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Microsporidia are unicellular eukaryotes occuring 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 singlecopy 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. Sprague, 1977) are small spore-forming unicellular eukaryotes with an obligate intracellular parasitic lifestyle. These parasites, characterized by 70S ribosomes and the absence of mitochondria, were thought to be very ancient (10). However, data accumulated from recent molecular phylogenies lend credit to a close relationship of these organisms with fungi (36). Several species are of medical and veterinary significance, infecting animals and humans (7). Three species from the Encephalitozoon genus (E. cuniculi, E. hellem, and E. intestinalis) are known to be involved in AIDSassociated pathologies (13). Disorders in immunocompetent individuals also have been reported. For example, E. intestinalis was found in travelers, not infected with human immunodeficiency virus, presenting with chronic diarrhea (33). Serological studies with blood donors and pregnant women revealed a prevalence of about 8% (37). Microsporidia (phylum MicrosporaMicrosporidia exhibit a remarkable invasion mechanism depending on the extrusion of a specific organelle called the polar tube, originally coiled within the spore. The polar tube discharges from the anterior pole of the spore like an everting glove finger (25) and then is used to transfer the sporoplasm inside a potential host cell. The whole process of in vitro spore germination is completed in less than 2 s (17). Very little information is available about the primary structure of polar tube proteins (PTPs) and the extent of interspecies sequence variability. Molecular characterization of the polar tube is therefore of importance for improving diagnostic and defining ther...