Deciphering the molecular principles of bacterial metabolosome biogenesis

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Pathogenic bacteria, such as Salmonella, thrive in mammalian intestines and can cause severe health issues in human, including food poisoning, massive gut inflammation and cardiovascular disease. There were estimated 535,000 cases of Salmonella gastrointestinal infections worldwide in 2017, and 91,857 cases in the EU in 2018. Salmonella cells produce a specialised nano-scale organelle, known as the bacterial microcompartment. These organelles provide a suite of unique metabolic advantages that allow Salmonella to become the predominant species in the hostile environment of the host gut. The organelle uses a shell that is made of thousands of proteins to sequester multiple enzymes used for 1,2-propanediol utilisation (Pdu). This unique structure allows the Pdu organelles to protect bacterial cells from toxic metabolites and to enhance the cell's metabolism. Although the importance of Pdu organelles for the metabolism of bacterial pathogens is appreciated, little is known about how bacterial cells generate and then modulate these organelles to confer adaptive cellular metabolism to survive in the sophisticated gut environment.We have recently reported the exact protein stoichiometry of Pdu organelles and have established a new structural model of the organelle. We have also developed systems for tagging Pdu proteins with fluorescent markers and depleting target proteins, so that we can track specific building proteins using microscopes and study their functions in bacterial cells. Using the developed systems, we have discovered that the cargo enzymes and shell proteins self-assemble independently in Salmonella. We have also shown that the locations and movement of Pdu organelles are confined within the bacterial cell. Standing on these exciting scientific and technical breakthroughs and an established research team with complementary expertise, we now aim to do an in-depth exploration of how Pdu organelles are synthesized and how the organisation of Pdu organelles is coordinated within the Salmonella cell. We will first determine the multi-step assembly that individual building proteins undergo to form higher-ordered assemblies, identify the proteins that make up the enzyme and shell assemblies, and elucidate how enzyme and shell assemblies associate together to form an intact organelle. In the second section of our programme, we will characterise the structures and functions of small linker proteins that drive the assembly of cargos to have a "liquid-like" dynamic phase and ascertain that the phase separation mechanism is vital for mediating the protein interactions and formation of a functional protein organelle. Finally, we will use state-of-the-art fluorescence microscopy to probe how the Pdu organelles are generated, located, and modulated to perform such important functions in bacterial cells.This ambitious and multidisciplinary research project has both fundamental and applied significance. Pdu MCPs represents an ideal model system for uncovering the principles of protein self-assembly and the generation of multi-protein complexes in biology. We will learn the basic physics and chemistry of how thousands of proteins assemble together to build a functional entity within a bacterial cell, and determine how the cell precisely and efficiently controls the formation and function of metabolic organelles. We anticipate that our findings will provide a deeper understanding of the biosynthesis and maintenance of natural bacterial organelles and protein assemblies. The research may inform strategies for the engineering of biological "factories" for the enhancement of cell metabolism and energy production in diverse biotechnological applications. Moreover, the essential protein-protein interactions that we find are required to mediate the assembly of Pdu organelles could represent novel therapeutic targets to disrupt the production of Pdu organelles and thus ablate the ability of Salmonella to thrive in the human gut.