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.

FIG. 1

Sequence differences in the rRNA ITS among E. hellem genotypes. Dots denote sequence identity to genotype 1A, and dashes depict nucleotide deletions. Genotypes 1A, 1B, and 1C = genotype 1 of Mathis et al.; genotype 2A = genotype 2 of Mathis et al.; genotype 2C = genotype 3 of Mathis et al. (21).

FIG. 2

Sequence diversity among E. hellem genotypes in the PTP gene. Dots denote sequence identity to genotype 1A (AF044915), and dashes depict nucleotide deletions. The repeat region is underlined, and the primer sequences used in direct genotyping PCR are double underlined. The numbers at the ends of lines are nucleotide positions in AF044915.

FIG. 3

Genotyping E. hellem isolates by PCR analysis of the PTP gene. Lanes 1 and 9, 100-bp ladders; lanes 2 and 5, genotype 1B; lanes 3 and 4, genotype 1A; lane 6, genotype 2B; and lanes 7 and 8, genotype 1C.