DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Bacterial Cell Remodeling by Viral Infection – BacVirRemodel

Bacterial Cell Remodeling by Viral Infection

Viral infection imposes a major challenge to the host cell. Bacterial viruses (phages or bacteriophages) use a variety of strategies to hijack cellular machineries and to restructure the bacterial cell. This project aims to study the molecular and cellular bases of host takeover used by bacteriophage SPP1 during infection of its host, the Gram-positive bacterium Bacillus subtilis.

Molecular and cellular mechanisms leading to conversion of a Gram-positive bacterium in a viral factory

Our previous work provided a cytological description of the bacteriophage SPP1 multiplication process in the bacterium Bacillus subtilis and identified most of its effectors and their interactions network. The present research proposal builds in this basic knowledge to dissect in real time the assembly, organization and dynamics of individual components of the virus genome replication and viral particles warehouses, as well as their relationship with host cellular factors, throughout the complete infection cycle. The present proposal has two main goals:<br /><br />- The first goal of the project is to investigate how the SPP1 DNA replication factory self-organizes, establishes its spatial localization, and the dynamics of its components at different stages of SPP1 genome replication. <br /><br />- The second goal of the project is to investigate the mechanisms that ensure formation of viral particles warehouses spatially distinct from genome replication factories.

We use different imaging approaches, including microfluidics, to follow the dynamics of viral proteins during infection of Bacillus subtilis by SPP1. Those studies are complemented by qPCR, by analyses of changes in the bacterial chromosome 3D topological organization (Hi-C), and by biochemical studies of viral macromolecular assemblies formed during the infectious process.

We showed that SPP1 infection leads to formation of a viral genome replication factory in the Bacillus subtilis cytoplasm. Phage mutants blocking different steps of SPP1 replication provided snapshots of the factory at distinct steps of its assembly. At late times of infection it was observed the formation of two warehouses of phage particles at neighbor, but distinct, regions to the replication factory. The temporal and spatial program of assembly of these foci confining viral biochemical reactions in the bacterial cytoplasm was analyzed by real time imaging.
We also showed that restructuration of the bacterial cytoplasm during SPP1 infection leads to changes of the bacterial nucleoid and of the host transcriptional machinery.

We have presently a detailed knowledge of the biochemical reactions leading to replication of the SPP1 genome and to assembly of its viral particles. Such knowledge combined with the imaging setup recently established to visualize these processes in the bacterial cell will be instrumental to follow the spatiotemporal dynamics of different viral proteins during the SPP1 infectious process. Correlation of the cellular behavior of phage proteins with their function and network of macromolecular interactions will provide a major advance to the understanding how the phage exploits the cellular environment to optimize the biochemical reactions leading to its multiplication.

Communications in Conferences:
- A. Labarde et al, Phages on MARSeille, Marseille, November 2015 (talk).
- A. Labarde et al, The 100th Centennial Celebration of Phage Research, Paris, April 2017 (poster, prize for best poster).
- A. Labarde et al, 19th International Conference on Bacilli & Gram-Positive Bacteria, Berlin, Germany, June 2017 (talk).

During the long co-evolution of viruses and cells, viruses exploited numerous ways to hijack cell machineries for their efficient multiplication and dissemination. In turn, cells developed defensive mechanisms to oppose virus attack. Studies of these encounters aimed to understand infection but also to dissect molecular and cell biology complex processes like signaling pathways, cell architecture and compartmentalization, traffic, protein folding and others. However, in contrast to eukaryotic viruses, the cell biology of bacterial viruses (phages or bacteriophages) infection remains largely uncharacterized. Recent progress that uncovered the bacterium complex architecture created the momentum to study how phages take advantage of the host cell organization to optimize their multiplication.

This research proposal brings together a team specialized in phage biology and another with extensive expertise in bacterial cell biology. The biological system under study is the well-characterized bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis. SPP1 is a model system for the lineage of tailed phages-herpesviruses that share common ancestry. We discovered that during infection SPP1 builds spatially independent factories for viral genome replication and assembly of viral particles in the bacterial cytoplasm. Our collaboration aims to characterize molecular and cellular mechanisms underlying formation of these factories. We will characterize their composition. Phage mutants will be used to arrest their assembly at specific steps to determine the order of recruitment of their individual components and to study transient states of the factories. Protein dynamics will be imaged using multidimensional live-cell microscopy and microfluidics to follow the complete infection cycle in real time. The nanoscopic organization of the factories will be investigated by super-resolution microscopy and collaborative cryo-electron tomography. The recruitment of the bacterial replisome to the viral genome replication factory and the transfer of viral DNA to the viral particle assembly factory will also be investigated. This work aims to unravel cellular and molecular mechanisms used by viruses to massively hijack cellular machineries converting the bacterium in a viral factory. Knowledge of these processes in molecular detail will be instrumental to understand remodeling of the bacterial cell and hijacking of its resources during viral infection.

Project coordination

Paulo TAVARES (Cnetre National de la Recherche Scientifique (CNRS), Délégation Régionale Ile-de-France Secteur Sud)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS (DR4) Cnetre National de la Recherche Scientifique (CNRS), Délégation Régionale Ile-de-France Secteur Sud
MICALIS UMR 1319 INRA

Help of the ANR 505,982 euros
Beginning and duration of the scientific project: February 2016 - 48 Months

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