The Human Microsporidian Encephalitozoon hellem Synthesizes Two Spore Wall Polymorphic Proteins Useful for Epidemiological Studies

摘要

Microsporidia are obligate intracellular fungus-related parasites considered as emerging opportunistic human pathogens. Their extracellular infective and resistance stage is a spore surrounded by a unique plasma membrane protected by a thick cell wall consisting of two layers: the electron-lucent inner endospore which contains chitin and protein components and the outer-electron-dense and mainly proteinaceous exospore. We identified the whole sequences of two spore wall proteins in the microsporidian species Encephalitozoon hellem, designated EhSWP1a and EhSWP1b. Isolation of the genes encoding these SWP1-like proteins was performed using degenerate oligonucleotides based on the amino acid sequence alignment of the previously reported Encephalitozoon cuniculi and Encephalitozoon intestinalis SWP1s. Sequences lacking the 5′ and 3′ ends were then identified by PCR and reverse transcription (RT)-PCR amplifications. The swp1a and swp1b genes encode proteins of 509 and 533 amino acids, respectively, which present an identical N-terminal domain of 382 residues and a variable C-terminal extension mainly characterized by a 26-amino-acid (aa) deletion/insertion containing glutamate- and lysine-rich repeats. Using polyclonal antibodies raised against recombinant polypeptides, we showed that EhSWP1a and EhSWP1b appear as dithiothreitol (DTT)-soluble bands of 55 and 60 kDa in size, respectively. Immunolocalization experiments by IFA and transmission electron microscopy (TEM) indicated that both proteins are present at the onset of sporogony and are specifically located to the spore wall exospore in mature spores. Analysis of four E. hellem human isolates revealed that the C-terminal regions of both EhSWP1a and EhSWP1b are polymorphic, which is of interest for epidemiological studies.