Differential molecular responses of hemolymph and hepatopancreas of swimming crab, Portunus trituberculatus, infected with Ameson portunus (Microsporidia).

Ameson portunus (Microsporidia) has caused serious economic losses to the aquaculture industry of swimming crab, Portunus trituberculatus. The hemolymph and hepatopancreas are the main immune organs of P. trituberculatus, and the main sites of A. portunus infection. Elucidating the response characteristics of hemolymph and hepatopancreas to microsporidian infection facilitates the development of microsporidiosis prevention and control strategy. This study performed comparative transcriptomic analysis of hemolymph (PTX/PTXA) and hepatopancreas (PTG/PTGA) of P. trituberculatus uninfected and infected with A. portunus. The results showed that there were 223 and 1309 differentially expressed genes (DEGs) in PTX/PTXA and PTG/PTGA, respectively. The lysosome pathway was significantly enriched after the invasion of the hemolymph by A. portunus. Also, immune-related genes were all significantly up-regulated in the hemolymph and hepatopancreas, suggesting that the invasion by A. portunus may activate host immune responses. Unlike hemolymph, antioxidant and detoxification-related genes were also significantly up-regulated in the hepatopancreas. Moreover, metabolism-related genes were significantly down-regulated in the hepatopancreas, suggesting that energy synthesis, resistance to pathogens, and regulation of oxidative stress were suppressed in the hepatopancreas. Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. The differential genes and pathways identified in this study can provide references for the prevention and control of microsporidiosis.

Microsporidia MB is found predominantly associated with Anopheles gambiae s.s and Anopheles coluzzii in Ghana.

A vertically transmitted microsporidian, Microsporidia MB, with the ability to disrupt Plasmodium development was reported in Anopheles arabiensis from Kenya, East Africa. To demonstrate its range of incidence, archived DNA samples from 7575 Anopheles mosquitoes collected from Ghana were screened. MB prevalence was observed at 1.8%. An. gambiae s.s constituted 87% of positive mosquitoes while the remaining were from An. coluzzii. Both sibling species had similar positivity rates (24% and 19%; p = 0.42) despite the significantly higher number of An. gambiae s.s analysed (An. gambiae s.s = 487; An. coluzzii = 94; p = 0.0005). The microsporidian was also more prevalent in emerged adults from field-collected larvae than field-caught adults (p < 0.0001) suggestive of an efficient vertical transmission and/or horizontal transfer among larvae. This is the first report of Microsporidia MB in Anopheles mosquitoes in West Africa. It indicates possible widespread among malaria vector species and warrants investigations into the symbiont's diversity across sub-Saharan Africa.

Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera.

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee's ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

Polar tube structure and three polar tube proteins identified from Nosema pernyi.

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.

Phosphatidic acid as a limiting host metabolite for the proliferation of the microsporidium Tubulinosema ratisbonensis in Drosophila flies.

Microsporidia are located at the base of the fungal evolutionary tree. They are obligate intracellular parasites and harness host metabolism to fuel their growth and proliferation. However, how the infestation of cells affects the whole organism and how the organism contributes to parasite proliferation remain poorly understood. Here, we have developed a Tubulinosema ratisbonensis systemic infection model in the genetically amenable Drosophila melanogaster host, in which parasite spores obtained in a mammalian cell culture infection system are injected into adult flies. The parasites proliferate within flies and ultimately kill their hosts. As commonly observed for microsporidia infecting insects, T. ratisbonensis preferentially grows in the fat body and ultimately depletes the host metabolic stores. We find that supplementing the fly diet with yeast does not benefit the host but the parasite, which increases its proliferation. Unexpectedly, fatty acids and not carbohydrates or amino acids are the critical components responsible for this phenomenon. Our genetic dissection of host lipid metabolism identifies a crucial compound hijacked by T. ratisbonensis: phosphatidic acid. We propose that phosphatidic acid is a limiting precursor for the synthesis of the parasite membranes and, hence, of its proliferation.

The invasive cell coat at the microsporidian Trachipleistophora hominis-host cell interface contains secreted hexokinases.

Microsporidia are obligate intracellular parasites causing significant disease in humans and economically important animals. In parallel to their extreme genetic reduction, Microsporidia have evolved novel mechanisms for exploiting host metabolism. A number of microsporidians confer secretion of otherwise cytosolic proteins by coding for signal peptides that direct entry into the endoplasmic reticulum. The human pathogen Trachipleistophora hominis encodes for four hexokinases, three of which have signal peptides at the N-terminus. Here, we localized hexokinase 2 and hexokinase 3 through developmental stages of T. hominis using light and electron microscopy. Both proteins were concentrated in an extracellular coat previously termed the plaque matrix (PQM). The PQM (containing hexokinases) was morphologically dynamic, infiltrating the host cytoplasm predominantly during replicative stages. Throughout development the PQM interacted closely with endoplasmic reticulum that was demonstrated to be active in membrane protein biosynthesis and export. The impact of hexokinase on the host metabolism was probed using the fluorescent analog of glucose, 2-NBDG, which displayed spatially restricted increases in signal intensity at the parasite/vacuole surface, coincident with hexokinase/PQM distribution. Gross metabolic aberrations, measured using metabolic profiling with the Seahorse XF Analyzer, were not detectable in mixed stage cocultures. Overall, these results highlight a role for the extended cell coat of T. hominis in host-parasite interactions, within which secreted hexokinases may work as part of a metabolic machine to increase glycolytic capacity or ATP generation close to the parasite surface.

Changes in the bioelement content of summer and winter western honeybees (Apis mellifera) induced by Nosema ceranae infection.

Proper bioelement content is crucial for the health and wellness of all organisms, including honeybees. However, the situation is more complicated in these important pollinators due to the fact that they change their physiology during winter in order to survive the relatively harsh climatic conditions. Additionally, honeybees are susceptible to many diseases such as nosemosis, which during winter can depopulate an entire colony. Here we show that summer bees have a markedly higher content of important bioelements such as: Al, Cu, P, V, (physiologically essential); Ca, K, Mg, (electrolytic); Cr, Se, Zn, (enzymatic); As, Hg, (toxic). In contrast, a markedly higher content of: Fe (physiologically essential); Mn, Ni, (enzymatic); Cd (exclusively toxic) were present in winter bees. Importantly, N. ceranae infection resulted in an increased honeybee bioelement content of: S, Sr (physiologically essential) and Pb (exclusively toxic), whereas the Nosema-free worker-bees had higher amounts of B and Si (physiologically essential). We propose that the shortages of Fe, Mn, Ni, and Na observed in Nosema-infected bees, could be the reason for the higher mortality of Nosema-infected bees throughout overwintering. In addition, a shortage of bioelements such as B and Si may be a reason for accelerated aging in foragers that is observed following N. ceranae infection. Therefore, in winter, bioelement content was more strongly affected by N. ceranae infection than during summer. We found a strong correlation between the bioelement content of bees and seasons (summer or winter) and also with Nosema infection. We conclude that the balance of bioelements in the honeybee is altered by both seasonal affects and by Nosema infection.

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.

Honey bee microRNAs respond to infection by the microsporidian parasite Nosema ceranae.

In order to study the effects of Nosema ceranae infection on honey bee microRNA (miRNA) expression, we deep-sequenced honey bee miRNAs daily across a full 6-day parasite reproduction cycle. Seventeen miRNAs were differentially expressed in honey bees infected by N. ceranae that potentially target over 400 genes predicted to primarily involve ion binding, signaling, the nucleus, transmembrane transport, and DNA binding. Based on Enzyme Code analysis, nine biological pathways were identified by screening target genes against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, seven of which involved metabolism. Our results suggest that differentially expressed miRNAs regulate metabolism related genes of host honey bees in response to N. ceranae infection.

Effector CD8 T cell immunity in microsporidial infection: a lone defense mechanism.

Microsporidia is a group of pathogens, which can pose severe risks to the immunocompromised population such as HIV-infected individuals. The expertise to diagnose these pathogens is limited and therefore their prevalence is believed to be much higher than what is currently known. In a mouse model of infections, it has been reported that CD8 T cells are the primary effector cells responsible for protecting the infected host. As the infection is acquired via per-oral route, CD8 T cells in the gut compartment apparently act as a first line of defense against the pathogens. Thus, generation of a robust CD8 T cell response that exhibits polyfunctional ability is critical for host survival. In this review, we describe the effector CD8 T cells generated during microsporidial infection and underline the factors that may be essential for the elicitation of protective immunity against this understudied but significant pathogen. Overall, this review will highlight the necessity for a better understanding of the development of the CD8 T cell response in gut associated lymphoid tissue (GALT) and provide some insights into therapies that may be used to restore defective CD8 T cell functionality in an immunocompromised situation.

Proteomic analysis of BmN cells (Bombyx mori) in response to infection with Nosema bombycis.

Nosema bombycis (N. bombycis, Nb) is an obligate intracellular parasite, which can cause pebrine disease in the silkworm. To investigate the effects of N. bombycis infection on the host cells, proteomes from BmN cells that had or had not been infected with N. bombycis at different infection stages were characterized with two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry, which identified 24 differentially expressed host proteins with significant intensity differences (P < 0.05) at least at one time point in mock- and N. bombycis infected cells. Notably, gene ontology analyses showed that these proteins are involved in many important biological reactions. During the infection phase, proteins involved in energy metabolism and oxidative stress had up-regulated expression. Two proteins participated in ubiquitin-dependent protein catabolic process had down-regulated expression. Quantitative real-time polymerase chain reaction was used to analyze the transcriptional profiles of these identified proteins. Taken together, the abundance changes, putative functions, and participation in biological reactions for the identified proteins produce a host-responsive protein model in N. bombycis-infected BmN cells. These findings further our knowledge about the effect of energy defect parasites on the host cells.

Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/ß-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/ß-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.

Effect of multiple parasitic infections on the tolerance to pollutant contamination.

The horizontally-transmitted acanthocephalan parasite Polymorphus minutus and the vertically-transmitted microsporidian parasite Dictyocoela roeselum have both been shown to influence on the antitoxic responses of mono-infected Gammarus roeseli exposed to cadmium. The present study investigates the effect of this co-infection on the antitoxic defence responses of naturally infected females exposed to cadmium stress. Our results revealed that, depending on the cadmium dose, bi-infection induced only slight, significant increased cell damage in G. roeseli as compared to non-infection. In addition, the antitoxic defence pattern of cadmium-exposed bi-infected hosts was similar to the pattern of cadmium-exposed D. roeselum-infected hosts. Reduced glutathione concentrations, carotenoid levels and γ-glutamylcystein ligase activity decreased, while metallothionein concentrations increased. This similar pattern indicates that host physiology can be controlled to some extent by microsporidia under stress conditions. It supports the hypothesis of a disruption of acanthocephalan effects in the presence of microsporidia. However, the global negative effects of bi-infection on host condition should be tested on more biological models, since competition between parasites depends on life history trade-off.

[Identification of a new serpin gene (NbSPN106) from Nosema bombycis].

OBJECTIVE: Serpins from pathogens have been implicated in evasion of the host immune system. We identified a new serpin protein (NbSPN106), analyzed its sequences, and detected using Western blotting. METHODS: Nosema bombycis proteins with an expect score less than 1 x 10(-5) were checked against MEROPS database (http://merops.sanger.ac.uk) by Local Alignment Search Tool search and confirmed with Database of Protein Families and HMMS. Multiple sequence alignments were performed using the ClustalX. The sequence encoding the mature protein was amplified by PCR, cloned into the pGEX4T-1 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay. RESULTS: A new serpin gene, named NbSPN106, was identified form Nosema Bombycis genome. The coding sequence of this gene is 1155 bp length with a putative signal peptide and contains the conserved serpin sequences. A specific band of approximately 45 kDa was recognized by the anti-NbSPN106 serum. CONCLUSIONS: The finding of serpins in Nosema bombycis raises new questions about their possible role in pathogenicity, which deserves further studies.

Whole body net ion fluxes, plasma electrolyte concentrations and haematology during a Loma salmonae infection in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum).

Loma salmonae infections of salmonids culminate in the development of branchial xenomas and subsequent focal hyperplasia of the lamellar or filament epithelium following xenoma rupture and spore release. The effects of this acute branchial disruption upon net ionic flux rates and plasma electrolyte concentrations were determined in juvenile rainbow trout given an experimental oral exposure to L. salmonae. Mean numbers of branchial xenomas peaked at week 5 post-exposure (PE), which coincided with a reduction in the specific growth rate, although there were no significant differences in mass, length or condition of Loma-exposed fish compared with unexposed controls. Following exposure, negative net whole body Na(+) and K(+) fluxes decreased, whereas net Cl(-) fluxes remained unchanged compared with non-exposed control fish. At week 3 PE during the initial branchial xenoma formation stage, there was a significant negative whole body net K(+) flux in Loma-exposed trout compared with other points during the exposure and subsequent infection. Additionally, Loma-exposed fish had marginally elevated plasma Na(+) and Cl(-) concentrations, whilst K(+) levels remained unchanged, compared with control fish. Although there was a progressive decrease in leucocrit, haematocrit remained unchanged over the course of the Loma exposure and subsequent infection. These results suggest that ionic compensation can occur at the gills during the development of xenomas during exposure to L. salmonae and the resultant infection, therefore allowing defence of plasma electrolyte concentrations, unlike the acute ionic disturbances seen with some other parasitic diseases.

Proteome of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis.

We report on the modification of the Aedes aegypti larval proteome following infection by the microsporidian parasite Vavraia culicis. Mosquito larvae were sampled at 5 and 15 days of age to compare the effects of infection when the parasite was in two different developmental stages. Modifications of the host proteome due to the stress of infection were distinguished from those of a more general nature by treatments involving hypoxia. We found that the major reaction to stress was the suppression of particular protein spots. Older (15 days) larvae reacted more strongly to infection by V. culicis (46% of the total number of spots affected; 17% for 5 days larvae), while the strongest reaction of younger (5 days) larvae was to hypoxia for pH range 5-8 and to combined effects of infection and hypoxia for pH range 3-6. MALDI-TOF results indicate that proteins induced or suppressed by infection are involved directly or indirectly in defense against microorganisms. Finally, our MALDI-TOF results suggest that A. aegypti larvae try to control or clear V. culicis infection and also that V. culicis probably impairs the immune defense of this host via arginases-NOS competition.

Differences in metabolic response to Loma salmonae infection in juvenile rainbow trout Oncorhynchus mykiss and brook trout Salvelinus fontinalis.

Routine and post-exercise metabolic rates were measured for juvenile rainbow trout Oncorhynchus mykiss and brook trout Salvelinus fontinalis infected with the microsporidium gill parasite Loma salmonae under laboratory conditions. Rainbow trout increased routine and post-exercise metabolic rate in response to infection compared with controls. Brook trout, on the other hand, lowered routine metabolic rate without effecting post-exercise metabolic rate compared to controls. The result of these 2 different strategies may either reflect defense of metabolic scope or a difference in the rate of recovery of the excess post-exercise oxygen consumption between the 2 species in response to the same infection.

Microsporidia: biology and evolution of highly reduced intracellular parasites.

Microsporidia are a large group of microbial eukaryotes composed exclusively of obligate intracellular parasites of other eukaryotes. Almost 150 years of microsporidian research has led to a basic understanding of many aspects of microsporidian biology, especially their unique and highly specialized mode of infection, where the parasite enters its host through a projectile tube that is expelled at high velocity. Molecular biology and genomic studies on microsporidia have also drawn attention to many other unusual features, including a unique core carbon metabolism and genomes in the size range of bacteria. These seemingly simple parasites were once thought to be the most primitive eukaryotes; however, we now know from molecular phylogeny that they are highly specialized fungi. The fungal nature of microsporidia indicates that microsporidia have undergone severe selective reduction permeating every level of their biology: From cell structures to metabolism, and from genomics to gene structure, microsporidia are reduced.

Differential metabolic response in AIDS-related chronic protozoal diarrhoea.

OBJECTIVE: As the pro-inflammatory cytokine tumour necrosis factor-alpha is greater in microsporidiosis than cryptosporidiosis, there may be a distinct metabolic response between the two organisms. DESIGN: Male HIV seropositive subjects with an untreated AIDS-defining diagnosis of microsporidiosis or cryptosporidiosis had measurement of oxygen consumption and carbon dioxide production by indirect calorimetry and body composition analysis to express resting energy expenditure (REE) and substrate oxidation per kilogram of metabolically active tissue. METHODS: Resting energy expenditure (REE), non-protein respiratory quotient (NPRQ), fat and carbohydrate oxidation were calculated from respiratory gas analysis. Fat, fat-free and appendicular muscle masses were measured by dual-energy X-ray absorptiometry. Subjects with protozoal diarrhoea were compared to other newly diagnosed, active opportunistic infections. Controls were asymptomatic HIV-seropositive men matched by peripheral CD4 count. RESULTS: Seven subjects with microsporidiosis and six with cryptosporidiosis were compared with 24 subjects with other AIDS-defining diagnosis (Pneumocystis carinii pneumonia, cytomegalovirus enteritis and Mycobacterium avium-intracellulare) and 10 controls free from secondary infection. Subjects with cryptosporidiosis had a decreased REE, a significantly increased NPRQ (P< 0.05), decreased fat oxidation (P < 0.05) and increased carbohydrate oxidation compared to microsporidiosis. Subjects with other AIDS diagnoses had an increased REE (P < 0.01) and fat oxidation and decreased carbohydrate oxidation compared to cryptosporidiosis, and a similar metabolic response to microsporidiosis. CONCLUSIONS: The metabolic response to cryptosporidiosis differs from microsporidiosis and associated weight loss may be mediated by different mechanisms. Metabolism in other AIDS diagnoses, including microsporidiosis, is compatible with a cachectic response.

Macronutrient intake and malabsorption in HIV infection: a comparison with other malabsorptive states.

BACKGROUND: Wasting is a major complication of HIV infection. The role of malabsorption in wasting is controversial. AIMS: To assess oral intake and malabsorption in a cohort of weight losing HIV infected patients, with or without chronic diarrhoea. METHODS: A prospective study using a predefined protocol for HIV infected patients was performed in a gastroenterology and nutrition unit in a university hospital. A retrospective comparison was made with HIV negative patients with malabsorption due either to small bowel disease or resection. Body weight and height, serum albumin, oral intake of macronutrients, faecal weight, and faecal fat were measured. RESULTS: Seventy nine weight losing HIV infected patients were studied. Among the 66 patients with more than 5% lipid malabsorption, wasting was significantly greater in patients with cryptosporidiosis (n = 22) than in patients with microsporidiosis (n = 18) who exhibited significantly more wasting than patients with no identified enteropathogen (n = 26) (body mass index 16.8 (14.0-20.7), 18.9 (16.5-21.3), 19.7 (15.9-23), respectively). When controlling for the level of lipid malabsorption, HIV infected patients had a significantly lower energy intake than HIV negative patients with chronic malabsorption. In HIV infected patients, but not in other categories of malabsorbers, body mass index correlated significantly with energy intake (r = 0.33, 95% confidence intervals 0.12 to 0.51). CONCLUSION: In weight losing HIV infected patients, reduced energy intake is superimposed on malabsorption and significantly contributes to wasting.