Glycosylation of the major polar tube protein of Encephalitozoon hellem, a microsporidian parasite that infects humans

The microsporidia are ubiquitous, obligate intracellular eukaryotic spore-forming parasites infecting a wide range of invertebrates and vertebrates, including humans. The defining structure of microsporidia is the polar tube, which forms a hollow tube through which the sporoplasm is transferred to the host cell. Research on the molecular and cellular biology of the polar tube has resulted in the identification of three polar tube proteins: PTP1, PTP2, and PTP3. The major polar tube protein, PTP1, accounts for at least 70% of the mass of the polar tube. In the present study, PTP1 was found to be posttranslationally modified. Concanavalin A (ConA) bound to PTP1 and to the polar tube of several different microsporidia species. Analysis of the glycosylation of Encephalitozoon hellem PTP1 suggested that it is modified by O-linked mannosylation, and ConA binds to these O-linked mannose residues. Mannose pretreatment of RK13 host cells decreased their infection by E. hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannose-binding molecule. A CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility to E. hellem infection compared to wild-type CHO cells. These data suggest that the O-mannosylation of PTP1 may have functional significance for the ability of microsporidia to invade their host cells.     

PCR for the diagnosis and species identification of microsporidia in patients with keratitis

Corneal scrapings from 30 patients with microbial keratitis were subjected to microsporidial PCR. PCR was positive for microsporidia in ten of 30 patients. The species was identified as Vittaforma corneae by sequencing in all ten patients. The remaining 20 patients were negative for microsporidia and showed the growth of other organisms (Acanthamoeba, fungi or bacteria).     

Opportunistic pneumonia caused by E. cuniculi in mice immunosuppressed with cyclophosphamide

Opportunistic fungal pneumonia is a cause of concern in immunocompromised patients due to its high morbidity and mortality rates. One such opportunistic agent affecting immunocompromised patients is the microsporidia called Encephalitozoon cuniculi. This study aimed to evaluate pneumonia caused by E. cuniculi in mice treated with the immunosuppressive agent cyclophosphamide (Cy). This study also aimed to describe the immune cells associated with the microsporidial pneumonia. C57BL/6 mice were infected intravenously with E. cuniculi spores and treated with Cy (75 mg/kg/week, intraperitoneally). Thirty days post-infection, the fungal burden (qPCR), histopathological lesions, cytokine production, and the phenotype of the immune cells in the lung parenchyma were evaluated. Histologically, interstitial pneumonia with lymphocytic infiltrate was observed in the infected animals. The infiltrate mainly consisted of CD8⁺ and CD4⁺ T lymphocytes, with reduced populations of B lymphocytes and macrophages. The production of tumor necrosis factor-alpha (TNF-α) was significant in the animals of the infected groups. Also, the fungal burden was higher in the Cy-treated animals, which was confirmed by the immunohistochemical observation of spores. These results demonstrated that E. cuniculi infection of C57BL/6 mice caused lymphocytic interstitial pneumonia (characterized by a predominant lymphocytic infiltrate), which was aggravated by Cy-induced immunosuppression. Thus, these results can be used to understand the different pathological, immunological, and therapeutic aspects of lymphocytic interstitial pneumonia.     

Apport de la PCR dans la recherche et l’identification des microsporidies intestinales chez les sujets infectés par le VIH

Aim

Intestinal microsporidiosis are among the most frequent opportunistic diseases in immunocompromised subjects. This study aimed to evaluate the contribution of PCR for a better detection and species identification of microsporidia in stool specimens of HIV-infected patients.

Patients and methods

Stool samples obtained from 119 HIV-infected Tunisian subjects were screened for intestinal microsporidiosis by light microscopy using Weber's modified Trichrome stain and by a PCR method using universal primers V1/PMP2 which amplified a common fragment of the small subunit rRNA gene of microsporidia. The obtained PCR products were then sequenced using an ABI PRISM 377 DNA sequencer.

Results

The results showed a better sensitivity of PCR in the detection of microsporidia with an infection rate of 14.3% significantly higher than that of 6.7% obtained by light microscopy (p = 0.03). As previously described, intestinal microsporidiosis was associated with low CD4 cell counts; 23.9% infection rate in patients having CD4 cell count under 200/mm³ against 5.6% in patients with higher CD4 cell count (p = 0.008). The sequencing of 15 out of the 17 positive PCR products has confirmed in all cases the species identified based on the PCR fragment size i.e., 250 pb for Enterocytozoon bieneusi (seven cases) and about 270 pb for Encephalitozoon intestinalis (nine cases); one case revealed a double infection.

Conclusion

PCR proved to be more effective than classical Trichrome stain for the diagnosis of intestinal microsporidiosis. Moreover, the ability of PCR to identify the species involved could also be useful for cases management.     

Monoclonal Antibodies against Enterocytozoon bieneusi of Human Origin

Enterocytozoon bieneusi is clinically the most significant among the microsporidia infecting humans, causing chronic diarrhea, wasting, and cholangitis in individuals with human immunodeficiency virus/AIDS. The lack of immune reagents is largely due to the absence of methods for laboratory propagation of E. bieneusi. We recently described a procedure for the concentration and purification of spores from diarrheic stool of infected humans. Purified spores were used to immunize mice for production and screening of monoclonal antibodies (MAbs) against E. bieneusi. The eight immunoglobulin M MAbs generated and fully characterized did not cross-react with other human microsporidia or with other microorganisms normally present in stool. One of the MAbs, 2G4, reacted with E. bieneusi spores in stools from monkeys and humans, without background fluorescence, which makes it an ideal diagnostic reagent. It also recognizes intracellular stages of the parasite and will be suitable for determining tissue distribution of E. bieneusi in infected hosts. At least two immunodominant antigens of E. bieneusi of 33,000 and 35,000 Da exist, which were recognized by rabbit and mouse antisera. The availability of MAbs against E. bieneusi will simplify considerably the diagnosis of this infection in humans and will provide tools for epidemiologic investigations regarding the true prevalence of the infection in various human and mammalian populations and the environmental sources of infection.     

Prevalence and diversity of Encephalitozoon spp. and Enterocytozoon bieneusi in wild boars (Sus scrofa) in Central Europe

From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveyed at 29 randomly selected localities (both forest areas and enclosures) across four Central European countries: Austria, the Czech Republic, Poland, and the Slovak Republic. Isolates were genotyped by PCR amplification and characterization of the internal transcribed spacer (ITS) region using Enterocytozoon and Encephalitozoon-specific protocols. PCR revealed 16 mono-infections of Encephalitozoon cuniculi, 33 mono-infections of Enterocytozoon bieneusi and 5 concurrent infections of both Encephalitozoon cuniculi and Enterocytozoon bieneusi out of 460 faecal samples. Two genotypes (I and II) were revealed by sequence analysis of the ITS region of Encephalitozoon cuniculi. Eleven genotypes, five previously found in other hosts including domestic pigs (D, EbpA, EbpC, G and Henan-I) and six novel (WildBoar1–6), were identified in Enterocytozoon bieneusi. No other microsporidia infection was found in the examined faecal samples. Prevalence of microsporidia at the locality level ranged from 0 to 58.8 %; the prevalence was less than 25 % at more than 86 % of localities. Enterocytozoon bieneusi was detected as a predominant species infecting Eurasian wild boars (Sus scrofa). The present report is the most comprehensive survey of microsporidia infections in wild boars within the Czech Republic and selected Central European countries.     

B-1 cell decreases susceptibility to encephalitozoonosis in mice

Encephalitozoon cuniculi is an opportunist intracellular pathogen of mammals. The adaptive immune response is essential to eliminate E. cuniculi, but evidence is mounting that the response initiated by the innate immune response may ultimately define whether or not the parasite can survive. B-1 cells may act as antigen-presenting cells or differentiate into phagocytes, playing different roles in many infection models. However, the role of these cells in the dynamics of Encephalitozoon sp. infections is still unknown. To investigate the role of B-1 cells in E. cuniculi infection, BALB/c and BALB/c XID (B-1 cells deficient) mice were infected with E. cuniculi spores. Cytometric analyses of peritoneal cells showed that B-1 cells and macrophages increased significantly in infected BALB/c mice compared to uninfected controls. Despite the increase in the number of CD4⁺ and CD8⁺ lymphocytes in XID mice, these animals were more susceptible to infection as evidenced histologically with more prominent inflammatory lesions and parasite burden. Pro-inflammatory cytokines increased in both infected BALB/c and BALB/c XID mice. To confirm B-1 cells role in encephalitozoonosis, we adoptively transferred B-1 cells to BALB/c XID mice and this group showed few symptoms and microscopic lesions, associated with an increased in cytokines. Together, these results suggest that B-1 cells may increase the resistance of BALB/c mice to encephalitozoonosis, evidencing for the first time the important role of B-1 lymphocytes in the control of microsporidia infection.     

Histopathological evaluation of ocular microsporidiosis by different stains

Background  

There is limited data on comparing stains in the detection of microsporidia in corneal biopsies. Hence we wanted to evaluate various stains for their ability to detect microsporidia in corneal tissue sections.     

Morphological characterization and <Emphasis Type="Italic">HSP70</Emphasis>-, <Emphasis Type="Italic">IGS</Emphasis>-based phylogenetic analysis of two microsporidian parasites isolated from <Emphasis Type="Italic">Antheraea pernyi</Emphasis>

Two microsporidian isolates were extracted from single infected egg-laying tussah silk moth (Antheraea pernyi) in Liaoning Province, China. The microsporidia were subsequently grown in silk moth larvae, isolated, and subjected to morphological characterization (by light and transmission electron microscopy) and phylogenetic analysis (based on conserved genes). One type of spore was long-axis-oval in shape, measuring 4.71 × 1.95 μm, and the other type was short-axis-oval, measuring 3.64 × 2.17 μm. These dimensions were markedly different from those reported in the spores of the common microsporidia infecting A. pernyi, namely, Nosema pernyi (4.36 × 1.49 μm). A neighbor-joining phylogenetic tree based on HSP70 indicated that these microsporidia belonged to Nosema species and were closely related with Nosema bombycis and Nosema ceranae. Furthermore, in the phylogenetic tree based on the intergenic spacer (IGS) region, the long-axis-oval isolates were closely related and tended to form a clade away from the short-axis-oval isolates and N. pernyi isolates. The microsporidia isolated from A. pernyi clustered in one group. Nosema bombycis, Nosema spodopterae, and Endoreticulatus spp. appeared to be genetically distant from N. pernyi. The two isolates from A. pernyi fell in the Nosema group, but their spores differed from those of the spores of the common A. pernyi parasite N. pernyi, both in morphological and genetic aspects. The two isolates were designated Nosema sp. Ap (L) and Nosema sp. Ap (S). IGS was found to be informative in ascertaining phylogenetic relationships among species, and even closely related strains, of microsporidia.     

Induction of Resistance by Listeria monocytogenes Cell Wall Fraction

A crude cell wall fraction of Listeria monocytogenes was prepared by sonic disruption and differential centrifugation of viable, washed cultures. When injected into mice, this sterile, crude cell wall fraction protected mice against an intraperitoneal challenge with 18 to 85 50% mean lethal dose of L. monocytogenes. Resistance was greatly enhanced when bacterial endotoxin (lipopolysaccharide) was injected along with the cell wall fraction. Resistance was measured both by enumerating the bacteria in the livers and spleens of vaccinated and control mice and by survival studies. Two major lines of evidence suggest that the resistance induced by cell wall fraction is at least in part specific. Unlike non-specific resistance, the cell wall fraction-induced resistance was relatively long-lived, (i.e., it was demonstrable 6 weeks after the last injection of cell wall fraction and lipopolysaccharide). In addition, cell wall fraction protected against challenge with L. monocytogenes, but not against challenge with S. typhimurium.     

Critical aspects of the Nosema spp. diagnostic sampling in honey bee (Apis mellifera L.) colonies

Nosemosis is one of the most widespread of the adult honey bee diseases and causes major economic losses to beekeepers. Two microsporidia have been described infecting honey bees worldwide, Nosema apis and Nosema ceranae, whose seasonality and pathology differ markedly. An increasing prevalence of microsporidian infections in honey bees has been observed worldwide during the last years. Because nosemosis has detrimental effects on both strength and productivity of the infected colonies, an accurate and reliable method to evaluate the presence of Nosema in honey bee colonies is needed. In this study a high degree of variability in the detection of microsporidia depending on the random subsample analyzed was found, suggesting that both sample size and the time of collection (month and day of sampling) notably affect the diagnosis.     

Concurrent Infection of the Urinary Tract with Encephalitozoon cuniculi and Enterocytozoon bieneusi in a Renal Transplant Recipient

A urinary tract coinfection, caused by Encephalitozoon cuniculi genotype II and Enterocytozoon bieneusi genotype D, was identified in an HIV-seronegative renal transplant recipient kept under lifelong immunosuppression. To our knowledge, this is the first report describing concurrent infection with these two microsporidia species in organ transplant recipients.     

Germination of phagocytosed E. cuniculi spores does not significantly contribute to parasitophorous vacuole formation in J774 cells

The obligate intracellular microsporidia have developed a unique invasion mechanism to infect their host cells. Spores explosively evert a tube-like structure and extrude the infectious spore content through this organelle into the host cell. Spores from species of the genus Encephalitozoon were also shown to be efficiently internalized by phagocytosis, which led to the hypothesis that spore germination from inside a phagosome might contribute to the infection process. Here, we challenge this hypothesis by quantifying Encephalitozoon cuniculi infection rates of J774 cells that were incubated with the phagocytosis inhibitor cytochalasin D. We demonstrate that the invasion rate in cytochalasin D-treated cells is identical to untreated controls, although phagocytic uptake of E. cuniculi spores was less than 10% of control samples. This study suggests that germination of phagocytosed spores is not a significant infection mode for E. cuniculi.     

Gastric LTi cells promote lymphoid follicle formation but are limited by IRAK-M and do not alter microbial growth

Lymphoid tissue inducer (LTi) cells are activated by accessory cell IL-23, and promote lymphoid tissue genesis and antibacterial peptide production by the mucosal epithelium. We investigated the role of LTi cells in the gastric mucosa in the context of microbial infection. Mice deficient in IRAK-M, a negative regulator of TLR signaling, were investigated for increased LTi cell activity, and antibody mediated LTi cell depletion was used to analyze LTi cell dependent antimicrobial activity. H. pylori infected IRAK-M deficient mice developed increased gastric IL-17 and lymphoid follicles compared to wild type mice. LTi cells were present in naive and infected mice, with increased numbers in IRAK-M deficient mice by two weeks. Helicobacter and Candida infection of LTi cell depleted rag1^−/− mice demonstrated LTi-dependent increases in calprotectin but not RegIII proteins. However, pathogen and commensal microbiota populations remained unchanged in the presence or absence of LTi cell function. These data demonstrate LTi cells are present in the stomach and promote lymphoid follicle formation in response to infection, but are limited by IRAK-M expression. Additionally, LTi cell mediated antimicrobial peptide production at the gastric epithelium is less efficacious at protecting against microbial pathogens than has been reported for other tissues.     

Infections of Stentor roeseli and S. polymorphus (Ciliophora,Heterotrichida) by microsporidia

In cells of the ciliated protozoa Stentor polymorphus and S. roeseli, infections by microsporidia were observed. In both stentors, the microsporidia were maintained in the laboratory and new cells could be infected when transferred to a culture of parasitized cells. The parasites were more pathogenic on S. roeseli than on S. polymorphus, which harbors green algal endocytobionts.     

Identification of Encephalitozoon intestinalis in Travelers with Chronic Diarrhea by Specific PCR Amplification

With the use of Weber’s modified trichrome and Uvitex 2B techniques, spores of microsporidia were detected in the stools of four travelers presenting clinically with chronic diarrhea. The general health of these patients was not impaired, and human immunodeficiency virus screening was negative. Immune evaluation, including the study of lymphocytic subpopulations, assay of serum immunoglobulins, and an intradermal multitest, showed normal results. Molecular identification of microsporidian species was based on the PCR amplification of a small-subunit rRNA sequence followed by HinfI endonuclease restriction. Encephalitozoon intestinalis microsporidiosis was thus shown in two of the four patients examined. In two patients, therapy based on albendazole made stools devoid of microsporidian spores without influence on the intestinal disorders. The pathogenic role of E. intestinalis in immunocompetent individuals remains to be demonstrated.     

In vitro cultivation of microsporidia of clinical importance

Although attempts to develop methods for the in vitro cultivation of microsporidia began as early as 1937, the interest in the culture of these organisms was confined mostly to microsporidia that infect insects. The successful cultivation in 1969 of Encephalitozoon cuniculi, a microsporidium of mammalian origin, and the subsequent identification of these organisms as agents of human disease heightened interest in the cultivation of microsporidia. I describe the methodology as well as the cell lines, the culture media, and culture conditions used in the in vitro culture of microsporidia such as Brachiola (Nosema) algerae, Encephalitozoon cuniculi, E. hellem, E. intestinalis, Enterocytozoon bieneusi, Trachipleistophora hominis, and Vittaforma corneae that cause human disease.     

Peritoneal T lymphocyte regulation by macrophages

The T cell composition of the peritoneal cavity (PerC) in naïve BALB/c, C57BL/6, DBA/2J, and B-1 B cell-defective BALB.xid mice was investigated. The BALB.xid PerC T cell pool had a high CD4:CD8 T cell ratio relative to the other strains whose ratios were similar to those found in their lymph node and spleen. All mice had significant representation of T cells with an activated (CD25⁺, GITR^hi, CD44^hi, CD45RB^lo, CD62L^lo) phenotype and low numbers of Foxp3⁺ T_reg cells in their PerC. Despite a phenotype indicative of activation, peritoneal T cell responses to CD3 ligation were very low for C57BL/6 and BALB.xid, but not BALB/c, mice. Enzyme inhibition and cytokine neutralization studies revealed active suppression of the T cell response mediated by the macrophages that represent a significant portion of PerC leucocytes. Driven by IFNγ to express iNOS, macrophages suppressed T cell activation in vitro by arginine catabolism. Although BALB/c T cells were also in a macrophage-dense environment their limited IFNγ production failed to trigger suppression. This difference between BALB/c and BALB.xid PerC T cells suggests a role for xid in shaping macrophage-mediated immune regulation.     

A Powerful DNA Extraction Method and PCR for Detection of Microsporidia in Clinical Stool Specimens

The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biopsy samples by light and/or electron microscopy. Limited information about the specificity and sensitivity of PCR for the detection microsporidia in clinical stool specimens is available. To establish a sensitive and specific method for the detection of microsporidia in clinical samples, we studied clinical stool specimens of 104 randomly selected human immunodeficiency virus-infected patients with diarrhea to compare light microscopy and PCR. Fluorochrome Uvitex 2B staining was used for light microscopy. To raise the sensitivity of PCR, we used a powerful and fast DNA extraction method including stool sedimentation, glass bead disruption, and proteinase K and chitinase digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1-SI500, and the nature of the PCR products was confirmed by Southern blot hybridization. Microsporidiosis was diagnosed by light microscopy in eight patients. Ten patients tested positive for microsporidiosis by PCR. Enterocytozoon bieneusi was found in seven cases, and Encephalitozoon intestinalis was found in four cases. In one case a double infection with E. bieneusi and E. intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagnosis and species differentiation of intestinal microsporidiosis in HIV patients.