Genetic and Immunologic Characterization of Seven Encephalitozoon hellem Human Strains
Over the last few decades microsporidia have emerged as opportunistic pathogens. Recent studies suggest a high presence of human-related microsporidia in nature, including immunocompetent individuals as well as different animal hosts. Encephalitozoon hellem is the third most frequent microsporidia causing clinical disease in humans. It was first isolated from ocular lesions in three AIDS patients [3] and subsequently, it has been found to cause respiratory, urogenital and disseminated microsporidiosis [6]. Until recently this microsporidia was considered exclusive of human hosts but it has recently been reported in avian hosts. To our knowledge, five reports exist in non-human hosts, four in psittacine birds [2,9,12,13] and the other in an ostrich [11]. The zoonotic potential of this microsporidia has been highlighted by the fact that it has been isolated not only in animals with clinical symptoms but also in a clinically normal pet bird [12].
Immunochemical and genetic characterization has proved to be useful in elucidating the epidemiology and the zoonotic potential of microsporidia. Genotype differentiation based on variation in the internal transcriber spacer (ITS) sequence of the rDNA and in the immunochemical characteristics has been achieved in three of the human microsporidia (Enterocytozoon bieneusi, Encephalitozoon cuniculi and E. hellem). In the case of E. hellem, three ITS genotypes have been described. ITS genotype 1 was the most frequently reported in humans and also the one found in avian hosts [7,12]. The immunochemical profiles of the three ITS genotypes are also differentiable [7].
To contribute to the knowledge of the epidemiology of E.hellem, we have immunochemically and genetically characterized 7 strains of E. hellem isolated from American, Italian and Spanish AIDS patients.
MATERIALS AND METHODS
Microorganisms
Seven strains of E. hellem were cultured on E6 monolayers and the spores were Percoll purified to be used for the immunochemical and genetic characterization [1]. All the strains were isolated from AIDS patients, but from different geographical regions; one (CDC: V213) [15] is from the United States, another from Spain (EHSV-96) [8] and 5 from Italy (MIPV-4–94 [10]; PV-5–95 [10]; MIPV-6–95; PV-7–95; PV-8–95).
SDS-PAGE and immunoblotting
One (μg of protein or 108 spores were suspended in the sample buffer (10%SDS, 9M urea, 0.1 M Tris-HC1) and heated 15 min at 65°C. Samples were loaded both onto 4–20% polyacrilamide (PA) linear resolving gels (Mini-gels: Ready Gel, BIO-RAD®) and on 12% PA gels with a 4% stacking gel. Afterwards, the gels were subjected to electrophoresis. A constant current of 200V was applied during 45 min. A continuous buffer system was used (95 mM Glycine, 12 mM Tris, 20 mM SDS). The separated proteins were either silver stained [14] or electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (0.2 μm, BIO-RAD), following the manufacturer's intructions for Western blot (BIO-RAD). The transfer buffer contained 25 mM Tris, 192 mM glycine and 20% (v/v) methanol, pH 8.3. For the transfer of proteins to PVDF membranes, the protein contents were increased to nearly three times those used for gels that were silver stained. The membranes were subsequently reacted for 1 hr at room temperature with a 1:400 dilution of rabbit anti-E.hellem serum. After appropriate washes, the membranes were treated at room temperature for 1 hr with a 1:3000 dilution of goat anti-rabbit peroxidase conjugated IgG (SIGMA). Hydrogen peroxidase (30%) and diaminobenzidine (0.005%) were used as the substrate and chromogen, respectively.
DNA extraction
DNA was extracted from 108 purified spores of all seven E. hellem isolates under study by using the Fast DNA Spin Kit (Biol0l). DNA concentration was measured at 260 nm. PCR. PCR was performed by using the primer pair A/B designed by Hollister et al [4], which amplifies a fragment of 208 bp including the ITS region. The forward primer A (5′ TTGTACACACCGCCCGTCG-3′) was designed on the basis of the sequence from positions 1186 to 1204 of the small-subunit (SSU) rDNA and the reverse primer B (5′CCGATAATGCCAATCAATCC-3′) on the sequence of the nucleotides from positions 1375 to 1394 of the large-subunit rDNA. PCR amplification was done by using a 25 μ1 reaction mix containing 50 ng of DNA, 0.2 mM of each dNTP, 0.2 μM each of the A and B primers, 1 unit of Taq DNA polymerase (Roche Molecular, Biochemicals) and the PCR buffer, including a final concentration of 1.5 mM MgCl2. After an initial step of 5 min at 80°C, a total of 35 cycles were performed as follows: 30s at 98°C (except for the first cycle which was made at 94°C), 30 s at 55°C and 1.5 min at 72°C. When the cycles were completed, an additional step of 9 min at 72°C was carried out and the reaction products were kept at 4°C. Polyacrylamide gel electrophoresis. A 10 μ1 aliquot from each PCR reaction, diluted 1:100 in sterile H2O containing Bromophenol Blue was run on a polyacrylamide (19:1) gel at 8% in TBE at a constant current of 130V during 22 hours. The PCR products were visualized by silver staining.
Sequencing
PCR products were purified using the kit Bioclean (Bio-tools) and were subsequently sequenced in each direction twice, in a ABI Prism 377 DNA sequencer (PE-Biosystems). Sequences were aligned with MegAlign software (DNAstar) to assess sequence homology and difference.
RESULTS AND DISCUSSION
When purified spores from all the 7 human isolates of E. hellem were compared using silver-stained polyacrylamide gel electrophoretic banding patterns, a similar but not identical pattern was observed (Fig1, 2). Differences that were confirmed by several runs in both kinds of gels used, gradient and fixed concentrations, were also observed, and more clearly, in the Western blot (Fig 3). The PV-7–95 strain from Italy showed a difference in the spacing of a double band in the 19- to 21-kDa and in the 15–17 kDa range and in a triple band in the 22–24 kDa range, along with a band at approximately 36 kDa which, in the other isolates, appeared at 38kDa. The other isolates looked very similar, with only slight differences. In this line, the American strain showed bands in the immunoelectrophoretic profile at 17 kDa and 18 kDa, not shared by the others and in the case of the Spanish strain, bands at 20, 29, 30, 32 and 43 kDa were not observed in the others.
The comparison of the rDNA fragments and sequences, which include the ITS of the seven strains analysed here, is shown in figures 4 and 5. It becomes evident that the six strains - MIPV4, PV-5–95, PV-6–95, PV-8–95, CDC:0291 and EHSV-96 - correspond to genotype 1 described by Katiyar et al. [5], as no differences exist in the ITS or the adjacent segments of rDNA. Li contrast, the sequence of the PV-7–95 strain is more similar to genotype 2 described by Mathis et al. [7];. However, it is not identical since, at the edge of the ITS, it has only one of the three CTTT repeats existing in genotype 2. It also differs from other sequences described as genotype 3 [7]. Thus, the sequence found for PV-7–95 must be considered a new genotype derived from genotype 2.
Although further differences could be established by the antigenic profiles, the greater ones matched the two different ITS genotypes. Differentiation of E.hellem genotypes by immunochemical as well as genetic characteristics has been described by others [7,12]. Mathis et al [7] found differences by Western blot analysis, particularly in the 55–60 kDa (double band with different spaces or single band) and 23–30 kDa (double band with different spaces). Under our experimental conditions no clear differences were observed in the 55–60 kDa range, but bearing in mind that molecular weights are approximate estimations, the differences observed in the 23–30 kDa [7] and in the 22–24 kDa range in our study could have the same origin.
Thus, the PV-7–95 strain is clearly differentiated and the isolates may show different genotypes even from the same location. Moreover, genotype 1 is also common in Europe (in our case, found in 5 out of 6 isolates) and the CTTT repeat is also prone to vary, producing new genotypes, such as that detected in PV-7–95. Further studies should be made to assess the existing level of variability and its epidemiological significance. Finally, genotype 1 is the one most frequently reported in humans [12] and the one observed in avian hosts. As clinically normal domestic birds have been reported to shed spores of E. hellem in their feces, epidemiological and comparative studies of this microsporidia from these hosts should be conducted to elucidate their zoonotic potential and to understand the significance of different genotypes. At present, while research is being done, severe immunossuppresed patients should be aware of the zoonotic risks of clinically normal avian pets.
ACKNOWLEDGEMENT
We are deeply thankful to Dr. G. S. Visvesvara for providing the E. hellem culture CDC:V213.
We are grateful to L. Hamalainen and Brian Crilly for help in the preparation of the manuscript.
This work was supported by grants from the Fundación San Pablo-CEU (09/98; 01/99) and from the Fundación Caja Madrid.
