Functional analysis of apical organelle proteins in Plasmodium sporozoites

L’infection par Plasmodium, parasite responsable du paludisme, débute par l’inoculation de sporozoïtes invasifs par un moustique Anopheles femelle. Les sporozoïtes infectent des cellules du foie pour s’y développer en mérozoïtes qui une fois libérés dans la circulation sanguine vont se développer dans les érythrocytes. L’invasion des cellules implique la sécrétion d’organites apicaux (micronèmes et rhoptries) et la formation d’une structure particulière (jonction mobile) conduisant à l’internalisation du parasite au sein d’une vacuole parasitophore. Les mécanismes moléculaires impliqués dans l’invasion des hépatocytes par les sporozoïtes restent méconnus. Les organites sécrétoires apicaux contiennent des protéines spécifiquement exprimées chez le sporozoïte (dont des protéines à domaine 6-cystéines), et des protéines également exprimées chez les mérozoïtes (comme AMA1, les RONs ou CLAMP) impliquées dans la formation de la jonction mobile. Nos travaux ont révélé le rôle essentiel d’une protéine à domaine 6-cystéines, B9, dans l’invasion des hépatocytes par les sporozoïtes. L’utilisation d’une stratégie de mutagénèse conditionnelle a permis de démontrer le rôle d’AMA1, RON2, RON4 et CLAMP chez les sporozoïtes, pour l’invasion des hépatocytes chez l’hôte mammifère mais également pour la colonisation des glandes salivaires du moustique. Nos résultats confortent l’hypothèse d’une compartimentation fonctionnelle des protéines micronémales chez les sporozoïtes. Ce travail contribue à une meilleure compréhension des mécanismes moléculaires mis en jeu lors de l’invasion des hépatocytes et identifie de nouvelles cibles potentielles pour bloquer l’invasion du foie par Plasmodium.Infection with Plasmodium, the parasite responsible for malaria, begins with the inoculation of invasive sporozoites by a female Anopheles mosquito. Sporozoites infect liver cells and develop into merozoites which, once released into the bloodstream, undergo asexual multiplication cycles in red blood cells. Host cell invasion involves the secretion of apical organelles (micronemes and rhoptries) and the formation of a unique structure (moving junction) that leads to the internalization of the parasite within a parasitophorous vacuole. However, the molecular mechanisms involved during invasion of hepatocytes by sporozoites remain unknown. Apical secretory organelles contain proteins specifically expressed in sporozoites (including 6-cysteine domain proteins), and proteins also expressed in merozoites (such as AMA1, RONs or CLAMP), which are involved in the formation of the moving junction. Our work revealed the essential role of a 6-cysteine domain protein, B9, in the invasion of hepatocytes by sporozoites. Using a conditional mutagenesis strategy, we also demonstrated the role of AMA1, RON2, RON4 and CLAMP in sporozoites, not only for hepatocyte invasion in the mammalian host but also for salivary gland colonization in the mosquito. Our results support the hypothesis of a functional compartmentalization of microneme proteins in Plasmodium sporozoites. This work contributes to a better understanding of the molecular mechanisms involved during infection of hepatocytes and identifies new potential targets to block malaria liver infection

Analyse fonctionnelle de protéines des organites apicaux chez les sporozoïtes de Plasmodium

Infection with Plasmodium, the parasite responsible for malaria, begins with the inoculation of invasive sporozoites by a female Anopheles mosquito. Sporozoites infect liver cells and develop into merozoites which, once released into the bloodstream, undergo asexual multiplication cycles in red blood cells. Host cell invasion involves the secretion of apical organelles (micronemes and rhoptries) and the formation of a unique structure (moving junction) that leads to the internalization of the parasite within a parasitophorous vacuole. However, the molecular mechanisms involved during invasion of hepatocytes by sporozoites remain unknown. Apical secretory organelles contain proteins specifically expressed in sporozoites (including 6-cysteine domain proteins), and proteins also expressed in merozoites (such as AMA1, RONs or CLAMP), which are involved in the formation of the moving junction. Our work revealed the essential role of a 6-cysteine domain protein, B9, in the invasion of hepatocytes by sporozoites. Using a conditional mutagenesis strategy, we also demonstrated the role of AMA1, RON2, RON4 and CLAMP in sporozoites, not only for hepatocyte invasion in the mammalian host but also for salivary gland colonization in the mosquito. Our results support the hypothesis of a functional compartmentalization of microneme proteins in Plasmodium sporozoites. This work contributes to a better understanding of the molecular mechanisms involved during infection of hepatocytes and identifies new potential targets to block malaria liver infection.L’infection par Plasmodium, parasite responsable du paludisme, débute par l’inoculation de sporozoïtes invasifs par un moustique Anopheles femelle. Les sporozoïtes infectent des cellules du foie pour s’y développer en mérozoïtes qui une fois libérés dans la circulation sanguine vont se développer dans les érythrocytes. L’invasion des cellules implique la sécrétion d’organites apicaux (micronèmes et rhoptries) et la formation d’une structure particulière (jonction mobile) conduisant à l’internalisation du parasite au sein d’une vacuole parasitophore. Les mécanismes moléculaires impliqués dans l’invasion des hépatocytes par les sporozoïtes restent méconnus. Les organites sécrétoires apicaux contiennent des protéines spécifiquement exprimées chez le sporozoïte (dont des protéines à domaine 6-cystéines), et des protéines également exprimées chez les mérozoïtes (comme AMA1, les RONs ou CLAMP) impliquées dans la formation de la jonction mobile. Nos travaux ont révélé le rôle essentiel d’une protéine à domaine 6-cystéines, B9, dans l’invasion des hépatocytes par les sporozoïtes. L’utilisation d’une stratégie de mutagénèse conditionnelle a permis de démontrer le rôle d’AMA1, RON2, RON4 et CLAMP chez les sporozoïtes, pour l’invasion des hépatocytes chez l’hôte mammifère mais également pour la colonisation des glandes salivaires du moustique. Nos résultats confortent l’hypothèse d’une compartimentation fonctionnelle des protéines micronémales chez les sporozoïtes. Ce travail contribue à une meilleure compréhension des mécanismes moléculaires mis en jeu lors de l’invasion des hépatocytes et identifie de nouvelles cibles potentielles pour bloquer l’invasion du foie par Plasmodium

Conditional Gene Deletion in Mammalian and Mosquito Stages of Plasmodium berghei Using Dimerizable Cre Recombinase

International audienc

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

International audienceAsexual 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 Plasmo-dium, 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 deletion in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymati-cally 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

Plasmodium sporozoites on the move: Switching from cell traversal to productive invasion of hepatocytes

International audienceParasites of the genus Plasmodium, the etiological agent of malaria, are transmitted through the bite of anopheline mosquitoes, which deposit sporozoites into the host skin. Sporozoites migrate through the dermis, enter the bloodstream, and rapidly traffic to the liver. They cross the liver sinusoidal barrier and traverse several hepatocytes before switching to productive invasion of a final one for replication inside a parasitophorous vacuole. Cell traversal and productive invasion are functionally independent processes that require proteins secreted from specialized secretory organelles known as micronemes. In this review, we summarize the current understanding of how sporozoites traverse through cells and productively invade hepatocytes, and discuss the role of environmental sensing in switching from a migratory to an invasive state. We propose that timely controlled secretion of distinct microneme subsets could play a key role in successful migration and infection of hepatocytes. A better understanding of these essential biological features of the Plasmodium sporozoite may contribute to the development of new strategies to fight against the very first and asymptomatic stage of malaria

The claudin-like apicomplexan microneme protein is required for gliding motility and infectivity of Plasmodium sporozoites.

Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the micronemes and the rhoptries. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during invasion by Toxoplasma gondii tachyzoites and in Plasmodium falciparum asexual blood stages. CLAMP is also expressed in Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, but its role in this stage is still unknown. CLAMP is essential for Plasmodium blood stage growth and is refractory to conventional gene deletion. To circumvent this obstacle and study the function of CLAMP in sporozoites, we used a conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. We successfully deleted clamp gene in P. berghei transmission stages and analyzed the functional consequences on sporozoite infectivity. In mosquitoes, sporozoite development and egress from oocysts was not affected in conditional mutants. However, invasion of the mosquito salivary glands was dramatically reduced upon deletion of clamp gene. In addition, CLAMP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes. This severe phenotype was associated with major defects in gliding motility and with reduced shedding of the sporozoite adhesin TRAP. Expansion microscopy revealed partial colocalization of CLAMP and TRAP in a subset of micronemes, and a distinct accumulation of CLAMP at the apical tip of sporozoites. Collectively, these results demonstrate that CLAMP is essential across invasive stages of the malaria parasite, and support a role of the protein upstream of host cell invasion, possibly by regulating the secretion or function of adhesins in Plasmodium sporozoites

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

International audiencePlasmodium 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 was 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. Three-dimensional electron microscopy data showed that sporozoites enter salivary gland cells through a ring-like structure and by forming a transient vacuole. The absence of a functional AMA1-RON complex led to an altered morphology of the entry junction, associated with epithelial cell damage. 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 efficiently and safely enter the mosquito salivary glands to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies

A global metagenomic map of urban microbiomes and antimicrobial resistance

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.Funding: the Tri-I Program in Computational Biology and Medicine (CBM) funded by NIH grant 1T32GM083937; GitHub; Philip Blood and the Extreme Science and Engineering Discovery Environment (XSEDE), supported by NSF grant number ACI-1548562 and NSF award number ACI-1445606; NASA (NNX14AH50G, NNX17AB26G), the NIH (R01AI151059, R25EB020393, R21AI129851, R35GM138152, U01DA053941); STARR Foundation (I13- 0052); LLS (MCL7001-18, LLS 9238-16, LLS-MCL7001-18); the NSF (1840275); the Bill and Melinda Gates Foundation (OPP1151054); the Alfred P. Sloan Foundation (G-2015-13964); Swiss National Science Foundation grant number 407540_167331; NIH award number UL1TR000457; the US Department of Energy Joint Genome Institute under contract number DE-AC02-05CH11231; the National Energy Research Scientific Computing Center, supported by the Office of Science of the US Department of Energy; Stockholm Health Authority grant SLL 20160933; the Institut Pasteur Korea; an NRF Korea grant (NRF-2014K1A4A7A01074645, 2017M3A9G6068246); the CONICYT Fondecyt Iniciación grants 11140666 and 11160905; Keio University Funds for Individual Research; funds from the Yamagata prefectural government and the city of Tsuruoka; JSPS KAKENHI grant number 20K10436; the bilateral AT-UA collaboration fund (WTZ:UA 02/2019; Ministry of Education and Science of Ukraine, UA:M/84-2019, M/126-2020); Kyiv Academic Univeristy; Ministry of Education and Science of Ukraine project numbers 0118U100290 and 0120U101734; Centro de Excelencia Severo Ochoa 2013–2017; the CERCA Programme / Generalitat de Catalunya; the CRG-Novartis-Africa mobility program 2016; research funds from National Cheng Kung University and the Ministry of Science and Technology; Taiwan (MOST grant number 106-2321-B-006-016); we thank all the volunteers who made sampling NYC possible, Minciencias (project no. 639677758300), CNPq (EDN - 309973/2015-5), the Open Research Fund of Key Laboratory of Advanced Theory and Application in Statistics and Data Science – MOE, ECNU, the Research Grants Council of Hong Kong through project 11215017, National Key RD Project of China (2018YFE0201603), and Shanghai Municipal Science and Technology Major Project (2017SHZDZX01) (L.S.