Kyle, J. E. | bioRxiv Science

bioRxiv2018

Integrated systems biology and imaging of the smallest free-living eukaryote Ostreococcus tauri

Ostreococcus tauri is an ancient phototrophic microalgae that possesses favorable genetic and cellular characteristics for reductionist studies probing biosystem design and dynamics. Here multimodal bioimaging and multi-omics techniques were combined to interrogate O. tauri cellular changes in response to variations in bioavailable nitrogen and carbon ratios. Confocal microscopy, stimulated Raman scattering, and cryo-soft x-ray tomography revealed whole cell ultrastructural dynamics and composition while proteomic and lipidomic profiling captured changes at the molecular and macromolecular scale.\n\nDespite several energy dense long-chain triacylglycerol lipids showing more than 40-fold higher abundance under N deprivation, only a few proteins directly associated with lipid biogenesis showed significant expression changes. However, the entire pathway for starch granule biosynthesis was highly upregulated suggesting much of the cellular energy is preferentially directed towards starch over lipid accumulation. Additionally, three of the five most downregulated and five of the ten most upregulated proteins during severe nitrogen depletion were unnamed protein products that warrant additional biochemical analysis and functional annotation to control carbon transformation dynamics in this smallest eukaryote.

systems biology↗

bioRxiv2017

Listeria monocytogenes virulence factors are secreted in biologically active Extracellular Vesicles

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteriaare implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Cell-free EV preparations were toxic to the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC can restrain LLO activity. Using immunogold electron microscopy we detect LLO localization at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes that colocalize with LLO during infection. Our findings demonstrate that L. monocytogenes utilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.

microbiology↗