Functional characterization of a putative aquaporin from Encephalitozoon cuniculi, a microsporidia pathogenic to humans

Abstract

The microsporidia are a group of obligate intracellular parasitic protists that have been implicated as both human and veterinary pathogens. The infectious process of these organisms is believed to be dependent upon the rapid influx of water into spores, presumably via aquaporins (AQPs), transmembrane channels that facilitate osmosis. An AQP-like sequence of the microsporidium Encephalitozoon cuniculi (EcAQP), when cloned and expressed in oocytes of Xenopus laevis, rendered these oocytes highly permeable to water. No permeability to the solutes glycerol or urea was observed. Pre-treatment of EcAQP-expressing oocytes with HgCl(2) failed to inhibit their osmotic permeability, as predicted from EcAQP's lack of mercury-sensitive cysteine residues near the NPA motifs which line the AQP aqueous pore. EcAQP exhibits sequence identity to AQP A of Dictyostelium discoideum (26%) and human AQP 2 (24%). Further study of AQPs in microsporidia and their potential inhibitors may yield novel therapeutic agents for microsporidian infections.

Fig. 1

EcAQP-injected Xenopus laevis oocyte swelling assay. After 1 min, EcAQP-injected oocytes had swelled an average of 8.0 vs. 1.8% water-injected controls. Inset: Altered water permeability (P⪡0.001) of EcAQP-injected oocytes (87.3±8.6 μm/s; n=12) vs. water-injected (19.3±2.0 μm/s; n=15). Legend: closed marker/bar, EcAQP-injected; open marker/bar, water-injected. (Mean±S.E.M; for clarity, error bars are only displayed every 15 s.).

Fig. 2

Swelling of EcAQP oocytes is not inhibited by H_gCl₂. Permeability differences between H_gCl₂-treated and -untreated oocytes were not significant (where n>1) for all tested concentrations (0.1 μM, n=2; 1 μM, n=1; 10 μM, n=5; 200 μM, n=4; 1 mM, n=2; untreated, n=12).

Fig. 3

An unrooted phylogenetic tree of aquaporins (AQP) including EcAQP. This is based on CTREE alignment of protein sequences of EcAQP (GenBank accession no. NP_586002), closest BLAST match AQP A of Dictyostelium discoideum (BAA85158), human AQPs 0-9 (NP_036196, NP_932766, NP_000477, NP_004916, P55087, NP_001642, Q13520, NP_001161, O94778, NP_066190, respectively), plant aquaporins of Arabidopsis thaliana (P25818) and Nicotiana tabacum (CAA69353), parasitic protist aquaporins of Leishmania major (AAS73184), Plasmodium falciparum (CAC88373), Toxoplasma gondii (CAE46485), Trypanosoma cruzi (AAM76680), and AQP 2 of the yeast Saccharomyces cerevisiae (AAD10058). Stars indicate aquaglyceroporins.

Fig. 4

Alignment of EcAQP, human AQPs 1 (accession no. NP_932766), 2 (NP_000477), and AQP A of Dictyostelium discoideum (BAA85158). Highlighted in black is the residue position at which the presence of a cysteine confers mercury-sensitivity to AQP 1; gray highlights are the NPA motifs thought to line the water-conducting pore of each AQP monomer. Asterisks indicate fully conserved residues; two dots, conservation of strong groups; one dot, conservation of weak groups.