Microsporidian invasion apparatus: identification of a novel polar tube protein and evidence for clustering of ptp1 and ptp2 genes in three Encephalitozoon species.

Microsporidia are unicellular eukaryotes occurring as obligate intracellular parasites which produce resistant spores. A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell. The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for Encephalitozoon cuniculi and E. hellem. Our immunological investigations provided evidence for an additional PTP in E. cuniculi, termed PTP2. The corresponding gene was sequenced and then expressed in Escherichia coli. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The single copy ptp1 and ptp2 genes of E. cuniculi were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infecting Encephalitozoon species (ptp2 in E. hellem and ptp1 and ptp2 in E. intestinalis) were facilitated by conserved gene clustering. PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail. Unlike PTP1, this protein is devoid of large tandem repeats. The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly. The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.

Polymorphism of the gene encoding a major polar tube protein PTP1 in two microsporidia of the genus Encephalitozoon.

Isolates of 2 microsporidian species from the genus Encephalitozoon (E. cuniculi and E. hellem) were compared by analysis of DNA amplified from a gene region encoding the repeat domain of a polar tube protein (PTP1). Sequence data obtained for 11 E. cuniculi isolates from 5 different mammalian hosts well support the existence of 3 previously designated strains. Strain type III was characterized by a lack of a 78 bp repeat, producing an amplicon of reduced size. Strain type II differed from strain type I by 3 nucleotide substitutions so that AvalI digestion of the corresponding PCR products provided distinct restriction patterns. Surprisingly, the comparison of 2 human isolates of E. hellem belonging to the same rDNA ITS genotype shows a high level of heterogeneity through numerous point mutations and variation in PTP1 repeat number. Further characterization of additional E. hellem isolates based on PTP1 sequence polymorphisms should be of interest for tracing sources of infection.