Biology subjects

Briquet, S.

Publications and source records attributed to Briquet, S..

8 recordsLinked to original sources

bioRxiv2026

The CLAMP-Linked Invasion Protein (CLIP) plays an essential role in Plasmodium berghei zoites

Apicomplexan parasites such as Toxoplasma and Plasmodium spp. rely on the sequential secretion of parasite apical organelles, called micronemes and rhoptries, to invade host cells. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during host cell invasion in Toxoplasma and Plasmodium zoites. Previous studies have shown that CLAMP is essential in Plasmodium merozoites for erythrocyte invasion and also in sporozoites for the invasion of the mosquito vector salivary glands and of mammalian host hepatocytes. In Toxoplasma gondii tachyzoites, CLAMP forms a complex with two other microneme proteins, the Secreted Protein with an Altered Thrombospondin Repeat (SPATR) and the CLAMP-Linked Invasion Protein (CLIP). Both SPATR and CLIP are also expressed in Plasmodium sporozoites, and downregulation of SPATR impacts sporozoite infectivity in P. berghei. In contrast, the role of CLIP in sporozoites remains unknown. To study the function of CLIP, we used a CRISPR-assisted conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. Deletion of clip in P. berghei blood stages impaired parasite growth and prevented erythrocyte invasion by merozoites. Upon deletion of clip gene in P. berghei transmission stages, sporozoite development in mosquitoes was not affected, but invasion of the mosquito salivary glands was dramatically reduced. In addition, CLIP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes, associated with a defect in gliding motility, recapitulating the phenotype of CLAMP-deficient parasites. Collectively, our data demonstrate that CLIP plays an essential role in host cell invasion by P. berghei merozoites and sporozoites, and support a conserved role of the CLAMP-CLIP-SPATR complex in invasive stages of apicomplexan parasites.

microbiology↗

bioRxiv2026

A structure-based epitope tagging approach identifies vulnerable sites on the malarial P36-P52 protein complex for antibody-mediated neutralization of Plasmodium sporozoites

Malaria is caused by apicomplexan parasites of the genus Plasmodium, which are transmitted through the bite of Anopheles mosquitoes that inject sporozoites (SPZs) into the skin. SPZs migrate to and infect the liver for an initial round of replication. SPZs and liver stages have long been considered as ideal targets for malaria vaccines. The main SPZ surface protein, the circumsporozoite protein (CSP), is the target of currently approved malaria vaccines and prophylactic antibody therapies. Studies in rodent malaria models have shown that anti-CSP antibodies exert their protective effect mainly in the skin, but some of the most potent anti-CSP monoclonal antibodies show additional protective effects in the vasculature and liver. Other SPZ proteins involved at different steps of the infection process may thus represent additional targets for antibody-mediated neutralization. Three 6-cysteine (6-Cys) domain proteins (P36, P52 and B9) play an essential role during SPZ invasion of hepatocytes, yet their molecular function and whether they can be targeted by neutralizing antibodies remains unknown. Here, to fill this gap, we combined an integrative structural biology approach with functional experiments in the P. berghei rodent malaria model. AlphaFold-based structural modeling followed by experimental validation via electron microscopy and small-angle X-ray scattering indicated that the P36-P52 heterodimer displays a head-to-tail architecture with an interaction interface that is largely conserved among Plasmodium species. The structural models supported the rational design of an epitope tagging approach, which, combined with neutralizing assays, revealed that antibodies against P36 and P52 can efficiently block invasion of hepatocytes by SPZs in culture conditions. The data show that the inhibitory activity of antibodies heavily depends on epitope position and revealed that antibody-exposed vulnerable sites lie on the membrane-distal side of the P36-P52 heterodimer. In contrast, antibodies targeting B9 had no inhibitory effect on SPZ invasion, irrespective of epitope positioning. These data show that the invasion step could be targeted by antibodies and indicate that the P36-P52 complex may be considered as a potential target for the development of next generation pre-erythrocytic malaria vaccines or therapeutic antibodies.

microbiology↗

bioRxiv2024

Specialized S-type ribosomes of Plasmodium yoelii enhance host-to-vector malaria transmission

Unlike most eukaryotes, Plasmodium species only encode 4-5 ribosomal DNA loci, making them an exceptional model system for genetic studies of ribosome specialization. Plasmodium ribosomes are characterized as A-type and S-type, defined by differing temporal expression patterns and sequence variation. The two S-type rRNAs (S1 & S2) are most abundant in developing mosquito stages, yet maintain low levels of abundance in blood stages. Two previous, conflicting studies found that one or either S-type rRNA was essential to mosquito-stage development, but these experiments were hampered by technical constraints. Therefore, we used the DiCre recombinase to generate transgenic parasites with clean deletions of the S1, S2, or both S-type rDNAs (S-type null) to characterize their roles and interplay in both mosquito- and blood-stage parasites. Contrary to previous conclusions, the presence of either or both S-type rRNAs was not required for sporozoite development, yet promoted host-to-vector transmission, oocyst maturation, and productive sporogony. Unexpectedly, we found that S-type ribosomes have different, opposing impacts on blood stage development despite being at low abundances. Deletion of S1 rDNA nearly ablated the first wave of parasitemia without impacting transmissible male gametocyte counts. Reciprocally, the deletion of S2 rDNA reduced counts of transmissible male gametocytes without impacting parasitemia, which was phenocopied by the S-type null line. Because the LSU portion of S1 rDNA could not be deleted, we assessed the S-type SSU rDNAs for potentially dominant roles. Introduction of extra plasmid-based copies of either S1 or S2 SSU rDNA resulted in either increased parasitemia or male gametogenesis, respectively. Yet, both decreased transmission and impacted early mosquito-stage development. These results indicate that driving functions of Plasmodiums S-type ribosomes occur during blood stages, with S-type SSUs contributing distinct, separable functions. To the best of our knowledge, this is the first example of specialized ribosomes driving cellular outcomes at low abundances. Author SummaryMalaria parasites (Plasmodium spp.) have specialized ribosomes with distinct temporal expression patterns and ribosomal RNA (rRNA) sequence variation. While previous work has demonstrated the importance of the S1 or S2 types of ribosomes to mosquito-stage development, technical limitations led to conflicting conclusions and prevented the investigation of whether the S-type ribosomes were essential for parasite development. Here, we used a DiCre recombinase system to overcome past limitations and generated clean deletions of both S-type rDNA genes, as well as the first Plasmodium line with both S-type rDNA genes deleted. Surprisingly, we found that Plasmodium yoelii parasites do not require either S-type ribosome for mosquito-stage development, when these rRNAs are most abundant. Instead, we found that S-type ribosomes contribute distinct functions during blood-stage development when these ribosomes make up only a small fraction of the total ribosomes present. In these stages, we found that S1 and S2 ribosomes have different, opposing functions that work cooperatively to promote transmission from the mammalian host to the mosquito vector.

microbiology↗

bioRxiv2024

Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei

Malaria is caused by protozoan parasites of the genus Plasmodium and remains a global health concern. The parasite has a highly adaptable life cycle comprising successive rounds of asexual replication in a vertebrate host and sexual maturation in the mosquito vector Anopheles. Genetic manipulation of the parasite has been instrumental for deciphering the function of Plasmodium genes. Conventional reverse genetic tools cannot be used to study essential genes of the asexual blood stages, thereby necessitating the development of conditional strategies. Among various such strategies, the rapamycin-inducible dimerisable Cre (DiCre) recombinase system emerged as a powerful approach for conditional editing of essential genes in human-infecting P. falciparum and in the rodent malaria model parasite P. berghei. We previously generated a DiCre-expressing P. berghei line and validated it by conditionally deleting several essential asexual stage genes, revealing their important role also in sporozoites. The advent of CRISPR enabled targeted genome editing with higher accuracy and specificity and greatly advanced genome engineering in Plasmodium spp. Here, we developed new P. berghei parasite lines by integrating the DiCre cassette and a fluorescent marker in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR-Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked-out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirm the role of CLAMP during invasion of erythrocytes.

microbiology↗

bioRxiv2022

The AMA1-RON complex drives Plasmodium sporozoite invasion in the mosquito and mammalian hosts

Plasmodium sporozoites that are transmitted by blood-feeding female Anopheles mosquitoes invade hepatocytes for an initial round of intracellular replication, leading to the release of merozoites that invade and multiply within red blood cells. Sporozoites and merozoites share a number of proteins that are expressed by both stages, including the Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck Proteins (RONs). Although AMA1 and RONs are essential for merozoite invasion of erythrocytes during asexual blood stage replication of the parasite, their function in sporozoites is still unclear. Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to safely enter the mosquito salivary glands without causing cell damage, to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies.

microbiology↗

bioRxiv2021

Plasmodium-encoded murine IL-6 impairs liver stage infection and elicits long-lasting sterilizing immunity

Plasmodium sporozoites inoculated by Anopheles mosquitoes into the skin of the mammalian host migrate to the liver before infecting hepatocytes. Previous work demonstrated that early production of IL-6 in the liver is detrimental for the parasite growth, contributing to the acquisition of a long-lasting immune protection after immunization with live attenuated parasites. Considering that IL-6 ais a critical pro-inflammatory signal, we explored a novel approach whereby the parasite itself encodes for the murine IL-6 gene. We generated transgenic P. berghei parasites that express murine IL-6 during liver stage development. Though IL-6 transgenic sporozoites develop into exo-erythrocytic forms in cultured hepatocytes in vitro and in vivo, these parasites were not capable of inducing a blood stage infection in mice. Furthermore, immunization of mice with transgenic IL-6-expressing P. berghei sporozoites elicited a long-lasting CD8+ T cell-mediated protective immunity against a subsequent infectious sporozoite challenge. Collectively, this study demonstrates that parasite-encoded IL-6 attenuates parasite virulence with abortive liver stage of Plasmodium infection, forming the basis of a novel suicide vaccine strategy to elicit protective antimalarial immunity. SummaryIL-6 was shown to control Plasmodium parasite development in the liver. Here, Belhimeur et al. generated a murine IL-6 transgenic Plasmodium berghei. These parasites show an arrest in hepatocyte development and protect mice against homologous and heterologous parasite challenge in a CD8-dependent manner.

immunology↗

bioRxiv2021

Plasmodium sporozoites require the protein B9 to invade hepatocytes

Plasmodium sporozoites are transmitted to a mammalian host during blood feeding by an infected mosquito and invade hepatocytes for initial replication of the parasite in the liver. This leads to the release of thousands of merozoites into the blood circulation and initiation of the pathogenic blood stages of malaria. Merozoite invasion of erythrocytes has been well characterized at the molecular and structural levels. In sharp contrast, the molecular mechanisms of sporozoite invasion of hepatocytes are poorly characterized. Here we report a new role during sporozoite entry for the B9 protein, a member of the 6-cysteine domain protein family. Using genetic tagging and gene deletion approaches in rodent malaria parasites, we show that B9 is secreted from sporozoite micronemes and is required for productive invasion of hepatocytes. Structural modelling indicates that the N-terminus of B9 forms a beta-propeller domain structurally related to CyRPA, a cysteine-rich protein forming an invasion complex with Rh5 and RIPR in P. falciparum merozoites. We provide evidence that the beta-propeller domain of B9 is essential for protein function during sporozoite entry and interacts with P36 and P52, both also essential for productive invasion of hepatocytes. Our results suggest that, despite using distinct sets of parasite and host entry factors, Plasmodium sporozoites and merozoites may share common structural modules to assemble protein complexes for invasion of host cells.

microbiology↗

bioRxiv2020

The dimerisable Cre recombinase allows conditional genome editing in the mosquito stages of Plasmodium berghei

Asexual blood stages of the malaria parasite are readily amenable to genetic modification via homologous recombination, allowing functional studies of parasite genes that are not essential in this part of the life cycle. However, conventional reverse genetics cannot be applied for the functional analysis of genes that are essential during asexual blood-stage replication. Various strategies have been developed for conditional mutagenesis of Plasmodium, including recombinase-based gene deletion, regulatable promoters, and mRNA or protein destabilization systems. Among these, the dimerisable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene targeting in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin-binding protein. Rapamycin-induced heterodimerization of the two components restores recombinase activity. We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle.

microbiology↗