Comment la protéine de phage Vp16PDF tue-t-elle les bactéries? Décodage du mécanisme moléculaire sous-jacent

How does the phage protein Vp16PDF kill bacteria? Decoding the underlying molecular mechanism

• Auteur / Autrice : Xiaofen Wu
• Direction : Carmela Giglione, Thierry Meinnel
• Type : Projet de thèse
• Discipline(s) : Biochimie et biologie structurale
• Date : Inscription en doctorat le 10/11/2020
• Etablissement(s) : université Paris-Saclay en cotutelle avec NO
• Ecole(s) doctorale(s) : École doctorale Structure et Dynamique des Systèmes Vivants
• Partenaire(s) de recherche : Laboratoire : I2BC - Institut de Biologie Intégrative de la Cellule
• Partenaire(s) de recherche : Equipe de recherche : RNA-binding proteins in gene expression and cell differentiation
• Partenaire(s) de recherche : référent : Faculté des sciences d'Orsay

Mots clés

Mots clés libres

Phage

Bactéries

Mécanisme moléculaire

Phage

Bacteria

Molecular mechanism

Résumé

Les peptides déformylases (PDFs) sont des enzymes qui font partie d'un processus essentiel appelé excision de la méthionine N-terminale (NME) impliqué dans la biogénèse des protéines au niveau du ribosome. Les PDFs retirent le groupe formyl de la N-formyl-methionine, qui se trouve au début de toutes les chaînes naissantes procaryotes. Le processus NME est la première modification que subissent toutes les protéines. La déformylation touche 95% des protéines d'un protéome bactérien et il est suggéré de contribuer directement à la stabilité des protéines. Le séquençage récent à haut débit de milliers de génomes a fortement contribué à révolutionner notre perception de la distribution des PDF entre les règnes du vivant, révélant des PDFs putatifs dans tous les organismes, y compris les virus. En particulier, des études de virus dans des échantillons microbiens océaniques ont permis de retrouver des gènes PDFs inhabituels comme la famille la plus abondante dans la plupart des génomes de phages marines. Pourquoi tant de phages portent des gènes PDFs dans leur génome reste inconnu. Des comparaisons de séquences avec des PDFs connus révèlent que les PDFs viraux présentent une conservation élevée dans les trois motifs qui construisent le site catalytique; cependant, les PDF viraux ne présentent pas d'extension C-terminal par rapport aux différents PDFs actifs provenant d'autres organismes. Dans la mesure où cette extension C-terminale s'est avérée importante pour la liaison PDF-ribosome et est requise pour l'activité déformylase in vivo de la PDF d' E. coli, il était difficile de savoir si les PDF phages découverts pourraient supporter une activité classique de déformylase. La caractérisation récente de plusieurs PDF de phages marins a fourni la première preuve que les PDFs des phages présentent une activité de déformylase à la fois in vitro et in vivo. Parmi eux, le Vp16PDF est le plus intriguant puisqu'il s'agit à ce jour de la déformylase active la plus courte connue. Fait intéressant, notre analyse in vivo préliminaire a révélé que l'expression de Vp16PDF dans E. coli, même à de faibles concentrations, présentait un effet bactéricide grave à une température < 37 ° C. Néanmoins, rien n'est connu sur la manière dont ces PDF phages affectent la biogenèse des protéines virales et de l'hôte. Dans le cadre de ce projet de thèse, le projet que nous proposons aidera à comprendre pourquoi les phages possèdent des PDFs. Ainsi, il s'agira d'élucider le mécanisme de fonctionnement de ces protéines et à révéler les voies biologiques impliquées. Plus particulièrement, l'étudiant évaluera l'hypothèse fascinant que la PDF du phage puisse contribuer à la lyse de son hôte

State-of-the-art and preliminary data Peptide deformylases (PDFs) are enzymes involved in an essential and conserved co-translational process called N-terminal methionine excision (NME), during which PDFs remove the formyl moiety from N-formyl methionine, found at the beginning of all prokaryotic nascent chains. NME is the first occurring N-terminal Protein Modification (NPM)1,2. PDFs act on the nascent chain at the level of the ribosome exit tunnel, a central hub for a number of proteins (Ribosome-associated Protein Biogenesis factors, RPBs) involved not only in NPMs but also in protein folding and translocation2. Deformylation involves 95% of the proteins of a bacterial proteome3 and it is suggested to directly contribute to protein stability4. Recent high-throughput sequencing of thousands of genomes has strongly contributed to revolutionizing our perception of the distribution of PDFs among kingdoms, revealing putative PDFs in all organisms, including viruses. In particular, studies of viruses within oceanic microbial samples retrieved unusual PDF genes as the most abundant family in most phage genomes5. Why so many phages carry PDF genes in their genome remains unknown. The evidence that viral proteins also undergo NME led Team15 to initially propose that encoded phage PDFs could be important for viral fitness. Nonetheless, nothing is known about how these phage PDFs impact viral and host protein biogenesis. Sequence comparisons with known PDFs reveal that viral PDFs show high conservation in the three motifs that build the catalytic site; however, viral PDFs do not display a C-terminal extension when compared to the different active PDFs from other organisms5. Inasmuch as this C-terminal extension was shown to be important for PDF-ribosome binding and is required for the in vivo deformylase activity of E. coli PDF6,7, it was unclear whether the discovered phage PDFs might support a classical deformylase activity. The recent characterization in my team and that of Prof. Segall of several marine phage PDFs provides the first evidence that phage PDFs display deformylase activity both in vitro and in vivo8,9. Among them, Vp16PDF is the most intriguing since it is to date the shortest known active deformylase. Large-scale N-terminomics characterization revealed that Vp16PDF has substrate specificity similar to that of other bacterial PDFs9. On the other hand, Giglione's team showed by biochemical and structural data, combined with site-directed mutagenesis analyses, that Vp16PDF significantly differs from previously characterized PDFs in terms of their properties, which can be related to its few infrequent peculiarities (9 and unpubl. data). Interestingly, Giglione's team's preliminary in vivo analysis revealed that expression of Vp16PDF in E. coli, even at low concentrations, exhibited a severe bactericidal effect at temperatures below 37°C. Of note, such a noxious effect of Vp16PDF was independent of the presence of the bacterial endogenous PDF and strictly relied on its PDF activity (unpubl. data). Moreover, preliminary characterization of this phenotype revealed that it caused cell lysis and affected outer membrane integrity (unpubl. data). Hence, the expression of Vp16PDF mimics the loss of the co-translational chaperone Trigger Factor (TF), which was shown to be critical in Outer Membrane Proteins (OMP) biogenesis. Interestingly, Giglione's team also found that Vp16PDF expressing cells were more sensitive to SecA-inhibitors and induced the formation of intracellular aggregates suggesting that Vp16PDF might specifically interfere with sec-dependent translocation (unpubl. data). Accordingly, Vp16PDF-induced lethality showed a strong genetic link with genes encoding cellular factors involved in nascent pre-secretory protein targeting (unpubl. data). Moreover, overexpression of some of these factors, such as the TF and certain Sec proteins, overcame this lethality (unpubl. data). In contrast, no effect was observed when conditional mutant and deletion strains of other general membrane-targeting and translocation pathways were investigated for potential in vivo links with PDF. This included the SRP and YidC pathways for inner membrane biogenesis and the Tat pathway for the export of folded proteins and protein complexes10. Outline the objectives, originality, and novelty of the project The proposed project has an overall objective to unravel the role of viral PDFs, particularly focusing on the characterization of Vp16PDF and its interplay with both the host bacterial PDF and with known RPBs. Vp16PDF is derived from bacteriophages originally isolated from V. parahaemolyticus strain 16 by Prof. Segall 11. Both phage and host are widely distributed in marine environments and V. parahaemolyticus is one of the major causes of human gastroenteritis after ingestion of bacteria-contaminated seafood. The different codon usage between Vp16 phage and V. parahaemolyticus, and the phage genome comparison indicating that the Vp16 phage clusters most closely with E. coli mycophagous P2 and 186, suggested the possibility that the phage may be a relative newcomer in V. parahaemolyticus. Hitherto, no other host examined by Prof. Segall was found sensitive to this phage11. The originality and innovative nature of this project, in relation to the state-of-the-art of field, will rely on the search for answers to new key unresolved questions related to phage PDFs. During this, the student will focus on the development of a specific WP (WP1), which is part of a vast joint project where a few WPs are already carried on in Giglione's lab and others will be developed by the student. In this project, we propose to address several timely questions through 4 specific coherent and structured objectives (see below WP). All proposed objectives will address how Vp16PDF influences the identified downstream pathways or other yet uncovered routes and which structural elements of the protein are involved. Moreover, the surmised contribution of Vp16PDF in host lysis will be explored. Least but not least, Vp16PDF will be also used as a novel experimental tool to challenge PDF-ribosome interactions including that of the host counterpart for which many aspects remain elusive. A tightly integrated combination of in vivo and in vitro techniques, several developed by the teams involved, will be used to provide complementary and interdependent information. Collectively, this project has the ultimate goal to draw a unified picture of the co-translation events involving phage and bacterial PDFs, from a macroscopic to a molecular level in the most integrative manner.