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Summary

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>

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