIGNIFICANCE: Microsporidia are obligate intracellular parasites that infect a wide variety of hosts, including humans. The polar filament is a unique invasion organelle for microsporidia, and it is also one of the important indexes of microsporidian taxonomy. The polar tube is deformed from the primitive polar filament in mature spores. During the germination, the polar filament turns into a polar tube, like "reversing a finger of gloves", through which pathogenic sporoplasm is transported to host cells to complete infection. Since the structure of the polar filament and polar tube has changed, what about their protein composition? In this study, it was the first time to purify the polar filament and the polar tube from microsporidium Nosema bombycis that was infected silkworm Bombyx mori, which provided new insights for studying the invasion organelle of microsporidia. Comparing the fine structure of polar filament and polar tube, we found that their structure was obviously different. Therefore, the protein composition of these two structures is supposed to be varied. In this case, the proteome of these two structures was comparatively analyzed. A total of 881 and 1216 proteins were respectively identified from the polar filament and polar tube. Ten potential novel polar tube proteins (PTPs) were screened, providing a reference for the novel PTPs identification. Compared with the polar filament, there were 35 upregulated and 41 downregulated proteins on the polar tube. GO and KEGG pathway analysis of all proteins from the polar filament and polar tube provided us with a profound understanding for the microsporidian germination process, which was of great significance for clarifying the infection mechanism of microsporidia.