Functional Dissection of the Apicomplexan Glideosome Molecular Architecture

SummaryThe glideosome of apicomplexan parasites is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45. We identify GAP40, a polytopic protein of the IMC, as an additional glideosome component and show that GAP45 is anchored to the PM and IMC via its N- and C-terminal extremities, respectively. While the C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. The orthologous Plasmodium falciparum GAP45 can fulfill this dual function, as shown by transgenera complementation, whereas the coccidian GAP45 homolog (designated here as) GAP70 specifically recruits the glideosome to the apical cap of the parasite

Identification de nouvelles protéines du tube polaire et de la paroi sporale chez différentes espèces microsporidiennes. Essais de tranfection d'Encephalitozoon cuniculi

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 réalisé

The Microsporidian Polar Tube and Its Role in Invasion

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Identification de nouvelles protéines du tube polaire et de la paroi sporale chez différentes espèces microsporidiennes (Essais de tranfection d'Encephalitozoon cuniculi)

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 réalisésCLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

Versatility in the acquisition of energy and carbon sources by the Apicomplexa

Members of the phylum Apicomplexa are motile and rapidly dividing intracellular parasites, able to occupy a large spectrum of niches by infecting diverse hosts and invading various cell types. As obligate intracellular parasites, most apicomplexans only survive for a short period extracellularly, and, during this time, have a high energy demand to power gliding motility and invasion into new host cells. Similarly, these fast-replicating intracellular parasites are critically dependent on host-cell nutrients as energy and carbon sources, noticeably for the extensive membrane biogenesis imposed during growth and division. To access host-cell metabolites, the apicomplexans Toxoplasma gondii and Plasmodium falciparum have evolved strategies that exquisitely reflect adaptation to their respective niches. In the present review, we summarize and compare some recent findings regarding the energetic metabolism and carbon sources used by these two genetically tractable apicomplexans during host-cell invasion and intracellular growth and replication

Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites

Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis

Microsporidian polar tube proteins: Highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups

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Plasticity between MyoC- and MyoA-Glideosomes: An Example of Functional Compensation in Toxoplasma gondii Invasion

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Identification of two new polar tube proteins related to polar tube protein 2 in the microsporidian Antonospora locustae.

International audienceMicrosporidia are obligate intracellular eukaryotic parasites with a broad host spectrum characterized by a unique and highly sophisticated invasion apparatus, the polar tube (PT). In a previous study, two PT proteins, named AlPTP1 (50 kDa) and AlPTP2 (35 kDa), were identified in Antonospora locustae, an orthoptera parasite that is used as a biological control agent against locusts. Antibodies raised against AlPTP2 cross-reacted with a band migrating at ~70 kDa, suggesting that this 70-kDa antigen is closely related to AlPTP2. A blastp search against the A. locustae genome database allowed the identification of two further PTP2-like proteins named AlPTP2b (568 aa) and AlPTP2c (599 aa). Both proteins are characterized by a specific serine- and glycine-rich N-terminal extension with elastomeric structural features and share a common C-terminal end conserved with AlPTP2 (~88% identity for the last 250 aa). MS analysis of the 70-kDa band revealed the presence of AlPTP2b. Specific anti-AlPTP2b antibodies labelled the extruded PTs of the A. locustae spores, confirming that this antigen is a PT component. Finally, we showed that several PTP2-like proteins are also present in other phylogenetically related insect microsporidia, including Anncaliia algerae and Paranosema grylli