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Rapid translational responses as a novel mechanism to repair cellular damage caused by the bacterial injectisome in animal and plant host cells

Reference	BB/X001261/1
Principal Investigator / Supervisor	Dr Betty Chung
Co-Investigators / Co-Supervisors	Professor John Carr
Institution	University of Cambridge
Department	Pathology
Funding type	Research
Value (£)	604,367
Status	Current
Type	Research Grant
Start date	24/01/2023
End date	23/01/2026
Duration	36 months

Abstract

Our Research Program comprises two work packages (WP), corresponding to our key hypotheses (see Objectives). WP1. To explore the hypothesis that in plants (as in mouse macrophages) injectisome penetration induces specific changes in translation of polysome-associated mRNAs, Arabidopsis seedlings will be inoculated with WT P. syringae or mutants lacking functional T3SS. T3SS-dependent versus T3SS-independent changes in mRNA translation dynamics will be investigated over 0-480 min time-courses using ribosome profiling by ultraHD-RiboSeq and RNA-Seq. Transgenic Arabidopsis expressing antigen-tagged ribosomal protein RPL18 controlled by cell type-specific promoters will allow selective polysome purification by immunoprecipitation for RNA-Seq & ultraHD-RiboSeq to identify T3SS-induced changes in cell type-specific mRNA translation. WP2. We will test the hypothesis that T3SS-induced effects on translation or transcription are triggered not only by effectors but also by injectisome penetration/pore formation in membranes of animal cells (mouse macrophages) and Arabidopsis protoplasts. Using protoplasts & macrophages permits synchronised experiments (not possible in whole organisms/tissues) to compare temporal changes in plant and animal cells following cocultivation with WT and T3SS-defective Pseudomonas & Salmonella, and exposure to the respective (PopB/D:PcrV & SipB/SipC) pore-forming T3SS components & ionophore controls (gramicidin & tyrocidine). Translational activation over time in response to these agents will be analysed as in WP1 by RNA-Seq & ultraHD-RiboSeq with cell-specific effects delineated by immunoprecipitation of antigen-tagged polysomes. Specifically-activated plant mRNAs (hypothesised to include transcription factors that engender transcriptome remodelling) will be characterised and their functions e.g. cell repair, tested using knockout/knockdown in transgenic/mutant plants or via protoplast transfection.

Summary

When animal and plant cells are assaulted by microbes, this causes changes in the production of proteins by the attacked cells. Proteins are large biological molecules with many different functions that include, among other things, structural proteins, signal molecules (including hormones), enzymes that catalyse specific chemical reactions, immune factors (including antibodies), and 'transcription factors'. Transcription factors control access to protein-making instructions archived in a cell's DNA by switching on synthesis of specific 'messenger RNA' molecules that transport copies of these instructions to 'polysomes'. Polysomes contain the complex machinery that 'translates' the data contained in each messenger RNA to guide assembly of a specific protein. Projecting from the surfaces of many disease-causing bacteria are tiny needle-like structures called 'injectisomes'. Injectisomes are nano-syringes that inject substances called 'effectors' into host cells to promote disease by, for example, inhibiting resistance. Injectisomes are used not only by bacteria that attack animals and humans, but also by bacteria that attack plants, as well as by food poisoning bacteria that colonise the edible parts of plants. Thus, understanding how cells respond to penetration by injectisomes is important for development of improved medical treatments, for enhancing protection of crops against bacterial diseases, and increasing food safety. Macrophages are white blood cells that attack invading microbes. The project leader discovered that when macrophages are penetrated by the injectisomes of attacking Salmonella bacteria, the host cell polysomes selectively increase synthesis of specific transcription factors, which in turn changes the pattern of copying of messenger RNAs from the macrophage's DNA. This reprogramming of protein synthesis activity (translation) by polysomes is remarkably rapid, occurring within seconds to minutes of penetration, and its discovery raises important new questions about how host cells respond to bacterial attack. The key questions we will answer are: - Are the polysomes of host cells responding to the effectors injected by the bacteria or to the shock of penetration? - Plant-attacking bacteria also use injectisomes are part of their armoury, so does the rapid change in polysome activity also occur when plant cells are attacked, either by bacteria that cause disease in plants, or by food poisoning bacteria that colonise plants? - How similar are the responses to bacterial and plant cells and can we separate the responses caused by the shock of penetration versus those caused by the effectors? - Is the rapid change in polysome activity an early step in mounting resistance to bacterial attack and repair of the cell, or does it mark a stage in the destruction of the host cell?

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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