Identification of a novel spore wall protein (SWP26) from microsporidia Nosema bombycis

Microsporidia are obligate intracellular parasites related to fungi with resistant spores against various environmental stresses. The rigid spore walls of these organisms are composed of two major layers, which are the exospore and the endospore. Two spore wall proteins (the endosporal protein-SWP30 and the exosporal protein-SWP32) have been previously identified in Nosema bombycis. In this study, using the MALDI-TOF-MS technique, we have characterised a new 25.7-kDa spore wall protein (SWP26) recognised by monoclonal antibody 2G10. SWP26 is predicted to have a signal peptide, four potential N-glycosylation sites, and a C-terminal heparin-binding motif (HBM) which is known to interact with extracellular glycosaminoglycans. By using a host cell binding assay, recombinant SWP26 protein (rSWP26) can inhibit spore adherence by 10%, resulting in decreased host cell infection. In contrast, the mutant rSWP26 (rΔSWP26, without HBM) was not effective in inhibiting spore adherence. Immuno-electron microscopy revealed that this protein was expressed largely in endospore and plasma membrane during endospore development, but sparsely distributed in the exospore of mature spores. The present results suggest that SWP26 is a microsporidia cell wall protein that is involved in endospore formation, host cell adherence and infection in vitro. Moreover, SWP26 could be used as a good prospective target for diagnostic research and drug design in controlling the silkworm, Bombyx mori, pebrine disease in sericulture.     

SWP25, A Novel Protein Associated with the Nosema bombycis Endospore1

ABSTRACT. Microsporidia are eukaryotic, obligate intracellular, spore-forming parasites. The resistant spores, which harbor a rigid cell wall, are critical for their host-to-host transmission and persistence in the environment. The spore wall comprises two major layers: the exospore and the endospore. In Nosema bombycis, two spore wall proteins have been characterized—an endosporal protein, SWP30, and an exosporal protein, SWP32. Here, we report the identification of the third spore wall protein of N. bombycis, SWP25, the gene of which has no known homologue. SWP25 is predicted to posses a signal peptide and a heparin-binding motif. Immunoelectron microscopy analysis showed that this protein is localized to the endospore. This characterization of a new spore wall protein of N. bombycis may facilitate our investigation of the relationship between N. bombycis and its host, Bombyx mori.     

SWP5, a spore wall protein, interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis

Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. The microsporidian invasion process involves the extrusion of a unique polar tube into host cells. Both the spore wall and the polar tube play an important role in microsporidian pathogenesis. So far, five spore wall proteins (SWP1, SWP2, Enp1, Enp2, and EcCDA) from Encephalitozoon intestinalis and Encephalitozoon cuniculi and five spore wall proteins (SWP32, SWP30, SWP26, SWP25, and NbSWP5) from the silkworm pathogen Nosema bombycis have been identified. Here we report the identification and characterization of a spore wall protein (SWP5) with a molecular mass of 20.3 kDa in N. bombycis. This protein has low sequence similarity to other eukaryotic proteins. Immunolocalization analysis showed SWP5 localized to the exospore and the region of the polar tube in mature spores. Immunoprecipitation, mass spectrometry, and immunofluorescence analyses revealed that SWP5 interacts with the polar tube proteins PTP2 and PTP3. Anti-SWP5 serum pretreatment of mature spores significantly decreased their polar tube extrusion rate. Taken together, our results show that SWP5 is a spore wall protein localized to the spore wall and that it interacts with the polar tube, may play an important role in supporting the structural integrity of the spore wall, and potentially modulates the course of infection of N. bombycis.     

Effect of Paranosema locustae (Microsporidia) on the behavioural phases of Locusta migratoria (Orthoptera: Acrididae) in the laboratory

The ability of parasites to modify the behaviour of their hosts is a wide spread phenomenon, but the effects of microsporidian parasites on locust behaviour remain unexplored. Here the frequencies of directional changes (ND) and jumping (NJ) per minute of gregarious locusts infected with 2000 spores of the microsporidian parasite Paranosema locustae were significantly different from those of untreated locusts 10 and 16 days after infection, being similar to values for solitary nymphs. In contrast, the behaviour of locusts inoculated with the lower doses of 200 spores/locust was sometimes like that of solitary nymphs. At other times, behaviour was intermediate between solitary and gregarious, i.e. transitional. The rearing density did not affect the turning and jumping behaviour of infected locusts, and their behaviours were similar to those of solitary locusts at 10–16 days after infection. Our study demonstrates that infection with P. locustae may lead gregarious locusts to change some of their behaviour to that typical of solitary locusts.     

Effects of a combined infection with Paranosema locustae and Beauveria bassiana on Locusta migratoria and its gut microflora

Even though Paranosema locustae is widely used in China as a biological agent for controlling grasshoppers,the mortality rate is initially quite low.This study sought to determine whether the simultaneous use of P.locustae and Beauveria bassiana would be a more effective control strategy.Additionally,changes in the intestinal microbial communities of migratory locusts infected with the two pathogens were analyzed to investigate the roles of gut microbes in pathogen-host interactions.The mortality rate of locusts inoculated with B.bassiana and P.locustae simultaneously was not significantly higher than expected but the mortality rates of locusts inoculated with B.bassiana 3,6,and 9 days after inoculation with P.locustae were significantly higher than if their effects were additive,indicating synergism.A MiSeq analysis found that Weissella was the most common bacterium,representing 41.48%and 51.62%of the total bacteria in the mid-and hindguts,respectively,and the bacterial declines were greatest during dual infections with B.bassiana and P.locustae.The appropriately timed combined application of P.locustae and B.bassiana was more effective against locusts than either treatment alone.Moreover,the combined inoculation of the two pathogens changed the gut microflora of locusts,indicating the potential relevancy of their synergistic effects on locust control.     

Effect of Paranosema (Nosema) locustae (Microsporidia) on morphological phase transformation of Locusta migratoria manilensis (Orthoptera: Acrididae)

Field and laboratory studies demonstrated that Paranosema (Nosema) locustae had significant effects on the morphological phase transformation of Locusta migratoria manilensis (Meyen 1835). In the field, spraying P. locustae on gregarious locusts caused a substantial population reduction by 16 days after treatment, with most of the surviving locusts being phase solitaria. However, the effects of P. locustae on locust phase transformation began before direct mortality had caused a substantial reduction in locust density: locust numbers were still high at day 10, but locusts had already transformed to phase transiens. Laboratory assays showed that while a low dose of P. locustae had no effect on phase transformation, at a higher dose of 1×10⁵ spores/mL, locusts had F/C ratios that were significantly (P<0.05) more solitaria than untreated locusts, with locusts having ratios that were either phase solitaria or on the solitaria side of phase transiens. In a second laboratory experiment that analysed the effects of locust density on phase transformation by P. locustae, there was no obvious effect of density on female locusts 10 days later as all were solitaria at all locust densities. At day 16, female locusts were transiens at higher densities, but were solitaria at 4/cage. With males there were lesser effects. These results provide new evidence for P. locustae having sub-lethal effects on locust phase transformation at a wide range of locust densities.     

Mass production of Glomus mosseae spores

Five crops inoculated with Glomus mosseae were grown for 10 weeks and the development of mycorrhizal infection and sporulation were assessed. Infected roots from pot cultures of different ages were used to examine the host effect on the development of mycorrhizae. The effectiveness of each host was assessed by measuring spore numbers. For all hosts, the percentage of root length infected increased rapidly up to 10 weeks after sowing. Infectivity of root inocula increased with increasing percentage of root length infected with the inoculum for all crops, except where large numbers of mature spores (1755) had been produced on barley. The highest spore numbers were achieved in the rhizosphere of barley plants, followed by chickpea and beans. The lowest spore numbers were found in the rhizosphere of corn and okra plants. The type of the crop as well as the harvest date greatly influenced the size of the spore population and the extent of root colonization of G. mosseae.     

Laboratory assessment of the potential of Paranosema locustae to control immature stages of Schistocerca gregaria and Oedaleus senegalensis and vertical transmission of the pathogen in host populations

We tested the effects of Paranosema locustae spores in wheat bran formulation on the immature stages of Schistocerca gregaria and Oedaleus senegalensis under laboratory conditions. Younger instars were the most sensitive to the pathogen. While 100% infection was recorded in younger instar nymphs, older instars were less sensitive, with 16–27% of the inoculated nymphs remaining uninfected at the end of the experiment. Mortality of each instar increased with increased spore concentration. Immature survival time was significantly reduced by the pathogen and none of the nymphs inoculated as first, second, and third instar nymphs developed to adulthood (6–30% and 55–74% of nymphs inoculated as fourth and fifth instar, respectively). Sublethal effects such as delayed host growth, reduced host size, and abnormal wing and leg development (37% of emerging adults) were noted. Almost half the infected adults showed morphological abnormalities at emergence. Moreover, infection in S. gregaria and O. senegalensis by P. locustae did not affect female oviposition. However, 60% of S. gregaria and 52% of O. senegalensis progeny clearly showed infection by P. locustae with infection intensity of 1.08±0.27×10¹ and 1.19±0.32×10² spores/nymph, respectively. In view of the mortality rates, immature survival, host growth, and abnormal development in the P. locustae treatments, and the high prevalence of the pathogen in offspring from infected parents, it can be expected that the reduction in the impact of the two acridid species in the field will be considerable.     

Differential susceptibility of two locust pests to the microsporidium Paranosema (Antonospora) locustae at suboptimal rearing temperature

Paranosema (Antonospora) locustae (Canning) is a microsporidium with an extensive host range including >100 reported insect hosts from the order Orthoptera. The susceptibility of two species of locusts (Orthoptera: Acrididae) – the migratory locust, Locusta migratoria subsp. migratorioides (Fairmaire & Reiche), and the desert locust, Schistocerca gregaria Forsskål – to P. locustae was compared under laboratory conditions at a decreased temperature of 27 °C to enhance susceptibility to infection. Locusta migratoria was found highly vulnerable as infection percentages exceeded 70% at 10⁴, 10⁵, and 10⁶ spores per insect, and mortality increased with increasing dosage. Conversely, P. locustae spores were not found in S. gregaria throughout the experiment. Only at 10⁷ spores per insect, as many as 20% of S. gregaria were infected. This suggests that the desert locust is resistant to P. locustae infection, as opposed to the migratory locust. The laboratory models of these parasite–host systems may be useful for elucidating mechanisms of insect immunity to microsporidia.     

Spore coat protein of Bacillus subtilis. Structure and precursor synthesis.

The coat protein of Bacillus subtilis spores comprises about 10% of the total dry weight of spores and 25% of the total spore protein. One protein with a molecular weight of 13,000 to 15,000 comprises a major portion of the spore coat. This mature spore coat protein has histidine at its NH2 terminus and is relatively rich in hydrophobic amino acids. Netropsin, and antibiotic which binds to A-T-rich regions of DNA and inhibits sporulation, but not growth, decreased the synthesis of this spore coat protein by 75%. A precursor spore coat protein with a molecular weight of 25,000 is made initially at t1 of sporulation and is converted to the mature spore coat protein with a molecular weight of 13,500 at t2 - t3. These data indicate that the spore coat protein gene is expressed very early in sporulation prior to the modifications of RNA polymerase which have been noted.     

EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall

Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.     

Identification of a Nosema bombycis (Microsporidia) spore wall protein corresponding to spore phagocytosis

Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion.     

A new species of Haplosporidium Caullery & Mesnil, 1899 in the marine false limpet Siphonaria lessonii (Gastropoda: Siphonariidae) from Patagonia

A new species of Haplosporidium Caullery & Mesnil, 1899 parasitising the pulmonate gastropod Siphonaria lessonii Blainville in Patagonia, Argentina, is described based on morphological (scanning and transmission electron microscopy) and sequence (small subunit ribosomal RNA gene) data. Different stages of sporulation were observed as infections disseminated in the digestive gland. Haplosporidium patagon n. sp. is characterised by oval or slightly subquadrate spores with an operculum that is ornamented with numerous short digitiform projections of regular height, perpendicular to and covering its outer surface. The operculum diameter is slightly larger than the apical diameter of the spore. Neither the immature nor mature spores showed any kind of projections of the exosporoplasm or of the spore wall. Regarding phylogenetic affinities, the new species was recovered as sister to an undescribed species of Haplosporidium Caullery & Mesnil, 1899 from the polychaete family Syllidae Grube from Japanese waters. The morphological characters (ornamentation of the operculum, spore wall structure, shape and size of spores, and the lack of spore wall projections) corroborate it as an as yet undescribed species of Haplosporidium and the first for the phylum in marine gastropods of South America. Siphonaria lessonii is the only known host to date.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Secondary leaf fall of Hevea brasiliensis: factors affecting the production, germination and viability of spores of Colletotrichum gloeosporioides

The optimum temperature for growth and sporulation of Colletotrichum gloeosporioides from Hevea brasiliensis was between 26 and 32 ^oC, whereas spore germination exceeded 90% between 21.5 and 30.5 ^oC. Germination decreased in culture after 3 days, and on exposure of spores to sunlight or oven heat (46 ^oC) for 10 min. Spore viability and germination were sensitive to atmospheric humidity; at 99% r.h. germination was half that at 100% r.h. and was negligible below 97% r.h. Germination decreased by up to 30% after 3 h storage at 80% r.h. Continuous light favoured spore production in vitro, but spores produced in the dark had a higher percentage germination. No differences were detected between the numbers of spores germinating on leaves of different ages, although there were slightly more on susceptible cultivars and in the presence of extracts of uninfected susceptible leaves. Extracts from, infected leaves depressed spore germination, as did concentrations above 5 times 10⁵ spores/ml. The highest % germination was observed when naturally infected leaves were dry-stored for up to 20 days and then incubated for 2 days in a moist chamber.     

Interactions of two insect pathogens, Paranosema locustae (Protista: Microsporidia) and Metarhizium acridum (Fungi: Hypocreales), during a mixed infection of Locusta migratoria (Insecta: Orthoptera) nymphs

Locusta migratoria nymphs were fed Paranosema locustae spores and/or surface-treated with Metarhizium acridum 3 (assay 1), 6 (assay 2) or 9 days (assay 3) post microsporidia application (p.m.a.). These three dates corresponded to the key phases of P. locustae development: (a) mass proliferation, (b) transition to sporogenesis and (c) onset of spore maturation, respectively. As a result, locust mortality due to mixed treatment increased slower, equally and faster, as compared to mortality expected from the combination of two pathogens in assays 1-3, respectively. However, a statistically significant difference in survival times was observed only in assay 3, indicating that only at the phase of spore maturation microsporidia drastically increase locust susceptibility to fungal infection. Analysis of perished nymphs showed that fungal treatment 3 days p.m.a. impeded development of microsporidia. Fungal sporulation on locust cadavers was not affected by co-occurring microsporidiosis.     

Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Increased survival of the honey bee Apis mellifera infected with the microsporidian Nosema ceranae by effective gene silencing

This study examined the control of nosemosis caused by Nosema ceranae, one of the hard-to-control diseases of honey bees, using RNA interference (RNAi) technology. Double-stranded RNA (dsRNA) for RNAi application targeted the mitosome-related genes of N. ceranae. Among the various mitosome-related genes, NCER_100882, NCER_101456, NCER_100157, and NCER_100686 exhibited relatively low homologies with the orthologs of Apis mellifera. Four gene-specific dsRNAs were prepared against the target genes and applied to the infected A. mellifera to analyze Nosema proliferation and honey bee survival. Two dsRNAs specifics to NCER_101456 and NCER_100157 showed high inhibitory effects on spore production by exhibiting only 62% and 67%, respectively, compared with the control. In addition, these dsRNA treatments significantly rescued the honey bees from the fatal nosemosis. It was confirmed that the inhibition of Nosema spore proliferation and the increase in the survival rate of honey bees were resulted from a decrease in the expression level of each target gene by dsRNA treatment. However, dsRNA mixture treatment was no more effective than single treatments in the rescue from the nosemosis. It is expected that the four newly identified mitosome-related target genes in this study can be effectively used for nosemosis control using RNAi technology.