Institute for Integrative Biology of the Cell
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Summary
- A genome-wide comprehensive analysis of nucleosome positioning in yeast
- Activity of MukBEF for chromosome management in E. coli and its inhibition by MatP
- Cooperation between two modes for DNA replication initiation in the archaeon Thermococcus barophilus
- JAK-STAT-dependent contact between follicle cells and the oocyte controls Drosophila anterior-posterior polarity and germline development
- Transposon sequencing reveals genes enabling insect gut colonization by the symbiont Caballeronia insecticola
- Protein-protein interactions: how to push forward the limits of the revolutionary AlphaFold2 programme?
- The stringent response is strongly activated in the high antibiotic producer, Streptomyces coelicolor.
- Composition of Poxvirus Core Revealed
- Schizosaccharomyces pombe as a fundamental model for research on mitochondrial gene expression: Progress, achievements and outlooks
- Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design
- Toxoplasma membrane inositol phospholipid binding protein TgREMIND is essential for secretory organelle function and host infection
Uncoupling programmed DNA cleavage and repair
scrambles the Paramecium somatic genome
No Ku – no cut: how the ciliate Paramecium tetraurelia protects its somatic genome from translocations and aberrant chromosome fragmentation during developmentally programmed rearrangements.
DNA double-strand breaks (DSBs) are a major threat to genome integrity. When incorrectly repaired, they can lead to genome rearrangements, chromosome instability, diseases, or cell death. Yet, key physiological processes, such as antibody gene assembly or meiotic recombination, rely on DSB induction. How living organisms cope with programmed DSBs is a major question in genome biology. The “Programmed genome rearrangements” team studies Paramecium to dissect developmentally programmed DNA elimination mechanisms. During each sexual cycle, tens of thousands of germline Internal Eliminated Sequences (IESs), scattered all along chromosomes, undergo precise excision. This process requires DSB introduction at IES boundaries, followed by error-free stitching of flanking DNA fragments by the non-homologous end joining (NHEJ) pathway. The team previously reported that the presence of a specialized variant of the NHEJ factor Ku is a pre-requisite for the activation of DNA cleavage, indicating that the two steps of the reaction are coupled. In this study, they engineered a DNA binding-deficient Ku mutant, which they characterized in collaboration with the IntGen team from the B3S department. They showed that the mutant Ku activates DNA cleavage, but is defective in repair. Unrepaired broken ends at IES boundaries are then trimmed and healed by telomere addition. By co-expressing wild-type Ku with the mutant, they were able to uncouple the cleavage and repair steps during IES excision. Using high throughput DNA sequencing and novel dedicated bioinformatic tools, they showed that chromosome translocations were largely favored under these conditions. This work demonstrates that coupling DNA cleavage and DSB repair ensures faithful genome assembly during programmed rearrangements.
More information: https://www.cell.com/cell-reports/fulltext/S2211-1247(24)00329-2
Contact: Mireille Bétermier mireille.betermier@i2bc.paris-saclay.fr – Julien Bischerour – julien.bischerour@i2bc.paris-saclay.fr
Transcription-induced DNA supercoiling clears RNA polymerase’s path in bacterial chromatin
Positive DNA supercoiling generated ahead of RNA polymerase during transcription elongation dislodges nucleoid-structuring protein H-NS from the DNA.
Bacterial chromosomal DNA is condensed more than 1000 times inside the cell. This condensation results from a slight underwinding of the double helix, leading to the formation of loops of negatively supercoiled DNA (plectonemes) and from interactions with structural proteins that help compact these loops. However, these chromatin-associated proteins obstruct transcription and need to be transiently removed to allow gene expression. This is the case for H-NS, an abundant protein that blocks the expression of numerous genes. In Gram-negative pathogenic bacteria such as Salmonella, H-NS represses the expression of the majority of virulence genes. Displacement of H-NS becomes necessary during the infection process, which relies on the function of virulence gene products.
In a study published in the journal Nature Communications, researchers from the Genome Biology department of the Institute for Integrative Biology of the Cell (I2BC) present evidence that binding of H-NS to DNA is destabilized, from a distance, by the positive supercoils forming ahead of approaching RNA polymerase. It is known that the process of transcription induces DNA supercoiling by forcing the rotation of the DNA axis during elongation. To explain the destabilization of H-NS:DNA complexes by positive supercoiling, the researchers propose a model in which H-NS oligomers play a scaffolding role in negatively supercoiled DNA by bridging opposite arms of the plectonemic structure. Accumulation of positive supercoils and concomitant DNA axis rotation ahead of the approaching RNA polymerase cause the H-NS-bound, negatively supercoiled plectoneme to unroll, leading to the collapse of the scaffold and allowing RNA polymerase to continue on its path.
More information: https://www.nature.com/articles/s41467-024-47114-w
Contact: Lionelo Bossi lionello.bossi@i2bc.paris-saclay.fr
A Fatty Acid Anabolic Pathway in Specialized-Cells Sustains a Remote Signal that Controls Egg Activation in Drosophila
While egg activation, i.e. oocyte to embryo transition, was not known to depend on physiological non-genital signal, this study shows that in Drosophila it depends on a very-long-chain-fatty-acid produced in the oenocytes (lipid metabolism specialized cells).
Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In Drosophila melanogaster, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated in vitro, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation.
More information: https://doi.org/10.1371/journal.pgen.1011186
Contact: Jacques Montagne jacques.montagne@i2bc.paris-saclay.fr
A highly conserved ligand-binding site for AccA transporters of antibiotic and quorum-sensing regulator in Agrobacterium leads to a different specificity
The highly conserved ligand binding site of the AccA transporters of antibiotic and quorum-sensing regulator in Agrobacterium is not linked to a conserved specificity.
Plants genetically modified by the pathogenic Agrobacterium strain C58 synthesize agrocinopines A and B, whereas those modified by the pathogenic strain Bo542 produce agrocinopines C and D. The four agrocinopines (A, B, C and D) serve as nutrients by agrobacteria and signaling molecule for the dissemination of virulence genes. They share the uncommon pyranose-2-phosphate motif, represented by the L-arabinopyranose moiety in agrocinopines A/B and the D-glucopyranose moiety in agrocinopines C/D, also found in the antibiotic agrocin 84. They are imported into agrobacterial cytoplasm via the Acc transport system, including the solute-binding protein AccA coupled to an ABC transporter. We have previously shown that unexpectedly, AccA from strain C58 (AccAC58) recognizes the pyranose-2-phosphate motif present in all four agrocinopines and agrocin 84, meaning that strain C58 is able to import agrocinopines C/D, originating from the competitor strain Bo542. Here, using agrocinopine derivatives and combining crystallography, affinity and stability measurements, modeling, molecular dynamics, in vitro and vivo assays, we show that AccABo542 and AccAC58 behave differently despite 75% sequence identity and a nearly identical ligand binding site. Indeed, strain Bo542 imports only compounds containing the D-glucopyranose-2-phosphate moiety, and with a lower affinity compared to strain C58. This difference in import efficiency makes C58 more competitive than Bo542 in culture media. We can now explain why Agrobacterium/Allorhizobium vitis strain S4 is insensitive to agrocin 84, although its genome contains a conserved Acc transport system. Overall, our work highlights AccA proteins as a case study, for which stability and dynamics drive specificity.
More information: https://portlandpress.com/biochemj/article-abstract/481/2/93/233802/A-highly-conserved-ligand-binding-site-for-AccA?redirectedFrom=fulltext
Contact: Solange Morera solange.morera@i2bc.paris-saclay.fr
Epigenetics: combining flexible and rigid regions into a single structure to ensure genome replication and stability
The AMIG team at I2BC, in collaboration with teams from the Institut Curie and the Synchrotron Soleil, shows that the CAF-1 protein combines in its spatial organization flexible regions and rigid modules to deposit histones on DNA and effectively couple this process to DNA synthesis.
Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.
More information: https://elifesciences.org/articles/91461
Contact: Françoise Ochsenbein francoise.ochsenbein@i2bc.paris-saclay.fr
A genome-wide comprehensive analysis of nucleosome positioning in yeast
We show that the regulation and compartmentalisation of nucleosomal organisation require the concomitant actions of local and global processes that are maintained actively by energy consuming factors.
In eukaryotic cells, the one-dimensional DNA molecules need to be tightly packaged into the spatially constraining nucleus. Folding is achieved on its lowest level by wrapping the DNA around nucleosomes. Their arrangement regulates other nuclear processes, such as tran- scription and DNA repair. Despite strong efforts to study nucleosome positioning using Next Generation Sequencing (NGS) data, the mechanism of their collective arrangement along the gene body remains poorly understood. Here, we classify nucleosome distributions of protein-coding genes in Saccharomyces cerevisiae according to their profile similarity and analyse their differences using functional Principal Component Analysis. By decomposing the NGS signals into their main descriptive functions, we compared wild type and chromatin remodeler-deficient strains, keeping position-specific details preserved whilst considering the nucleosome arrangement as a whole. A correlation analysis with other genomic proper- ties, such as gene size and length of the upstream Nucleosome Depleted Region (NDR), identified key factors that influence the nucleosome distribution. We reveal that the RSC chromatin remodeler—which is responsible for NDR maintenance—is indispensable for decoupling nucleosome arrangement within the gene from positioning outside, which inter- fere in rsc8-depleted conditions. Moreover, nucleosome profiles in chd1Δ strains displayed a clear correlation with RNA polymerase II presence, whereas wild type cells did not indicate a noticeable interdependence. We propose that RSC is pivotal for global nucleosome orga- nisation, whilst Chd1 plays a key role for maintaining local arrangement.
More information: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1011799
Contact: Arach GOLDAR <arach.goldar@i2bc.paris-saclay.fr>
Activity of MukBEF for chromosome management in E. coli
and its inhibition by MatP
The MukBEF condensin of E. coli loads at the replication fork and is unloaded at the Ter region by MatP.
Structural maintenance of chromosomes (SMC) complexes share conserved structures and serve a common role in maintaining chromosome architecture. In the bacterium Escherichia coli, the SMC complex MukBEF is necessary for rapid growth and the accurate segregation and positioning of the chromosome, although the specific molecular mechanisms involved are still unknown. Here, we used a number of in vivo assays to reveal how MukBEF controls chromosome conformation and how the MatP/matS system prevents MukBEF activity. Our results indicate that the loading of MukBEF occurs preferentially on newly replicated DNA, at multiple loci on the chromosome where it can promote long-range contacts in cis even though MukBEF can promote long-range contacts in the absence of replication. Using Hi-C and ChIP-seq analyses in strains with rearranged chromosomes, the prevention of MukBEF activity increases with the number of matS sites and this effect likely results from the unloading of MukBEF by MatP. Altogether, our results reveal how MukBEF operates to control chromosome folding and segregation in E. coli.
More information: https://elifesciences.org/articles/91185
Contact: Stéphane DUIGOU <stephane.duigou@i2bc.paris-saclay.fr>
Cooperation between two modes for DNA replication initiation in the archaeon Thermococcus barophilus
Demonstration that diverse physiological states influence the mode of DNA replication initiation in the archaeon Thermococcus.
The mechanisms underpinning the replication of genomic DNA have recently been challenged in Archaea. Indeed, the lack of origin of replication has no deleterious effect on growth, suggesting that replication initiation relies on homologous recombination. Recombination-dependent replication (RDR) appears to be based on the recombinase RadA, which is of absolute requirement when no initiation origins are detected. The origin of this flexibility in the initiation of replication and the extent to which it is used in nature are yet to be understood. We combined deep sequencing and genetics to elucidate the dynamics of oriC utilization according to growth phases. We discovered that in Thermococcus barophilus, the use of oriC diminishes from the lag to the middle of the log phase, and subsequently increases gradually upon entering the stationary phase. Although oriC demonstrates no indispensability, RadA does exhibit essentiality. Notably, a knockdown mutant strain provides confirmation of the pivotal role of RadA in RDR for the first time. Thus, we demonstrate the existence of a tight combination between oriC utilization and homologous recombination to initiate DNA replication along the growth phases. Overall, this study demonstrates how diverse physiological states can influence the initiation of DNA replication, offering insights into how environmental sensing might impact this fundamental mechanism of life.
More information: https://pubmed.ncbi.nlm.nih.gov/38421162/
Contact: Jacques OBERTO <jacques.oberto@i2bc.paris-saclay.fr>
JAK-STAT-dependent contact between follicle cells and the oocyte controls Drosophila anterior-posterior polarity and germline development
The authors identified a population of somatic cells in Drosophila follicles that elaborate filopodia penetrating the oocyte, thereby maintaining the contact between the two tissues. This is essential to control polarity and germline development of the future embryo.
The number of embryonic primordial germ cells is determined, in Drosophila, by the quantity of germ plasm, whose assembly starts in the posterior region of the oocyte during oogenesis. Here, we report that extending JAK-STAT activity in the posterior somatic follicular epithelium leads to an excess of primordial germ cells in the future embryo. We show that JAK-STAT signaling is necessary for the differentiation of approximately 20 specialized follicle cells maintaining tight contact with the oocyte. These cells define, in the underlying posterior oocyte cortex, the anchoring of the germ cell determinant oskar mRNA. We reveal that the apical surface of these posterior anchoring cells extends long filopodia penetrating the oocyte. We identify two JAK-STAT targets in these cells that are each sufficient to extend the zone of contact with the oocyte, thereby leading to production of extra primordial germ cells. JAK-STAT signaling thus determines a fixed number of posterior anchoring cells required for anterior-posterior oocyte polarity and for the development of the future germline.
More information: https://doi-org.insb.bib.cnrs.fr/10.1038/s41467-024-45963-z
Contact: Marianne MALARTRE <marianne.malartre@i2bc.paris-saclay.fr>
Transposon sequencing reveals genes enabling insect gut colonization by the symbiont Caballeronia insecticola
High-througput genetic screens with Tn-seq in an insect gut bacterium reveals gut-derived nutrients consumed by the symbiont.
Caballeronia insecticola is a bacterium belonging to the Burkholderia sensu lato, able to colonize multiple environments like soils and the gut of the bean bug Riptortus pedestris. We constructed a saturated Himar1 mariner transposon library and revealed by transposon-sequencing (Tn-seq) that 498 protein-coding genes constitute the essential genome of C. insecticola for growth in free-living conditions. By comparing essential gene sets of C. insecticola and seven related Burkholderia s.l. strains, only 120 common genes were identified indicating that a large part of the essential genome is strain-specific. In order to reproduce specific nutritional conditions that are present in the gut of R. pedestris, we grew the mutant library in minimal media supplemented with candidate gut nutrients and identified several condition-dependent fitness-defect genes by Tn-seq. To validate the robustness of the approach, insertion mutants in six fitness genes were constructed and their growth-deficiency in media supplemented with the corresponding nutrient was confirmed. The mutants were further tested for their efficiency in R. pedestris gut colonization, confirming that gluconeogenic carbon sources, taurine and inositol, are nutrients consumed by the symbiont in the gut. Thus, our study provides insights about specific contributions provided by the insect host to the bacterial symbiont.
More information: https://doi.org/10.1093/ismeco/ycad001
Contact: Peter MERGAERT <peter.mergaert@i2bc.paris-saclay.fr>
Protein-protein interactions: how to push forward the limits of the revolutionary AlphaFold2 programme?
AlphaFold2 is revolutionising protein structure prediction and structural biology practices. However, it may prove less effective for certain protein assemblies, particularly when they depend on intrinsically disordered regions. In an article published in Nature Communications, researchers from the I2BC show that applying a fragmentation strategy to the protein partners of such assemblies very significantly improves AlphaFold2’s prediction capacity.
Mapping protein-protein interaction networks is essential for understanding the dynamics of cellular functions and their cross-regulation. Precise knowledge of interaction sites makes it possible to specifically perturb the proteins in these networks and understand the synergies and competitions that ensure cell function.
Unfortunately, a great amount of structural information is still lacking to provide a detailed understanding of the organisation of interaction networks. The AlphaFold2 artificial intelligence programme has demonstrated a remarkable ability to predict the structures of protein assemblies that have co-evolved over long time scales. Its performance remained poorly characterised for assemblies involving intrinsically disordered regions, which often mediate transient and dynamic interactions during evolution.
In a study published in the journal Nature Communications, researchers from the AMIG team at the Institut de Biologie Intégrative de la Cellule – I2BC (CNRS/CEA/UPSaclay, Gif-sur-Yvette) have shown that AlphaFold2 performs poorly if large disordered regions are used directly for prediction (40% success rate). A protein fragmentation strategy was found to be particularly well adapted to predicting the interfaces between folded domains and small protein motifs that fold on contact with the partner. Applied on a large scale using the Jean Zay HPC infrastructure on more than 900 complexes, this strategy achieved a success rate of almost 90%, a very encouraging result for the systematic screening of protein interaction networks. Nevertheless, the study calls for vigilance with regard to the risks of detecting false positives, which will be at the heart of future developments in artificial intelligence strategies such as AlphaFold2.
More information: https://www.nature.com/articles/s41467-023-44288-7
Contact: Jessica ANDREANI and Raphaël GUEROIS <jessica.andreani@i2bc.paris-saclay.fr> <raphael.guerois@i2bc.paris-saclay.fr>
The stringent response is strongly activated in the high antibiotic producer, Streptomyces coelicolor.
The stringent response controls positively antibiotic biosynthesis. Antibiotics are thus part of the stringent response.
In most bacteria, the stringent response (SR) was initially characterized as a response to nitrogen (N) limitation resulting into the depletion of aminoacylated tRNAs leading to the stalling of ribosomes on mRNA. The recruitment of the ppGpp synthetase, RelA, at the stalled ribosomes activates (p)ppGpp synthesis from GTP. ppGpp that is the mediator of the SR controls negatively, at the transcriptional and translational levels, the expression of most ribosomal proteins leading to the down regulation of the translational process and thus of growth.
The model strains Streptomyces coelicolor (SC) and Streptomyces lividans (SL), strong and weak producers of the same antibiotics, respectively, were grown in condition of phosphate (Pi) limitation or proficiency and the abundance of proteins of their translational apparatus was compared. This study revealed that the expression of RelA was induced in Pi limitation suggesting that, besides N limitation, Pi limitation also contributes to the triggering of the SR. Interestingly, most proteins of the translational apparatus had a similar or slightly higher abundance in SL than in SC, in Pi limitation whereas most of these proteins were far more abundant in SL than in SC, in Pi proficiency. This indicated an alleviation of the SR in Pi proficiency in SL, but not in SC. This suggested an alteration of Pi up-take and/or Pi-mediated regulation in SC whose molecular basis remain to be elucidated.
Interestingly, the production of specialized metabolites in SC (CDA, RED and ACT) is usually concomitant of phases of growth slow down and it is known that ppGpp controls positively the expression of their biosynthetic pathways. Their production could thus be considered as part of the SR. Indeed, these metabolites were proposed to regulate negatively, through different processes, the energetic metabolism and thus the generation of ATP, in SC, a process that might contribute to the slower growth rate of SC compared to SL.
More information: https://www.sciencedirect.com/science/article/pii/S0923250823001547?via%3Dihub
Contact: Marie-Joëlle VIROLLE <marie-joelle.virolle@i2bc.paris-saclay.fr>
Composition of Poxvirus Core Revealed
A collaboration between groups at I2BC, CSSB, MPI, and PEI, reveals the structure and the flexibility of A10 trimers that compose the palisade layer of the Vaccinia virus core encasing the viral genome.
Vaccinia virus is the model for the family of poxviruses, however, the structure of the viral particle used to propagate infection is poorly understood. The group of Emmanuelle Quemin at I2BC together with collaborators at CSSB in Hamburg, MPI for Biophysics in Frankfurt, and PEI in Langen, have revealed the composition and architecture of Vaccinia virus core that encases the viral genome. Their findings have been published in Nature Structural & Molecular Biology.
By combining cryo-electron tomography with subtomogram averaging and AlphaFold2, the authors were able to identify components of the core of Vaccinia virus. During entry, there is the fusion of the viral and cellular membranes that lead to the subsequent release of the viral core inside the host cell. As these are rare and fast events difficult to tackle by cellular cryo electron tomography, the researchers studied the core both in situ and in vitro, using virusal particles treated with detergents to access so-called “naked” cores containing the viral genome still. In parallel to the large dataset obtained in vitro, entering cores found inside cells were also analysed under native conditions at CSSB cryo-EM facility directed by Kay Grünewald in Hamburg, Germany,
The four groups involved focused more specifically on the outer layer of the core called the palisade that displayed a dense and organized surface of tubular protrusions that we refer to as stakes. Their study determined that these protrusions are trimers of the viral protein A10, previously known as one of the major core proteins. Here, the stakes appeared as more randomly organized than reported in other recent published work and have an inherent flexibility.
While some poxviruses can spread in human populations as recently exemplified with the Mpox virus multi-country outbreak, improving our understanding of Vaccinia virus and its core sub-structure is key to shed light on conserved mechanisms of poxvirus infection and pathogenicity.
More information: https://www.nature.com/articles/s41594-024-01218-5
Contact: Emmanuelle QUEMIN <emmanuelle.quemin@i2bc.paris-saclay.fr>
Schizosaccharomyces pombe as a fundamental model for research on mitochondrial gene expression: Progress, achievements and outlooks
This is a comprehensive and critical review on mitochondrial gene expression in fission yeast, presenting up-to-date knowledge, and emphasising numerous contributions of the unicellular model to both fundamental and biomedical research.
Schizosaccharomyces pombe (fission yeast) is an attractive model for mitochondrial research. The organism resembles human cells in terms of mitochondrial inheritance, mitochondrial transport, sugar metabolism, mitogenome structure, and dependence of viability on the mitogenome (the petite-negative phenotype). Transcriptions of these genomes produce only a few polycistronic transcripts, which then undergo processing as per the tRNA punctuation model. In general, the machinery for mitochondrial gene expression is structurally and functionally conserved between fission yeast and humans. Furthermore, molecular research on S. pombe is supported by a considerable number of experimental techniques and database resources. Owing to these advantages, fission yeast has significantly contributed to biomedical and fundamental research. Here, we review the current state of knowledge regarding S. pombe mitochondrial gene expression, and emphasise the pertinence of fission yeast as both a model and tool, especially for studies on mitochondrial translation.
More information: https://doi.org/10.1002/iub.2801
Contact: Nathalie BONNEFOY <nathalie.bonnefoy@i2bc.paris-saclay.fr>
Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design
Self-anchoring of large antigens onto Capsid-Like Particles derived from bacteriophage T5 paves the way for the development of a new vaccination platform that is highly immunogenic without the need for extrinsic adjuvant.
The constant need for immunization to prevent life-threatening diseases worldwide is urging the search for new vaccines. The promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. In this study, we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5 to design a novel Ag delivery platform. DNA-free T5 capsid-like particles (T5-CLP) were decorated with chimeric proteins formed of pb10 fused to the model antigen ovalbumin (Ova). SPR experiments demonstrated that these proteins retained picomolar affinity for T5-CLP, while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunisation with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8+ T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes a unique DNA-free bacteriophage capsid able to display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform.
More information: https://www.nature.com/articles/s41541-023-00798-5
Contact: Pascale BOULANGER <pascale.boulanger-biard@i2bc.paris-saclay.fr>
Toxoplasma membrane inositol phospholipid binding protein TgREMIND is essential for secretory organelle function and host infection
CApicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.
More information: doi: 10.1016/j.celrep.2023.113601
Contact: Stanislas TOMAVO <stanislas.tomavo@i2bc.paris-saclay.fr>