Genotyping Encephalitozoon hellem Isolates by Analysis of the Polar Tube Protein Gene

Abstract

To develop an alternative genotyping tool, the genetic diversity of Encephalitozoon hellem was examined at the polar tube protein (PTP) locus. Nucleotide sequence analysis of the PTP gene divided 24 E. hellem isolates into four genotypes, compared to two genotypes identified by analysis of the internal transcribed spacer of the rRNA gene. The four PTP genotypes differed from each other by the copy number of the 60-bp central repeat as well as by point mutations. A simple PCR test was developed to differentiate E. hellem genotypes based on the difference in the size of PTP PCR products, which should facilitate the genotyping of E. hellem in clinical samples.

Intraspecies Genotype Variability of the Microsporidian ParasiteEncephalitozoon hellem

“…Allelic variants that differ in size due to the number of repeats of a given unit of small insertions or deletions may be distinguished by PCR, followed by electrophoresis in either agarose or polyacrylamide gels (depending on the absolute difference in the number of nucleotides). Thus, genotyping for PTP and ITS alleles is easy to perform by quite simple methods (33), although for our material the latter proved more difficult to distinguish since alleles 1 and 2D differ by only 2 bp in length. Nevertheless, in many instances some alleles differ in point nucleotide changes.…”

“…Since many mutations may be lethal or severely affect the normal gene products, functional genes are expected to show lower levels of variability than noncoding regions. The spore wall protein gene of E. cuniculi and that of the PTP in both E. cuniculi and E. hellem are the two only known polymorphic genes in these species (24,33,34). Although it is still not clear which physiological mechanisms are involved, such polymorphisms may have an adaptive significance since these proteins are important candidates for early interaction with the host cell (24).…”

“…The alignment of this sequence (GenBank accession no. AY171241) indicates that it does not fit with any of the six sequences described (33) and suggests that PV7-95 has a genotype probably derived from genotype 2A (Xiao et al [33]) that is equivalent to genotype 2 of Mathis et al (22). Since the differences involve both point mutations and small insertion or deletions, the variants were detected both after PAGE or sequencing.…”

“…The fragment analyzed here has 490 to 620 bp, which is much longer than the maximum 300-bp length preferred for an accurate SSCA (13,14). The basic unit repeated in PTP alleles may have either 60 or 66 bp, both involving different variants due to point nucleotide mutations (33). Isolates MiPV4-94, PV5-95, PV6-95, and PV8 share allele 1C and CDC:V257 has allele 1A, whereas the sequence obtained for EHVS96 (AY024342) corresponds to allele 1B; these variants have eight, seven, and six repeats of the 60-bp unit, respectively (Fig.…”

Laboratory Identification of the Microsporidia

“…Although some nucleic acid-based methods have been developed, commercial products are not yet available (17,18,24,25). PCR detection of E. intestinalis in intestinal biopsy specimens from AIDS patients has been reported (4).…”