Science Advances: Bacterial cells form membraneless organelles through liquid-liquid separation to enhance drug resistance

Liquid-liquid phase separation (LLPS) is a key driving mechanism for many important life processes in cells
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In eukaryotic cells, LLPS can enrich various related biomolecules and help cells form various membraneless structures, such as P-granules, nucleoli, heterochromatins, and Stress-granules, etc.

Screenshot of the paper

Peking University Biomedical Frontier Innovation Center (BIOPIC), Beijing Future Gene Diagnosis Advanced Innovation Center (ICG), School of Life Sciences Researcher Bai Fan's research group once published in Molecular Cell in 2018 entitled "ATP-dependent dynamic protein" The research paper "aggregation regulates bacterial dormancy depth critical for antibiotic tolerance" reports a new type of intracellular structure in bacteria that can dynamically aggregate and disperse-protein precipitation aggregates (aggresomes)
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Aggresomes appear as small black spots distributed in bacterial cells in the bright field of the microscope, which are formed when cells encounter external pressure (nutrient deficiency, antibiotic attack), and promote cells to enter a dormant state; after the external environment improves, aggresomes are eliminated and the bacteria Regain growth

Figure 1 Using single-molecule high-resolution fluorescence microscopy imaging technology to study the formation mechanism of bacterial protein aggregates

The research team first selected three proteins (HslU, Kbl, AcnB) enriched in aggresomes as the research objects, and constructed fusion proteins with fluorescent labels for them
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By overexpressing HokB (a bacterial Toxin) in the bacteria, the ATP level in the bacterial cells is reduced, thereby artificially inducing the formation of aggresomes (Figure 1)

The researchers selected the HslU protein with the fastest response speed as the biomarker of aggresomes, and explored the spatiotemporal dynamic characteristics of aggresomes formation through single-molecule fluorescence tracking
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Using the Slimfield single-molecule tracking technology (Figure 1C), the researchers obtained the trajectory and average diffusion coefficient of a single HslU-EGFP molecule inside aggresomes, and found that the HslU protein can move freely inside aggresomes, which shows that the aggresome is not solid; and At the same time, the dynamic process of the formation of aggresomes was observed under a microscope, and it was found that in the early stage of the formation of aggresomes, there will be a collision-fusion process between different aggresomes in the cell.

Figure 2 Liquid-liquid separation drives the formation of bacterial protein precipitation aggregates

The research team used the Individual-Protein-Based Model (IPBM) to simulate the process of the gradual aggregation of protein precipitation droplets and phase separation to form larger membraneless organelles
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Through Monte-Carlo simulation of the diffusion-collision-aggregation process of bacterial intracellular proteins, the model can quantitatively explain and verify the results previously observed in the experiment (Figure 3)

Figure 3 The mathematical model simulates the fine process of aggresomes formation

The research team proved that reducing intracellular ATP in a variety of common bacteria can induce the production of aggresomes, suggesting that the formation of aggresomes may be a universal response of bacteria against external pressure
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In order to explore the biological functions of aggresomes, the research team used chemical small molecule blockade and gene knockout to construct a defective strain that prevents aggresomes from forming normally.

This study expands the application of the liquid-liquid phase separation mechanism in prokaryotes, and found that bacterial cells use phase separation to dynamically adjust the temporal and spatial distribution of proteins in the cytoplasm, and enhance the bacteria's ability to resist external environmental pressure and antibiotic resistance
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In particular, the response of the liquid-liquid phase separation to the intracellular ATP concentration revealed in this study is a brand-new regulatory mechanism that deserves further study

Dr.
Jin Xin, Peking University Biomedical Frontier Innovation Center, School of Life Sciences, PhD student Luo Xinwei, Dr.
Ji-Eun Lee from the Department of Physics, University of York, UK, and Dr.
Charley Schaefer are the co-first authors of this paper
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Fan Bai and Mark C.