Unravelling the secretion machinery for virulence factors in apicomplexan parasites

Informazioni relative al progetto

KissAndSpitRhoptry

ID dell’accordo di sovvenzione: 833309

DOI

10.3030/833309

Data della firma CE 12 Agosto 2019

Data di avvio 1 Ottobre 2020

Data di completamento 30 Settembre 2026

Finanziato da

• EXCELLENT SCIENCE - European Research Council (ERC)

Costo totale

€ 2 496 210,00

Contributo UE

€ 2 496 210,00

Coordinato da

INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
France

Periodic Reporting for period 3 - KissAndSpitRhoptry (Unravelling the secretion machinery for virulence factors in apicomplexan parasites)

Periodo di rendicontazione: 2023-10-01 al 2025-03-31

Sintesi del contesto e degli obiettivi generali del progetto

The phylum Apicomplexa comprises more than 5000 unicellular eukaryotes. It includes some of the most important pathogenic parasites of man and animals, the deadliest of which is the malaria parasite Plasmodium falciparum, responsible for half a million human deaths per year in many tropical and subtropical countries. Human pathogens also include Toxoplasma, a prominent cause of human congenital infections, and Cryptosporidium, one of four pathogens responsible for severe diarrhea in infants. Apicomplexa are strictly intracellular and their mechanisms of invasion and intracellular survival involve the secretion of virulence factors from secretory organelles called Rhoptries.
Rhoptries are large vesicles docked at the apical end of the parasite, ready for fusion with the parasite plasma membrane (PPM) upon contacting the host cell. In contrast with the exocytosis of synaptic and dense-core vesicles in mammals, the secretory material (proteins and lipids) of rhoptries is not released outside the cell, instead it is injected directly inside the host. In fact, rhoptries proteins act as bacterial effectors, but the mechanisms of their release and injection into the host are different from bacteria (genes encoding prokaryotic secretion systems are not conserved in Apicomplexa). Moreover, conventional exocytic factors described in mammals are not associated with rhoptry exocytosis. Overall, rhoptry secretion mechanisms are unknown and clearly distinct from those controlling secretion in bacteria and in classic eukaryotic model systems, i.e. yeast and animals.
KissAndSpitRhoptry aims to dissect the structure, the molecular components and the mechanistic steps that allow the parasite to inject virulence factors. The specific objectives are 1- to explore the mechanisms that trigger rhoptry exocytosis upon binding of the parasite to the host cell, 2- to provide insights into the machinery of fusion of rhoptries with the PPM, and 3- to decipher how the rhoptry content crosses the host membrane.
Understanding this essential mechanism will address an untouched topic of central importance, which will greatly impact our understanding of exocytosis in early-diverging eukaryotes, and offer new concepts in the transport of proteins across membranes by nanomachines. It may also have a major translational impact for the development of new strategies targeting Apicomplexa pathogens, in particular in feeding research on malaria treatment, prevention and eradication.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

We addressed rhoptry secretion mechanisms through an evolutionary angle., Apicomplexa form the superphylum Alveolata together with dinoflagellates and ciliates. Ciliates possess specialized secretory organelles known as trichocysts in Paramecium or mucocysts in Tetrahymena, that are used for defense or predation. Their exocytosis involves a rosette of intramembranous particles at the plasma membrane, whose formation depends on NDs genes (Non-Discharged). Paramecium ND mutants are unable to discharge the content of trichocysts. Remarkably, a comparable rosette is visible in Apicomplexa but, since its discovery in the 70’s, it has remained uncharacterized at the molecular level. Based on these comparative observations, we hypothesized that there is a common ancestry for the fusion machinery of secretory organelles in Alveolates.
We found two NDs, ND6 and ND9, conserved in Apicomplexa and showed that they are important for rhoptry secretion and invasion in Toxoplasma and P. falciparum. Moreover, one of them is required for rosette formation, linking the rosette with rhoptry secretion. Both ND proteins are part of a complex unique to Alveolata that comprised other proteins essential for rhoptry secretion and rosette formation in Toxoplasma and for mucocysts exocytosis in Tetrahymena.
Toxoplasma ND6 is present at the site of exocytosis in association with an apical vesicle (AV). The AV, absent in Paramecium and Tetrahymena, is sandwiched between the rosette and the tip of the rhoptry. Remarkably, the rosette is part of an elaborate structure, named “rhoptry secretory apparatus” (RSA) that connects the AV to the PPM. This unique arrangement and the RSA are conserved in P. falciparum and Cryptosporidium.
We extended the use of ciliate models to further uncover the mechanism of rhoptry secretion using transcriptomic profiling in Tetrahymena and homology search in Apicomplexa. We identified two novel mucocysts exocytic factors in Tetrahymena. The Toxoplasma orthologs, named CRMPs, are in complex together and are important for rhoptry secretion, but appear dispensable for the assembly of the RSA or the anchoring of the AV to the RSA. They transiently accumulate in proximity of the RSA just prior to invasion, exposing putative host cell‐binding domains toward the host cell; moreover, one of them is related to G protein‐coupled receptors. These features support a role for this complex in the signaling pathway that coordinates rhoptry content discharge with host contact.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

Our results support the existence of a common and unique secretory machinery in protists in which individual lineages diverged hundreds of millions of years ago to sustain radically different lifestyles. This machinery has evolved for predation/defense in the case of free-living ciliates, and host-cell invasion and intracellular replication for apicomplexan parasites. We discovered the existence of a new class of secretion machinery in pathogenic eukaryotes: the Rhoptry Secretion Apparatus (RSA). The RSA is a prime candidate to exert the mechanical force needed to bring opposing membranes in proximity and allowing their fusion. We described an apical vesicle (AV) that connects one or two rhoptries with the RSA. The structural association of the AV with the RSA prompted a rethinking about the mechanism of rhoptry secretion in Apicomplexa and suggested the need of two fusions events: the rhoptries with the AV, and the AV with the PPM. Certainly, further work is required to uncover the molecular players for each of these two fusion steps, however, structural features of the Nd proteins already offer valuable insights on the mechanisms involved in rhoptry exocytosis, such as the role of the GTP/GDP hydrolysis and calcium signaling. The apparent lack of an AV in Ciliates suggests that it might be important to coordinate secretion with the transfer of rhoptry proteins into the host cell cytoplasm, a hypothesis that required further investigations. Whether the RSA is also involved in breaching the host cell is an intriguing hypothesis.
The Tetrahymena-based in silico screening led us to the discovery of CRMP factors that likely link the recognition of the host cell to the activation of the rhoptry exocytic machinery. CRMPs cannot be considered bona fide GPCRs because they do not have all the features typical of these signaling receptors, but they may be divergent forms that have maintained similar activities. Uncovering their host ligands, as well as the signaling pathway downstream of their interaction with host, are future steps to develop strategies for blocking rhoptry exocytosis and subsequent invasion, contributing to further fight human infections caused by apicomplexans.
Overall, our comparative studies have proven to be transformative for revealing the signaling molecule for rhoptry secretion but also the fusion machinery shared between Apicomplexa and Ciliata. Our work bring new concepts and approaches to explore Apicomplexan biology.

image.jpg