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The microsporidian Enterocytozoon hepatopenaei (EHP) is a major threat to shrimp health worldwide. Severe EHP infections in shrimp cause growth retardation and increase susceptibility to opportunistic infections. EHP produces spores with a chitin wall that enables them to survive prolonged environmental exposure. Previous studies showed that polar tube extrusion is a prerequisite for EHP infection, such that inhibiting extrusion should prevent infection. Using a proteomic approach, polar tube protein 2 of EHP (EhPTP2) was found abundantly in protein extracts obtained from extruded spores. Using an immunofluorescent antibody against EhPTP2 for immunohistochemistry, extruded spores were found in the shrimp hepatopancreas (HP) and intestine, but not in the stomach. We hypothesized that presence of EhPTP2 might be required for successful EHP spore extrusion. To test this hypothesis, we injected EhPTP2-specific double-stranded RNA (dsRNA) and found that it significantly diminished EHP copy numbers in infected shrimp. This indicated reduced amplification of EHP-infected cells in the HP by spores released from previously infected cells. In addition, injection of the dsRNA into EHP-infected shrimp prior to their use in cohabitation with naïve shrimp significantly (p < 0.05) reduced the rate of EHP transmission to naïve shrimp. The results revealed that EhPTP2 plays a crucial role in the life cycle of EHP and that dsRNA targeting EHP mRNA can effectively reach the parasite developing in host cells. This approach is a model for future investigations to identify critical genes for EHP survival and spread as potential targets for preventative and therapeutic measures in shrimp.

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Nosema bombycis is a representative species of Microsporidia, and is the pathogen that causes pebrine disease in silkworms. In the process of infection, the polar tube of N. bombycis is injected into the host cells. During proliferation, N. bombycis recruits the mitochondria of host cells. The general transcriptional corepressor Ssn6 contains six tetratricopeptide repeats (TPR) and undertakes various important functions. In this study, we isolated and characterized Nbssn6 of the microsporidium N. bombycis. The Nbssn6 gene contains a complete ORF of 1182 bp in length that encodes a 393 amino acid polypeptide. Indirect immunofluorescence assay showed that the Ssn6 protein was mainly distributed in the cytoplasm and nucleus at the proliferative phase of N. bombycis. We revealed the interaction of Nbssn6 with polar tube protein 2 (Nbptp2) and the transcriptional repressor for RNA polymerase II (Nbtrrp2) by Co-IP and yeast two-hybrid assays. Results from RNA interference further confirmed that the transcriptional level of Nbptp2 and Nbtrrp2 was regulated by Nbssn6. These results suggest that Nbssn6 impacts the infection and proliferation of N. bombycis via interacting with the polar tube protein and transcriptional repressor for RNA polymerase II.

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Enterocytozoon hepatopenaei (EHP) infections are a global challenge for the Penaeid shrimp industry with a sharp rise in prevalence over the last 10 yr. EHP is known to cause sub-optimal growth, large size variation and reduced survival of shrimp. Molecular methods development has mainly focussed on 18S rRNA or spore wall protein 1 (SWP1). Due to the specificity and sensitivity issues with previously designed assays for both targets, new molecular assays are needed by the global shrimp industry and regulators to help manage the risks posed by EHP. This paper describes new real-time PCR (qPCR) methods developed for the novel EHP gene targets polar tube protein 2 (PTP2) and spore wall protein 26 (SWP26), whilst also presenting performance metrics of the new Shrimp MultiPathTM technology EHP assay. qPCR assays PTP2G and SWP26G show high amplification efficiency, a limit of detection (LOD) of between 1 and 4 copies, low assay variation and high diagnostic sensitivity (DSe) and specificity (DSp) compared to imperfect reference assays. Similar performance is seen with Shrimp MultiPathTM EHP showing an LOD of 8 copies, low assay variation and high DSe and DSp. These novel molecular targets for EHP and Shrimp MultiPathTM EHP strengthen global efforts to monitor and mitigate risks of EHP infections and outbreaks. Moreover, this study presents novel data on distribution of EHP in shrimp populations from South-East Asia and Latin America, and how sequence variations need to be considered when monitoring EHP in different geographies.

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Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = -3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming.

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BACKGROUND: Microsporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50-500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually. RESULTS: A novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability. CONCLUSIONS: Based on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.

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Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting ptp2 gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37 °C within 1 h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP.

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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.

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All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed-sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5-6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.

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Microsporidian spores contain a single polar filament that is coiled around the interior of the spore. Upon germination the polar tube (post-germination polar filament) is ejected by inversion into a host cell. The sporoplasm flows through the polar tube, directly infecting the cytoplasm of the cell. Various species of microsporidia display differences in the number of coils in the polar filament and in the amino acid sequence of the polar tube proteins (PTPs). Nosema pernyi is a lethal pathogen that causes microsporidiosis in the Chinese oak silkworm, Antheraea pernyi. In this study, we identified three PTPs in N. pernyi using RT-PCR and LC-MS/MS. Polar tube protein 3 was localized in the polar tube using immuno-histochemical staining and an immunofluorescence assay. Co-immunoprecipitation data and LC-MS/MS analysis revealed that some potential proteins, like immune related proteins in A. pernyi may interact with PTP3.

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The polar tube protein is the major component of polar tube, and can specifically locate on the polar tube of microsporidia and plays an important role in invasion host cell. In this study, we analyzed the potential O- and Nglycosylation sites in polar tube protein 1 from Nosema bombycis. NbPTP1 was successfully cloned to eukaryotic expression vector pMT/Bip/V5-His A, involved V5 and His tags. After transfection, NbPTP1 gene could be efficiently expressed in Drosophila S2 cells. In addition, Lectin blotting and beta elimination analysis showed that NbPTP1 expressed in Drosophila S2 cells was O-glycosylation. These studies provided a basis for understanding the relationship between glycosylation and function of NbPTP1, helped us to reveal the infection mechanism of microsporidia and established effective diagnosis and prevention methods for microsporidia.

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RNA interference (RNAi) is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene and is conserved in a wide range of eukaryotic organisms. The RNAi mechanism has provided unique opportunities for combating honey bee diseases caused by various parasites and pathogens. Nosema ceranae is a microsporidian parasite of European honey bees, Apis mellifera, and has been associated with honey bee colony losses in some regions of the world. Here we explored the possibility of silencing the expression of a N. ceranae putative virulence factor encoding polar tube protein 3 (ptp3) which is involved in host cell invasion as a therapeutic strategy for controlling Nosema parasites in honey bees. Our studies showed that the oral ingestion of a dsRNA corresponding to the sequences of N. ceranae ptp3 could effectively suppress the expression of the ptp3 gene in N. ceranae-infected bees and reduce Nosema load. In addition to the knockdown of ptp3 gene expression, ingestion of ptp3-dsRNA also led to improved innate immunity in bees infected with N. ceranae along with an improvement in physiological performance and lifespan compared with untreated control bees. These results strongly suggest that RNAi-based therapeutics hold real promise for the effective treatment of honey bee diseases in the future, and warrant further investigation.

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Microsporidia are highly specialized obligate intracellular, spore forming divergent fungi with a wide variety host range that includes most vertebrates and invertebrates. The resistant spores are surrounded by a rigid cell wall which consists of three layers: the electron-lucent chitin and protein inner endospore, the outer-electron-dense and mainly proteinaceous exospore and plasma membrane. Interestingly, microsporidia owns a special invasion organelle, called polar tube, coiled within the interior of the spore wall and attached to anchoring disk at the anterior end of spore. Spore wall and polar tube are the major apparatuses for mature spores adhering and infecting to the host cells. In this review, we summarize the research advances in spore wall proteins (SWPs) related to spore adherence and infection, and SWPs and deproteinated chitin spore coats (DCSCs) interaction associated with SWPs deposit processes and spore wall assembly. Furthermore, we highlight the SWPs-polar tube proteins (PTPs) interaction correlated to polar tube orderly orientation, arrangement and anchorage to anchoring disk. Based on results obtained, it is helpful to improve understanding of the spore wall assembly and polar tube orderly arrangement mechanisms and molecular pathogenesis of microsporidia infection. Also, such information will provide a basis for developing effective control strategies against microporidia.

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The polar tube protein is the major component of polar tube, and can specifically locate on the polar tube of microsporidia and plays an important role in invasion host cell. In this study, we analyzed the potential O- and Nglycosylation sites in polar tube protein 1 from Nosema bombycis. NbPTP1 was successfully cloned to eukaryotic expression vector pMT/Bip/V5-His A, involved V5 and His tags. After transfection, NbPTP1 gene could be efficiently expressed in Drosophila S2 cells. In addition, Lectin blotting and beta elimination analysis showed that NbPTP1 expressed in Drosophila S2 cells was O-glycosylation. These studies provided a basis for understanding the relationship between glycosylation and function of NbPTP1, helped us to reveal the infection mechanism of microsporidia and established effective diagnosis and prevention methods for microsporidia.

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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.

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