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Widespread Bacterial CORE Complex Executes Intra- and Inter-Kingdom Cytoplasmic Molecular Trade

Periodic Reporting for period 2 - BacterialCORE (Widespread Bacterial CORE Complex Executes Intra- and Inter-Kingdom Cytoplasmic Molecular Trade)

Reporting period: 2020-10-01 to 2022-03-31

Bacteria are the dominant and most abundant organisms on earth, residing in complex multi-species communities. Crucial for their capacity to flourish, is the need to maintain intricate molecular crosstalk with proximal prokaryotic and eukaryotic cells. However, as yet, we know very little about the basic principles controlling these multicellular interactions. We have identified a complex, herein termed CORE, composed of five membrane proteins that are highly conserved across the entire bacterial kingdom. We found that the CORE serves for biogenesis of extended membranous nanotubes, which in turn conduct a contact-dependent exchange of cytoplasmic molecules among bacteria belonging to different species, as well as between pathogenic bacteria and their human host cells, thus, facilitating both intra- and inter-kingdom crosstalk. The implications of such a continuous intercellular flow of cytoplasmic molecules via CORE are profound, indicating that in natural niches, such as soil, infected tissues, or human gut, the bacterial metabolic flexibility and versatility are considerably higher than previously appreciated. The objectives of the project are to elucidate the global molecular trafficking mediated by the CORE-nanotube, to reveal the structure and function of this newly identified organelle, and to determine the impact of the molecular flow mediated by the CORE-nanotube organelle on bacterial physiology and virulence.
On the first scientific period of the project, we have made significant progress towards achieving our aims according to our original plan. Our major results and progress are:
1) We revealed that the CORE provides the basis for a nanotube-organelle that connects pathogenic E. coli (EPEC) to its host, enabling host nutrient extraction (Pal et al., 2019 Cell, Cell cover, Cell featured article, Nature Highlights) (see Figure "Cell cover").
2) We discovered that the conserved CORE components serve dually for flagellar and nanotube assembly in diverse species belonging to different phyla (Bhattacharya et al., 2019 Cell Reports) (see Figure "Bs nanotubes").
3) We found that bacterial intercellular nanotube connection is enabled by the delivery of lytic enzymes over nanotubes form a donor bacterium to rapture the recipient bacterium cell wall (Baidya et al., 2020 Nature Communications) (see Figure "Bs Sa nanotube").
4) We have developed an array of biochemical and genetic strategies to identify and characterize CORE-associated components in EPEC and Bs. We have candidates that are now being explored in both species.
5) We established experimental systems and approaches to assess the global molecular cargo delivered by the CORE-nanotube apparatus, including proteins, RNA and metabolites.
6) We have made significant progress in addressing the structure the CORE-nanotube apparatus by in situ and in vitro Cryo-EM structural analysis.
In first scientific period of the project, we reported the discovery of a new bacterial CORE-nanotube organelle that mediates communication among bacteria and between pathogenic bacteria and human host cells. This is an entirely novel and unexpected concept. By the end of the project, we aim at obtaining a better understanding of this new organelle, including: (i) the identification of additional components composing the organelle, regulating its production or temporarily associating with it; (ii) gaining new insight as to the structure, function and cargo selection of the CORE-nanotube organelle; and (iii) elucidating the impact of this organelle on bacterial communities and virulence.

To achieve these aims, we will continue to develop new tools and suitable methodologies addressing these issues. In the longer term, we expect that our publications will attract other groups to launch related research projects. The outcome of this project and follow up studies are expected to revolutionize the way we view bacterial communities and host-pathogen interactions. The new knowledge may lead to the development of innovative methodologies, enabling to inhibit or promote intercellular molecular exchange. These methodologies have the potential to transform the entire field of bacteriology ranging from basic bacterial biology and host-pathogen interaction, through the control of infectious diseases and acquisition of antibacterial resistance, to food technology and industrial applications.
Fig 2. Nanotube connecting bacterial cells of B. subtilis, with the CORE depicted in red
Fig 3. Molecules of a cell wall lytic enzyme (whit dots) on nanotubes
Fig 1. Nanotubes emanate from pathogenic E. coli penetrating the host