Draft genome sequence of the Daphnia pathogen Octosporea bayeri: insights into the gene content of a large microsporidian genome and a model for host-parasite interactions

Background  

The highly compacted 2.9-Mb genome of Encephalitozoon cuniculi placed the microsporidia in the spotlight, encoding a mere 2,000 proteins and a highly reduced suite of biochemical pathways. This extreme level of reduction is not universal across the microsporidia, with genomes known to vary up to sixfold in size, suggesting that some genomes may harbor a gene content that is not as reduced as that of Enc. cuniculi. In this study, we present an in-depth survey of the large genome of Octosporea bayeri, a pathogen of Daphnia magna, with an estimated genome size of 24 Mb, in order to shed light on the organization and content of a large microsporidian genome.     

First Characterization in China of Encephalitozoon cuniculi in the Blue Fox (Alopex lagopus)

Encephalitozoon cuniculi is a microsporidian parasite that infects a wide range of vertebrates, including primates. It has recently emerged as an opportunistic parasite of patients infected with the human immunodeficiency virus. The blue fox (Alopex lagopus; also known as the arctic fox) is one of the most susceptible species for encephalitozoonosis. Here, we report an outbreak of encephalitozoonosis at a fox farm in China. The isolated parasites displayed the typical morphology of E. cuniculi as assessed by Masson's trichrome staining. Analysis of the internal transcribed spacer sequence indicated that the isolated parasite is a genotype III strain of E. cuniculi. Furthermore, phylogenetic analysis of the PTP1 gene verifies classification of this new strain (termed LN‐1) with other genotype III E. cuniculi strains, though the PTP3 and SWP1 sequences diverge from the reference strain. This is the first report of encephalitozoonosis in farmed blue foxes in China.     

Latent Microsporidiosis Caused by Encephalitozoon cuniculi in Immunocompetent Hosts: A Murine Model Demonstrating the Ineffectiveness of the Immune System and Treatment with Albendazole

Background

Microsporidia are obligate intracellular parasites causing severe infections with lethal outcome in immunocompromised hosts. However, these pathogens are more frequently reported as latent infections in immunocompetent individuals and raises questions about the potential risk of reactivation following induced immunosuppression.

Aims

To evaluate the possibility latent microsporidiosis, efficacy or albendazole, and reactivation, the authors monitored the course of E. cuniculi infection in immunocompetent BALB/c mice and immunodeficient SCID mice using molecular methods.

Methods

Mice were per orally infected with 10⁷ spores of E. cuniculi. Selected groups were treated with albendazole, re-infected or chemically immunosuppressed by dexamethasone. The presence of microsporidia in the host’s organs and feces were determined using PCR methods. Changes in numbers of lymphocytes in blood and in spleen after induction of immunosuppression were confirmed using flow cytometry analysis.

Results

Whereas E. cuniculi caused lethal microsporidiosis in SCID mice, the infection in BABL/c mice remained asymptomatic despite parasite dissemination into many organs during the acute infection phase. Albendazole treatment led to microsporidia elimination from organs in BALB/c mice. In SCID mice, however, only a temporary reduction in number of affected organs was observed and infection re-established post-treatment. Dexamethasone treatment resulted in a chronic microsporidia infection disseminating into most organs in BALB/c mice. Although the presence of E. cuniculi in organs of albendazole- treated mice was undetectable by PCR, it was striking that infection was reactivated by immunosuppression treatment.

Conclusion

Our results demonstrated that microsporidia can successfully survive in organs of immunocompetent hosts and are able to reactivate from undetectable levels and spread within these hosts after induction of immunosuppression. These findings stress the danger of latent microsporidiosis as a life-threatening risk factor especially for individuals undergoing chemotherapy and in transplant recipients of organs originating from infected donors.     

Toxoplasma gondii,Neospora caninum and Encephalitozoon cuniculi in Animals from Captivity (Zoo and Circus Animals)

Problems with parasitic infections are common in zoological gardens and circuses. In some animals it can lead to several disorders such as systemic disease, reproductive disorders (abortions and neonatal mortality), and even to death if severe illness is untreated. Thus, the aim of this study was to evaluate the prevalence of three common parasites in 74 animals from three zoos, and four circuses in Southern Italy. Antibodies to Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi were detected in 51%, 12%, and 20% of animals, respectively. Co‐infections of T. gondii and N. caninum were reported in seven animals (9%) and co‐infection of T. gondii and E. cuniculi in one animal. T. gondii, N. caninum and E. cuniculi seroprevalence differed in type of diet (P ≤ 0.0001; P ≤ 0.037 and P ≤ 0.004, respectively). T. gondii and E. cuniculi seroprevalence also differed in animal families (P ≤ 0.0001) and according to type of housing (P ≤ 0.003), respectively. Statistical differences were not found in other characteristics (gender, age, country of birth, origin, and contact with cats or dogs). This is the first serological study focusing on protozoan and microsporidian parasites in zoo and circus animals from Southern Italy and the first detection of antibodies to E. cuniculi in camels in Europe.     

What Does the Microsporidian <Emphasis Type="Italic">E. cuniculi</Emphasis> Tell Us About the Origin of the Eukaryotic Cell?

The relationship among the three cellular domains Archaea, Bacteria, and Eukarya has become a central problem in unraveling the tree of life. This relationship can now be studied as the completely sequenced genomes of representatives of these cellular domains become available. We performed a bioinformatic investigation of the Encephalitozoon cuniculi proteome. E. cuniculi has the smallest sequenced eukaryotic genome, 2.9 megabases coding for 1997 proteins. The proteins of E. cuniculi were compared with a previously characterized set of eukaryotic signature proteins (ESPs). ESPs are found in a eukaryotic cell, whether from an animal, a plant, a fungus, or a protozoan, but are not found in the Archaea and the Bacteria. We demonstrated that 85% of the ESPs have significant sequence similarity to proteins in E. cuniculi. Hence, E. cuniculi, a minimal eukaryotic cell that has removed all inessential proteins, still preserves most of the ESPs that make it a member of the Eukarya. The locations and functions of these ESPs point to the earliest history of eukaryotes.Reviewing Editor: Dr. Manyuan Long     

Establishing a New Species Encephalitozoon pogonae for the Microsporidian Parasite of Inland Bearded Dragon Pogona vitticeps Ahl 1927 (Reptilia,Squamata, Agamidae)

The microsporidium parasitizing Inland Bearded Dragons Pogona vitticeps, and developing primarily in macrophages within foci of granulomatous inflammation of different organs, is described as a new species Encephalitozoon pogonae. Establishing the new species was based on sequencing the ITS‐SSUrDNA region of the ribosomal gene and consequent SSUrDNA‐inferred phylogenetic analyses, as well as on comparison of pathogenesis, host specificity, and ultrastructure among Encephalitozoon species and isolates. The new species is closely related to E. lacertae and E. cuniculi. Analysis of the literature suggests that this microsporidium has been reported previously as an unidentified microsporidian species or isolate of E. cuniculi and may represent a common infection in bearded dragons. All stages of E. pogonae develop in parasitophorous vacuoles. Uninucleate spores on methanol‐fixed smears measured 2.1 × 1.1 μm, range 1.7–2.6 × 0.9–1.7 μm; on ultrathin sections spores measured 0.8–1.1 × 1.8–2.2 μm. Ultrastructural study revealed 3–6 polar filament coils, a mushroom‐shaped polar disk, and a polar sac embracing half of the volume occupied by the lamellar polaroplast. In activated spores, polar filament everted eccentrically. The overall morphology and intracellular development of E. pogonae were similar to other Encepahalitozoon spp. We also review the existing data on microsporidia infecting reptiles.     

Transient increase of ATP as a response to temperature up-shift in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift.     

An Investigation of the Route and Progression of Encephalitozoon cuniculi Infection in Adult Rabbits

Rabbits infected either orally or intratracheally with cell culture-grown Encephalitozoon cuniculi were monitored regularly for serum antibody levels and E. cuniculi in the urine. Their responses were compared with intravenously inoculated and uninoculated control rabbits. All rabbits receiving E. cuniculi developed serum antibodies, generally within 3 weeks, and excreted E. cuniculi by 6 weeks. In the acute stage of infection, the organs most affected were lung, kidney and liver; the brain and gut were unaffected. However, during chronic infection, the brain, kidney, and heart were the only organs found to be involved. Antibody levels were very high at this stage. Thus both the oral and tracheal routes may be normal routes of infection with E. cuniculi in adult rabbits.     

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus × domestica) with Erwinia amylovora

Background  

The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceaespecies, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora.     

Patterns of genome evolution among the microsporidian parasites Encephalitozoon cuniculi, Antonospora locustae and Enterocytozoon bieneusi

Background

Microsporidia are intracellular parasites that are highly-derived relatives of fungi. They have compacted genomes and, despite a high rate of sequence evolution, distantly related species can share high levels of gene order conservation. To date, only two species have been analysed in detail, and data from one of these largely consists of short genomic fragments. It is therefore difficult to determine how conservation has been maintained through microsporidian evolution, and impossible to identify whether certain regions are more prone to genomic stasis.

Principal Findings

Here, we analyse three large fragments of the Enterocytozoon bieneusi genome (in total 429 kbp), a species of medical significance. A total of 296 ORFs were identified, annotated and their context compared with Encephalitozoon cuniculi and Antonospora locustae. Overall, a high degree of conservation was found between all three species, and interestingly the level of conservation was similar in all three pairwise comparisons, despite the fact that A. locustae is more distantly related to E. cuniculi and E. bieneusi than either are to each other.

Conclusions/Significance

Any two genes that are found together in any pair of genomes are more likely to be conserved in the third genome as well, suggesting that a core of genes tends to be conserved across the entire group. The mechanisms of rearrangments identified among microsporidian genomes were consistent with a very slow evolution of their architecture, as opposed to the very rapid sequence evolution reported for these parasites.     

Multiyear Survey of Coccidia,Cryptosporidia, Microsporidia,Histomona, and Hematozoa in Wild Quail in the Rolling Plains Ecoregion of Texas and Oklahoma,USA

We developed nested PCR protocols and performed a multiyear survey on the prevalence of several protozoan parasites in wild northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) in the Rolling Plains ecoregion of Texas and Oklahoma (i.e. fecal pellets, bird intestines and blood smears collected between 2010 and 2013). Coccidia, cryptosporidia, and microsporidia were detected in 46.2%, 11.7%, and 44.0% of the samples (n = 687), whereas histomona and hematozoa were undetected. Coccidia consisted of one major and two minor Eimeria species. Cryptosporidia were represented by a major unknown Cryptosporidium species and Cryptosporidium baileyi. Detected microsporidia species were highly diverse, in which only 11% were native avian parasites including Encephalitozoon hellem and Encephalitozoon cuniculi, whereas 33% were closely related to species from insects (e.g. Antonospora, Liebermannia, and Sporanauta). This survey suggests that coccidia infections are a significant risk factor in the health of wild quail while cryptosporidia and microsporidia may be much less significant than coccidiosis. In addition, the presence of E. hellem and E. cuniculi (known to cause opportunistic infections in humans) suggests that wild quail could serve as a reservoir for human microsporidian pathogens, and individuals with compromised or weakened immunity should probably take precautions while directly handling wild quail.     

Potential biological control of Erwinia tracheiphila by internal alimentary canal interactions in Acalymma vittatum with Pseudomonas fluorescens

Aims

We aim to determine if Pseudomonas fluorescens is a viable biological control for Erwinia tracheiphila within the insect vector, Acalymma vittatum.

Methods and Results

Pseudomonas fluorescens secreted fluorescein and inhibited growth of E. tracheiphila in disc diffusion assays. To determine if this antagonism was conserved within the insect vector, we performed in vivo assays by orally injecting beetles with bacterial treatments and fluorescent in situ hybridization to determine bacterial presence within the alimentary canal.

Conclusions

Pseudomonas fluorescens inhibited the growth of E. tracheiphila on a nutrient‐limiting medium. In situ experiments demonstrated that P. fluorescens is maintained within the alimentary canal of the beetle for at least 4 days, and co‐occurred with E. tracheiphila. When beetles were first presented with Pseudomonas and then challenged with E. tracheiphila, E. tracheiphila was not recovered via FISH after 4 days. These data suggest that P. fluorescens has potential as a biological control agent to limit E. tracheiphila within the insect vector.

Significance and Impact of the Study

This is a novel approach for controlling E. tracheiphila that has the potential to decrease reliance on insecticides, providing a safer environment for pollinators and growers.     

Inhibition of human in vitro osteoclastogenesis by Equisetum arvense

Objectives

Equisetum arvense has long been used in traditional medicines to treat different disorders, including bone pathologies. In this study a hydromethanolic extract of E. arvense was assessed for its effects on human osteoclastogenesis.

Materials and methods

Osteoclast precursors were maintained in non‐stimulated and stimulated (presence of M‐CSF and RANKL) conditions, or in co‐cultures with osteoblasts. Cell cultures were treated with 0.00016–0.5 mg/ml of a hydromethanolic E. arvense extract.

Results

The extract did not affect spontaneous osteoclastogenesis. In osteoclast precursors committed to osteoclastogenesis (stimulated or co‐cultured with osteoblasts), E. arvense caused dose‐dependent inhibitory effect that became statistically significant at concentrations ≥0.004 mg/ml. This was observed using different osteoclast differentiation and activation markers. Cell response was associated with changes in relative contribution of MEK and NFkB signalling pathways, as well as PGE2 production. As there were differences in the response of osteoclast precursors maintained in the presence of inductive factors, or co‐cultured with osteoblastic cells, it seems that E. arvense extract had the ability to modulate osteoclastogenesis, either by acting directly on osteoclast precursor cells, and/or via osteoblasts.

Conclusions

Equisetum appeared to have a negative effect on human osteoclastogenesis, which is in line with its putative beneficial role in pathophysiological conditions associated with increased osteoclastic activity, and might suggest potential utility for treatment with bone regeneration strategies.     

Encapsulated <Emphasis Type="Italic">Escherichia coli</Emphasis> in alginate beads capable of secreting a heterologous pectin lyase

Background  

Production of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process.     

Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus

Background  

Although Aspergillus fumigatus is an important human fungal pathogen there are few expression systems available to study the contribution of specific genes to the growth and virulence of this opportunistic mould. Regulatable promoter systems based upon prokaryotic regulatory elements in the E. coli tetracycline-resistance operon have been successfully used to manipulate gene expression in several organisms, including mice, flies, plants, and yeast. However, the system has not yet been adapted for Aspergillus spp.     

Improving Salmonella vector with rec mutation to stabilize the DNA cargoes

Background  

Salmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in E. coli by mutating several genes including the recA, recE, recF and recJ. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in Salmonella enterica.     

Diversity of Microsporidia,Cryptosporidium and Giardia in Mountain Gorillas (Gorilla beringei beringei) in Volcanoes National Park,Rwanda

Background

Infectious diseases represent the greatest threats to endangered species, and transmission from humans to wildlife under increased anthropogenic pressure has been always stated as a major risk of habituation.

Aims

To evaluate the impact of close contact with humans on the occurrence of potentially zoonotic protists in great apes, one hundred mountain gorillas (Gorilla beringei beringei) from seven groups habituated either for tourism or for research in Volcanoes National Park, Rwanda were screened for the presence of microsporidia, Cryptosporidium spp. and Giardia spp. using molecular diagnostics.

Results

The most frequently detected parasites were Enterocytozoon bieneusi found in 18 samples (including genotype EbpA, D, C, gorilla 2 and five novel genotypes gorilla 4–8) and Encephalitozoon cuniculi with genotype II being more prevalent (10 cases) compared to genotype I (1 case). Cryptosporidium muris (2 cases) and C. meleagridis (2 cases) were documented in great apes for the first time. Cryptosporidium sp. infections were identified only in research groups and occurrence of E. cuniculi in research groups was significantly higher in comparison to tourist groups. No difference in prevalence of E. bieneusi was observed between research and tourist groups.

Conclusion

Although our data showed the presence and diversity of important opportunistic protists in Volcanoes gorillas, the source and the routes of the circulation remain unknown. Repeated individual sampling, broad sampling of other hosts sharing the habitat with gorillas and quantification of studied protists would be necessary to acquire more complex data.     

Effect of modeled reduced gravity conditions on bacterial morphology and physiology

Background  

Bacterial phenotypes result from responses to environmental conditions under which these organisms grow; reduced gravity has been demonstrated in many studies as an environmental condition that profoundly influences microorganisms. In this study, we focused on low-shear stress, modeled reduced gravity (MRG) conditions and examined, for Escherichia coli and Staphlyococcus aureus, a suite of bacterial responses (including total protein concentrations, biovolume, membrane potential and membrane integrity) in rich and dilute media and at exponential and stationary phases for growth. The parameters selected have not been studied in E. coli and S. aureus under MRG conditions and provide critical information about bacterial viability and potential for population growth.     

Enterococcal surface protein Esp is not essential for cell adhesion and intestinal colonization of Enterococcus faecium in mice

Background  

Enterococcus faecium has globally emerged as a cause of hospital-acquired infections with high colonization rates in hospitalized patients. The enterococcal surface protein Esp, identified as a potential virulence factor, is specifically linked to nosocomial clonal lineages that are genetically distinct from indigenous E. faecium strains. To investigate whether Esp facilitates bacterial adherence and intestinal colonization of E. faecium, we used human colorectal adenocarcinoma cells (Caco-2 cells) and an experimental colonization model in mice.     

Identifying the mechanism of Escherichia coli O157:H7 survival by the addition of salt in the treatment with organic acids

Aim

In this study, the effects of the addition of salt to treatment with acids (one of several organic acids and salt in various solutions including rich or minimal broth, buffer, or distilled water) on the reduction of Escherichia coli O157:H7 were investigated. The protein expression profiles corresponding to acid stress (acetic acid) with or without salt addition were studied using a comparative proteomic analysis of E. coli O157:H7.

Methods and Results

When acetic, lactic, or propionic acid was combined with 3% NaCl, mutually antagonistic effects of acid and salt on viability of E. coli O157:H7 were observed only in tryptone and yeast extract broth. After exposure to acetic acid alone or in combination with salt, approximately 851 and 916 protein spots were detected, respectively. Analysis of 10 statistically significant differentially expressed proteins revealed that these proteins are mainly related to energy metabolism.

Conclusions

When we compared protein expression of E. coli O157:H7 treated with acetic acid and the combination of the acid and salt, the differentially expressed proteins were not related to acid stress‐ and salt stress‐inducible proteins such as stress shock proteins.

Significance and Impact of the Study

According to these results, the increased resistance of E. coli O157:H7 to acetic acid after the addition of salt may not be the result of synthesis of proteins related to these phenomena; therefore, further research needs to be conducted to identify the mechanism of the mutually antagonistic effect of some organic acids and salt.