Periodic Reporting for period 4 - PROPHECY (Translational control in infection biology: riboproteogenomics of bacterial pathogens) Periodo di rendicontazione: 2023-05-01 al 2023-10-31 Sintesi del contesto e degli obiettivi generali del progetto The aim of PROPHECY was to better our understanding of bacterial infection biology. To achiebe this goal, physiological responses were studied in an integrated manner and at the host/pathogen interaction interface by combining host- and pathogen-focussed research methods. As a model bacterial pathogen, we made use of the enteropathogen Salmonella Typhimurium (Salmonella).While deep sequencing has enabled the study of gene expression at the transcript level, the depth of sequencing has so far proven to be unsatisfactory in case of the bacterial pathogen in an infection context. Moreover, the study of bacterial proteome changes upon infection remains highly unexplored because of the higher proteome complexity of the host cell compared to the pathogen. These challenges clearly stress the need for novel strategies. The use of complementary riboproteogenomics approaches enabled to study host-pathogen interactions for the first time from the integrative proteome perspective of the bacterial pathogen as well as the infected host. To achieve this goal we employed state-of-the-art proteomics, translatomics and (effector) interactomics and obtain unique insights into the great diversity of host-pathogen encounters. Our data highlighted novel bacterial virulence factors, therefore informing the future development of novel antibacterial treatment.Overall, by exploiting our proteogenomics toolset in combination with molecular (micro-)biology, cell biological and cell physiological approaches, we obtained answers to intriguing fundamental questions of host/pathogen interactions. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti The project commenced effectively on November 1th, 2018. Throughout the project, we obtained proof-of-concept data and established novel, state-of-the-art OMICS workflows and methodologies.Initially, we published an optimized proteogenomic workflow utilizing bacterial ribo-seq and proteomic data, employing Salmonella as a test case. To comprehensively detect novel, previously unannotated genomic elements, we conducted extensive analyses, including total translatome (ribo-seq), translation initiation (retapamulin-assisted ribo-seq), and shotgun proteomics. These analyses, based on reported transcriptomic efforts, provided a cross-section of infection stages and complementary genomic expression patterns. To delineate translated ORFs using ribo-seq data, we developed and applied a new gene discovery pipeline. Additionally, we interrogated matching proteomics datasets for protein evidence of the novel genes, strengthening the confidence in their true nature.A breakthrough concept emerging from these riboproteogenomics data was the omnipresence of protein variants that originate from the same gene but differ at their N-terminus due to alternative translation initiation (i.e. N-terminal proteoforms) and the wealth of unannotated small proteins expressed. These insights further shifted the paradigm in how we understand the relationship between a gene and resulting protein. The workflow also demonstrated great promise in experimentally-based bacterial genome annotation.Viewing the discovery of a large number of new and underexplored bacterial genomic elements encoding sORF-encoded polypeptides (SEPs), N-terminal proteoforms and virulence factors (effector proteins), we also set out to out to validate and functionally characterize a selection of these. Besides (real-time) expression and localization analysis, we developed a novel Ribo-seq method which specifically enabled the study of short transmembrane domain containing SEPs, a category frequently found amongst (uncharacterized) SEPs. Additionally, by applying an optimized proximity labelling strategy implemented in plant and for the first time in bacteria, protein/protein interactions were also studied at the pathogen/plant host (the interaction between Agrobacterium pathogen effectors and plant host) as well as bacterial predator/bacterial prey interaction interface (preprint; DOI 10.1101/2023.11.29.569176)Further, we evaluated and compared protein extraction methodologies for their efficacy in the extraction and concomitantly proteomics detection of SEPs, and optimized an experimental protocol that specifically enriches for SEPs. Building upon this knowledge, we investigated whether computational analysis and state-of-the-art riboproteogenomic approaches can shed light on the challenges faced in the identification of SEPs. Given the versatile biological functions SEPs have been shown to exert, this work provides an accessible protocol and analysis pipeline for the proteomics exploration of this fascinating class of small proteins.Finally, dual-proteome profiling was empowered by the development of an optimized hybrid library generation workflow for data-independent acquisition (DIA) mass spectrometry relying on the use of data-dependent and in silico predicted spectral libraries. This strategy significantly improved peptide detection, particularly relevant in profiling host-pathogen interactions. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) The established omics expertise across various research fields investigating host-microbe interaction biology has greatly contributed to the impact and visibility of the research within the broad scientific community. Undoubtedly, this expertise will be of key importance for the successful continuation, dissemination, and further exploitation of the PROPHECY legacy.More specifically, the simultaneous capture of the pathogens transcriptome, translatome and proteome during infection, provides an unprecedented complete view of the interaction between host and bacterial pathogen and revealed important differential regulations when comparing proteome- versus transcriptome-wide expression changes.With the ongoing development of dual Ribo-seq that allows the selective isolation of host or bacterial ribosomes, we expect to obtain translational maps of the bacterial translatome with high resolution in the context of infected hosts.By the implementation of the innovative interactomics and proxeome technologies developed, bacterial proteoform and SEP interaction maps besides (newly discovered) bacterial effector host protein interaction maps will be created, setting the stage for the functional characterization of selected (N-terminal) proteoforms, SEPs and other newly discovered gene products implicated in bacterial pathogenesis. Furthermore, the identification and characterization of new pathogen virulence factors will contribute to the development of therapeutics and diagnostics for multiple models of infectious diseases. prophecy-banner.png
