#	Pagina
attuale pagina	/open-h2020/projects/203239/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - Virocellsphere (Host-virus chemical arms race during algal bloom in the ocean at a single cell resolution)

Teaser

Summary

Phytoplankton are unicellular alga that form massive oceanic blooms covering thousands of square kilometers. Half of the photosynthetic activity on Earth is performed in the ocean by these microscopic organisms, and their influence on global cycles of carbon, nitrogen and sulfur is immense. The short life span of these organisms makes them a sensitive proxy for evaluating the impact of climate change on marine ecosystems. Microbial interactions that regulate the fate of algal blooms play a profound role in determining carbon and nutrient cycling in the ocean and feedback to the atmosphere. Some of the key mortality agents of algal cells are marine viruses, bacteria and grazers. Viral infection that leads to demise of a bloom will have a unique footprint on the environment and is predicted to influence the carbon export to the deep ocean, recycling of nutrients available to the rest of the marine organisms, biodiversity and even influence the atmosphere above the dying bloom. The latter occurs when dying algal cells produce volatile chemicals that are emitted to the atmosphere as aerosols and can modulate cloud formation.

Marine viruses are the most abundant biological entities in the marine environment and are hypothesized to be major ecological, evolutionary and biogeochemical engines. Algae-infecting viruses were estimated to turn-over more than a quarter of the total photosynthetically fixed carbon, thereby fueling microbial food webs, short-circuiting carbon transfer to higher trophic levels and promoting export to the deep sea. As major evolutionary drivers, marine viruses enhance the diversity of microbial life, affect species composition, and are responsible for widespread lateral gene transfer with their hosts. Despite the huge ecological importance of host-virus interactions, the ability to assess their ecological impact is limited to current approaches, which focus mainly on quantification of viral abundance and diversity. A major challenge in our current understanding of host-virus interactions in the marine environment is to decode the wealth of genomic and metagenomics data and translate it into cellular mechanisms that mediate host susceptibility and resistance to viral infection.
Extensive work conducted in our lab in the past few years focused on the molecular mechanisms underlying the interaction between E. huxleyi and its specific virus. By combining advanced cell biology, genome-enabled technologies and analytical chemistry approaches, we were able to identify several fundamental metabolic pathways that mediate these host-virus interactions.

The overarching objectives of our proposal are:
1. To characterize distinct cell-states within a population of infected host cells that respond differentially to viral infection.
2. To reveal the composition of the information-conveying chemicals, generated by virus-infected cells and their role in determining cell fate and susceptibility to infection.
3. Comprehensive characterization of phenotypic heterogeneity during host-virus interactions and its ecological significance during natural E. huxleyi blooms.

Work performed

We study the interaction between Emiliania huxleyi, a bloom-forming alga, and its specific virus (EhV), an ecologically important host-virus model system in the ocean. Almost all of our current understanding of the molecular mechanisms that govern host-virus interactions in the ocean, is derived from experiments carried out at the population level, assuming synchrony and uniformity of the cell populations and neglecting any heterogeneity. We quantified host and virus gene expression on a single-cell resolution during the course of infection, using automatic microfluidics that captures individual algal cells. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and allowed detection specific host response in a subpopulation of infected cell which otherwise masked by the majority of the infected population (Rosenwasser et al., 2019). Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and sensitive cells within heterogeneous populations in the marine environment (Frada et al., 2017).
Rewiring of host metabolic pathways by the virus during infection generates multiple metabolic states which may affect the infection outcome (i.e. cell death or resistance). Mapping these metabolic states into infection states might uncover unique strategies employed by the virus, which are essential for an optimal infection process. Gaining a continuous view into the infection process is highly challenging and is limited by current metabolomics approaches, which typically measure the average of the entire population at various stages of infection. We employ an innovative approach to study the metabolic basis of E. huxleyi-EhV interaction. We combined a classical method in virology, the plaque assay, with mass spectrometry imaging (MSI), an approach we termed â€˜in plaque-MSIâ€™ (Schleyer et al 2019). Taking advantage of the spatial characteristics of the plaque, we mapped the metabolic landscape induced during infection in a high spatiotemporal resolution, unfolding the infection process in a continuous manner. By using this method we were able to discover novel metabolites that are induced during infection and may now be used as biomarkers to track host-virus dynamics in natural blooms (Figure 1).

By using communication-conveying signals, microorganisms can acclimate to changing environmental conditions and synchronize population level behaviors. Extracellular vesicles (EVs) have recently been discovered as a new mode of communication across all domains of life. Little is known about the involvement of EVs in microbial interactions in the marine environment (Schatz and Vardi 2018). We investigate the signaling role of EVs produced during E. huxleyi-EhV interactions and found that EVs are highly produced during viral infection or when bystander cells are exposed to infochemicals derived from infected cells (Figure 2). These vesicles have a unique lipid composition and their cargo is composed of specific small RNAs that are predicted to target sphingolipid metabolism and cell-cycle pathways (Schatz et al. 2017). EVs can be internalized by E. huxleyi cells consequently leading to a faster viral infection dynamic. EVs can also prolong EhV half-life in the extracellular milieu. We propose that extracellular vesicles are exploited by viruses to sustain efficient infectivity and propagation across E. huxleyi blooms. This novel mode of communication may influence the fate of the blooms and, consequently, the composition and flow of nutrients in marine microbial food webs.

Despite the huge ecological importance of E. huxleyi-EhV interactions, the ability to assess their spatial and temporal dynamics and their possible impact on nutrien

Final results

Based on our recent findings we aim at deepening our understanding of E. huxleyi-EhV interactions. We are currently in the midst of developing single-cell whole transcriptome analyses of infected cells. This novel approach will enable discovery of the different phases of viral infection and genes associated with host response to the infection. We hope to shed light on the resistance mechanisms of the minority of host cells on one hand, and on the other to learn about the cellular programs that lead to the death of infected cells and, consequently, the demise of algal blooms. This analysis will be coupled to the newly-developed single-molecule-FISH (smFISH), a method that enables the detection and visualization of specific RNA molecules within single cells. smFISH allows to probe for expression of specific E. huxleyi and EhV genes within cells collected from natural populations, thus enabling specific detection of the level of infection within the E.huxleyi sub-population of phytoplankton. Using a metabolomics approach we are able to detect small molecules that are produced specifically during infection. Additionally, the metabolic composition of resistant and sensitive E. huxleyi strains is currently under investigation. These unique metabolic fingerprints will be used as biomarkers to track the different infection states and resistance phenotypes within natural samples from mesocosm experiments and those collected during scientific cruises. We have recently characterized extracellular vesicles produced by E. huxleyi during viral infection and used as a communication agent to modify the dynamic of infection. We are now expanding this new research field to enable detection of specific vesicles in the environment and to understand their ecological role in natural microbial assemblages.