Identification of new polar tube and spore wall proteins in different microsporidian species. First attempts for the transfection of the microsporidia Encephalitozoon cuniculi
Identification de nouvelles protéines du tube polaire et de la paroi sporale chez différentes espèces microsporidiennes. Essais de tranfection d'Encephalitozoon cuniculi
Résumé
Microsporidia are obligate intracellular parasites producing spores surrounded by a thick cell wall and containing the polar tube, a unique invasion apparatus coiled within the cytoplasm. Under stimuli, the polar tube is suddenly extruded allowing the transfer of the infectious content, named the sporoplasm, within the cytoplasm of a new host cell. Two polar tube proteins (PTP1 and PTP2), the genes of which are tandemly arranged, have been previously described in the three Encephalitozoon species. A third protein of higher size, PTP3, was only identified in E. cuniculi. To improve our knowledge of the polar tube composition and architecture, the search for new polar tube components has been undertaken. The comparison of E. cuniculi and Antonospora locustae genomes reveals significant gene order conservation (synteny). This syntenic relationship facilitated the characterisation of PTP1 and PTP2 proteins in A. locustae despite their high sequence divergences with those of E. cuniculi. In addition, the A. locustae genome is characterized by the presence of several ptp2 like genes. The ptp1 and ptp2 gene cluster organization is also conserved in Paranosema grylli. An E. cuniculi proteomic analysis of proteins expressed in late sporogonial stages was helpful in the identification of two new PTPs (PTP4 and PTP5) the genes of which are tandemly arranged. These proteins, presenting no homology with other known PTPs, have been also identified in A. locustae and P. grylli. PTP4 is localized at the end terminal region of extruded polar tubes suggesting a role in host cell interactions. Co-expression experiments in E. coli showed the existence of interactions between PTP1 and PTP2. Moreover, molecular data concerning spore wall components indicate the presence of SWP1 proteins in the exospore of both E. cuniculi and E. intestinalis. In the present study, we identified the whole sequences of two SWP1-like proteins in E. hellem. The high variability of their C-terminal regions is of interest for epidemiological studies. Finally, for functional genomic approaches, the development of a transfection strategy of the microsporidia E. cuniculi was initiated. Only few transient transformants have been obtained.
Les microsporidies, parasites intracellulaires obligatoires, forment des spores délimitées par une paroi épaisse, qui renferment un appareil invasif original, le tube polaire à l'origine du transfert du matériel infectieux dans une cellule hôte. PTP1 et PTP2, protéines du tube polaire ont été décrites au sein du genre Encephalitozoon. Pour améliorer nos connaissances sur la composition du tube polaire, de nouvelles PTP ont été recherchées. L'identification des protéines PTP1 et PTP2 chez Antonospora locustae a été facilitée par la conservation de l'organisation des gènes (synténie) entre E. cuniculi et A. locustae. L'analyse du protéome d'E. cuniculi a permis d'identifier deux nouvelles PTP conservées chez A. locustae. Des études de co-expression chez E. coli ont montré des interactions entre PTP1 et PTP2. Chez E. hellem, les séquences complètes de 2 protéines localisées au niveau de l'exospore ont été caractérisées. Enfin, des essais de transfection d'E. cuniculi ont été réalisés