Abstract
The protein P29 is a potential serological marker for post-treatment monitoring of cystic echinococcosis (CE) especially in young patients. We now have demonstrated that P29 is encoded in the Echinococcus genus by a single gene consisting of 7 exons spanning 1.2 kb of DNA. Variability of the p29 gene at inter- and intra-species level was assessed with 50 cDNA and 280 genomic DNA clones isolated from different E. granulosus s.l. isolates (E. granulosus sensu stricto (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6), E. canadensis (G7) and E. canadensis (G10)) as well as four E. multilocularis isolates. Scarce interspecies polymorphism at the p29 locus was observed and affected predominantly E. granulosus s.s. (G1), where we identified two alleles (A1 and A2) coding for identical P29 proteins and yielding in three genotypes (A1/A1, A2/A2 and A1/A2). Genotypic frequencies expected under Hardy-Weinberg equilibrium revealed a high rate of heterozygosity (47%) that strongly supports the hypothesis that E. granulosus s.s. (G1) is predominantly outbreeding. Comparative sequence analyses of the complete p29 gene showed that phylogenetic relationships within the genus Echinococcus were in agreement with those of previous nuclear gene studies. At the protein level, the deduced P29 amino acid (AA) sequences exhibited a high level of conservation, ranging from 97.9% AA sequence identity among the whole E. granulosus s.l. group to 99.58% identity among E. multilocularis isolates. We showed that P29 proteins of these two species differ by three AA substitutions without implication for antigenicity. In Western-blot analyses, serum antibodies from a human CE patient infected with E. canadensis (G6) strongly reacted with recombinant P29 from E. granulosus s.s. (G1) (recEg(G1)P29). In the same line, human anti-Eg(G1)P29 antibodies bound to recEcnd(G6)P29. Thus, minor AA sequence variations appear not to impair the prognostic serological use of P29.
Highlights
Human echinococcosis is a severe disease caused by infection with the metacestode stage of tape-worms belonging to the genus Echinococcus [1]
We experimentally addressed the genetic polymorphism of the p29 gene within a relatively large selection of members of the Echinococcus genus, in order to predict the reliability of this serological tool upon use for cystic echinococcosis (CE) in humans infected with different genotypes of E. granulosus sensu lato
Molecular characterization of p29 The exon/intron organization of p29 was characterized by comparing (i) the p29 cDNA sequences isolated from 5 Tunesian E. granulosus s.s. (G1) sheep isolates (732 nucleotides, deduced sequence of 238 amino acid (AA)) with (ii) the published genomic sequence
Summary
Human echinococcosis is a severe disease caused by infection with the metacestode stage of tape-worms belonging to the genus Echinococcus (family Taeniidae) [1]. E. granulosus sensu lato (E. granulosus s.l.) and E. multilocularis are the most common species infecting humans, hereby causing cystic echinococcosis (CE) or alveolar echinococcosis (AE), respectively. In the post-surgical or medical echinococcosis therapy program, the risk of recurrence represents the major problem [3,4,5]. In this respect, post-treatment follow- up of patients for several years is mandatory to detect potential recurrences as soon as possible. For CE, an agreement on which tests should be used, and how these tests should be applied and interpreted, is still lacking
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