First Complete Amino Acid Sequence of a Polar Tube Protein in a Microporidian Species, Encephalitozoon cuniculi

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First Complete Amino Acid Sequence of a Polar Tube Protein in a Microsporidian Species, Encephalitozoon cuniculi. FRkDERIC DELBAC. DANIELLE DAVID, GUY MkTENIER (JRFUSTYJYW.&S Pronstologie Moleculaire et Cellulaire des Parasites Oppormnisfes,UPEU CNRS 6023, UnrverstteBlatse Pascal, 631 77Aubiere Ceder. France.

Microsporidia exhibit a peculiar invasive process involving the e x m i o n of the polar tube. Very few information is available on polar tube components and mechanisms of extrusion and invasion. Great inter-species similarities in the structural organization of the polar tube let suggest that some of its components may be useful to identify possible common structural motifs of polar tube proteins (PTP) in order to determine new molecular probes for diagnosis purposes and some therapeutic strategies for the treatment of microsporidian infections. Recently, a proline rich 43-kDa PTP has been isolated in the fish microsporidian Glugea amencanus, but sequencing data are limited to a short N-terminal part [21. In the present study, we report the first complete amino acid sequence of a PTP in the mammal microsporidian Encephalitozoon cuniculi. M A T E W S AND METHODS. E. cuniculi was groan in vitro in MDCK cells. Proteins were separated by either SDS-PAGE as previously described [11 or hvo-dimensional electrophoresis and transferred onto PVDF membranes for western blotting. For isoelectrofocusing (EF), proteim were exbacted from spores in a buffer containing 9 M urea, 40 mM CHAPS and 5% 2mercaptoethanol. The ampholite combination was 40% pH 3-10 and 60% pH 4 - 6 5 A 55-kDa protein, reacting with antibodies directed against the polar tube, was excised kom two-dimensional gel and analyzed for microsequencing. After digestion with the endoproteaseLys C at 35 C for 18 h, peptides were separated by HF’LC on a DEAECIS column with acetonitnle gradient / 0.1% txifluoroacetic acid (TFA). Degenerate primers determined from the two peptides were used for PCR amplification: after denaturation of the DNA at 94 C for 5 min, 35 cycles were run as follows : denaturation at 94 C for 1 min, annealing at 54 C for 1 min and elongation at 72 C for 2 min. Amplified products were cloned into pCR2 TA cloning vectors (Invitrogen). Recombinant plasmids were sequenced by the Sanger method, using the ABI Prism 377 sequencer (Perkin Elmer). Gel readings were treated using the Staden package available on the french molecular biology server Infobiogen. 5’ and 3’ ends of the gene were determined using the single speclfic primer (SSP) PCR technique [3]. The amplified product of about 1 kbp was subcloned into the expression vector pQE30, 6XHis (QIAGEN) and exqxession was performed in the Eschenchia coli M15 strain. Recombinant protein was purified by nickel chelate chromatography then injected in mice. Corresponding anbsera were tested in western blotting, immunofluorescence assays (FA) and electron microscopy immunogold labelling. RESULTS AND DISCUSSION. In order to identify Encephnlitozoon cunicufi PTPs, polyclonal and monoclonal antibodies were first produced. Western blot analysis of SDS-PAGE separated proteins allowed to demonstrate PTP multiplicity with at least two proteins of 55 and 35 kDa in size. Antisem anti55 kDa and -35 kDa, and a monoclonal antibody (Ec 102), reacting with three protein bands ( 5 5 , 3 5 and 28 m a ) , were tested in FA. Both antibodies decorated the polar tube either within the spore or wfien extruded. The 55-kDa PTP was separated by twodimensional electrophoresis and two peptide fragments of 15 and 20 amino acids were sequenced.

Using degenerate primers determined fiom each peptide, a 1-kbp DNA fragment was PCR amplified, cloned then sequenced. Hybridization of E. cuniculi chromosomal bands separated by pulsed field gel electrophoresis (molecular karyotype comprising 11 chromosomes) indicates a location of the gene on chromosome 6. In addition, Southern blot analysis of E. cuniculi genomic DNA digested with m e r e n t restriction endonucleases shows that the P I P gene exists as a single copy per haploid genome. To ascertain the assignment of the protein to the polar tube, the corresponding gene was subcloned in an e?