Biology subjects

Lebrun, M.

Publications and source records attributed to Lebrun, M..

6 recordsLinked to original sources

bioRxiv2026

Cryptosporidium sequentially remodels its single rhoptry into the host interface

The apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease in young children. Within the intestinal epithelium, Cryptosporidium establishes a unique intracellular niche in the apical brush border of enterocytes. A structurally complex interface between host and parasite acts as a holdfast and enables protein and metabolite transport. How the parasite invades its host cell and builds the interface is poorly understood. Here, we reveal parasite invasion with high temporal and spatial resolution using rigorous molecular markers. We find that sequential discharge of specialized secretory organelles anchors the parasite within the host prior to internalization. The single Cryptosporidium rhoptry persists beyond its initial pre-invasion discharge, acting as a conduit for the secretion of multiple waves of effector proteins. Ultimately, the rhoptry membrane gives rise to the feeder organelle that separates host from parasite cytoplasm. These findings lead us to propose a mechanistic model of Cryptosporidium invasion and intracellular parasitism.

microbiology↗

bioRxiv2025

Cardiovirus-Mediated PKR Inhibition Results from Nucleocytoplasmic Trafficking Disruption

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), know as PKR, is a key antiviral kinase activated by double-stranded RNA (dsRNA) typically produced during viral replication. Upon activation, PKR phosphorylates eIF2, leading to the inhibition of translation and viral replication. However, many viruses have evolved mechanisms to counteract PKR activity. In Cardioviruses, the Leader protein (L), a short peptide cleaved from the N-terminus of the viral polyprotein, not only inhibits PKR but also blocks interferon production and disrupts nucleocytoplasmic trafficking (NCT). L disrupts NCT by recruiting host RSK kinases to the nuclear pore complex (NPC), where RSK phosphorylates FG-nucleoporins, thereby impairing NCT. L mutations that affect NCT disruption also impact its ability to inhibit PKR, suggesting a mechanistic link. Recombinant TMEV and EMCV viruses designed to disrupt NCT through different mechanisms exhibited some extent of PKR inhibition, supporting the link between NCT disruption and PKR inhibition. Immunostaining and live-cell imaging revealed that L-induced NCT disruption redistributes a fraction of PKR to the nucleoli, where PKR remains inactive. This suggests that nucleolar sequestration contributes to PKR inhibition. Additionally, L-mediated NCT disruption leads to the release of nuclear RNA-binding proteins (nRBPs) into the cytosol, which may bind or modify viral dsRNA, further preventing PKR activation. Collectively, these results highlight nucleocytoplasmic trafficking as a critical regulatory mechanism governing PKR activation. Thus, beyond the specific action of cardiovirus L protein, our study reveals that interference with host nucleocytoplasmic transport can significantly impact the subcellular localization and functional regulation of immune effectors such as PKR. Author SummaryProtein kinase R (PKR) is a crucial component of the host innate immune response. It is activated by double-stranded RNA (dsRNA) typically produced during viral replication and triggers a shutdown of mRNA translation. This antiviral mechanism limits viral propagation by inhibiting both host and viral protein synthesis. However, many viruses have developed mechanisms to inhibit PKR, allowing them to escape immune detection. PKR downregulation facilitates viral replication whereas uncontrolled PKR activation can lead to autoimmune disorders. Therefore, PKR activity must be tightly regulated to maintain immune homeostasis. Using recombinant viruses which target the nuclear pore complex, we show that nucleocytoplasmic trafficking of cellular components is critical for regulation of PKR activity. Infection of cells with Theilers murine encephalomyelitis virus triggers an efflux of nuclear RNA binding proteins which likely compete with PKR for dsRNA binding and thereby block PKR activity. Moreover, upon TMEV infection as well as during mitosis, PKR is detected in the nucleoli where it is thought to interact with structured RNAs without being activated. Our data highlight an important link between nucleocytoplasmic trafficking and PKR activity.

immunology↗

bioRxiv2024

Perforation of the host cell plasma membrane during Toxoplasma gondii invasion requires rhoptry exocytosis

Toxoplasma gondii is an obligate intracellular parasite, and the delivery of effector proteins from the parasite into the host cell during invasion is critical for invasion itself and for parasite virulence. The effector proteins are released from specialized apical secretory organelles known as rhoptries. While much has been learned recently about the structure and composition of the rhoptry exocytic machinery and the function of individual rhoptry effector proteins that are exocytosed, virtually nothing is known about how the released proteins are translocated across the host cell plasma membrane. Previous electrophysiology experiments reported an unanticipated observation that invasion by T. gondii is preceded by a transient increase in host cell plasma membrane conductance. Here, we confirm this electrophysiological observation and propose that the conductance transient represents a parasite-induced perforation in the host cell plasma membrane through which rhoptry proteins are delivered. As a first step towards testing this hypothesis, and to provide higher throughput than patch clamp electrophysiology, we developed an alternative assay to detect the perforation. This assay utilizes high-speed, multi-wavelength fluorescence imaging to enable simultaneous visualization of host cell perforation and parasite invasion. Using this assay, we interrogated a panel of mutant parasites conditionally depleted of key invasion-related proteins. Parasites lacking signaling proteins involved in triggering rhoptry secretion (e.g., CLAMP) or components of the rhoptry exocytic machinery (e.g., Nd9, RASP2) are defective in their ability to induce the perforation. These data are consistent with a model in which the perforating agents that disrupt host cell membrane integrity during invasion - and may thereby provide the conduit for delivery of rhoptry effector proteins - are stored within the rhoptries themselves and released upon contact with the host cell.

microbiology↗

bioRxiv2024

The malaria parasite PP1 phosphatase controls the initiation of the egress pathway of asexual blood-stages by regulating the rounding-up of the vacuole

A sustained blood-stage infection of the human malaria parasite P. falciparum relies on the active exit of merozoites from their host erythrocytes. During this process, named egress, the infected red blood cell undergoes sequential morphological events: the rounding-up of the surrounding parasitophorous vacuole, the disruption of the vacuole membrane and finally the rupture of the red blood cell membrane. These events are coordinated by two intracellular second messengers, cGMP and calcium ions (Ca2+), that control the activation of their dedicated kinases, PKG and CDPKs respectively, and thus the secretion of parasitic factors that assist membranes rupture. We had previously identified the serine-threonine phosphatase PP1 as an essential enzyme required for the rupture of the surrounding vacuole. Here, we address its precise positioning and function within the egress signaling pathway by combining chemical genetics and live-microscopy. Fluorescent reporters of the parasitophorous vacuole morphology were expressed in the conditional PfPP1-iKO line which allowed to monitor the kinetics of natural and induced egress, as well as the rescue capacity of known egress inducers. Our results underscore a dual function for PP1 in the egress cascade. First, we provide further evidence that PP1 controls the homeostasis of the second messenger cGMP by modulating the basal activity of guanylyl cyclase alpha and consequently the PKG-dependent downstream Ca2+ signaling. Second, we demonstrate that PP1 also regulates the rounding-up of the parasitophorous vacuole, as this step is almost completely abolished in PfPP1-null schizonts. Strikingly, our data show that rounding-up is the step triggered by egress inducers, and support its reliance on Ca2+, as the calcium ionophore A23187 bypasses the egress defect of PfPP1-null schizonts, restores proper egress kinetics and promotes the initiation of the rounding-up step. Therefore, this study places the phosphatase PP1 upstream of the cGMP-PKG signaling pathway, and sheds new light on the regulation of rounding-up, the first step in P. falciparum blood stage egress cascade. AUTHOR SUMMARYMalaria caused by Plasmodium falciparum infections remains a major human threat in endemic countries. Its proliferation within the host relies on the iteration of red blood cell invasion, multiplication and release of newly formed parasites in the blood circulation. This last step, named egress, is tightly regulated by a signaling pathway controlled by phospho-regulation. The phosphatase PP1 is a conserved pleiotropic enzyme that regulates various biological processes in mammals and controls the replication and egress mechanisms in P. falciparum. Indeed, PP1-depleted parasites are unable to egress from the erythrocytes and remain trapped within a vacuole in the host cell. Here, using fluorescent reporters of the surrounding vacuole, and pharmacological inducers of the egress pathway, we analyzed natural and induced egress by time-lapse video-microscopy. Our results underscore a dual function of PP1 during egress and identify the phosphatase as an early regulator of this essential process.

microbiology↗

bioRxiv2022

In situ structure and priming mechanism of the rhoptry secretion system in Plasmodium revealed by cryo-electron tomography

Apicomplexan parasites secrete the contents of rhoptries into host cells to permit their invasion and establishment of an infectious niche. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is positioned at the cell apex and associates with an enigmatic apical vesicle (AV), which docks one or two rhoptries at the site of exocytosis. The interplay among the rhoptries, the AV, and the parasite plasma membrane for secretion remains unclear. Moreover, it is unknown if a similar machinery exists in the deadly malaria parasite Plasmodium falciparum. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct AV docking the tips of the two rhoptries to the RSA. We also discover two new organizations: one in which the AV is absent with one of the two rhoptry tips docks directly to the RSA, and a second in which the two rhoptries fuse together and the common tip docks directly to the RSA. Interestingly, rhoptries among the three states show no significant difference in luminal volume and density, suggesting that the exocytosis of rhoptry contents has not yet occurred, and that these different organizations likely represent sequential states leading to secretion. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to each state, including the opening of a gate-like density in the rhoptry-fused state. These conformational changes of the RSA uncover structural details of a priming process for major rhoptry secretion, which likely occur after initial interaction with a red blood cell. Our results highlight a previously unknown step in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

microbiology↗

bioRxiv2020

Co-option of Plasmodium falciparum PP1 for egress from host erythrocytes

Asexual proliferation of the Plasmodium parasites that cause malaria follow a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. A phosphoproteomic screen and chemical-genetic analysis provided evidence for a HECT E3 protein-ubiquitin ligase, as well as a fusion protein with guanylyl cyclase and phospholipid transporter domains, as functional targets of PfPP1. Extracellular phosphatidylcholine stimulates PfPP1-dependent egress. Parasite PfPP1 acts as a master regulator that can integrate multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.

microbiology↗