Periodic Reporting for period 2 - PROPHECY (Translational control in infection biology: riboproteogenomics of bacterial pathogens) Berichtszeitraum: 2020-05-01 bis 2021-10-31 Zusammenfassung vom Kontext und den Gesamtzielen des Projekts The aim of PROPHECY is to better our understanding of bacterial infection biology. Therefore, physiological responses will be studied in an integrated manner by combining host- and pathogen-focussed research methods. For this, we will make use of Salmonella Typhimurium (Salmonella) as a model for studying bacterial pathogenesis.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 based on complementary riboproteogenomics approaches which enable to study host-pathogen interactions for the first time from the integrative proteome perspective of the bacterial pathogen as well as the infected host. We employ state-of-the-art proteomics, translatomics and (effector) interactomics to obtain unique insights into the great diversity of host-pathogen encounters Obtained knowledge will highlight novel bacterial virulence factors and inform the development of novel antibacterial treatment.By exploiting our proteogenomics toolset in combination with molecular biology, genetics, cell biological and cell physiological approaches, we strive to obtain answers to intriguing fundamental questions of host/pathogen interactions. Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse The project started effectively on November 1th, 2018. During this reporting period significant progress was made in the project, proof-of-concept data obtained and novel OMICS workflows and methodologies established, the latter of key importance for the successful continuation of the project.An optimized proteogenomic workflow was published that uses bacterial ribo-seq and proteomic data using Salmonella as test-case. This workflow aids the identification of unannotated (small) proteins and alternative protein forms raised upon alternative translation initiation (i.e. N-terminal proteoforms), overall demonstrating its great promise to assist in experimentally-based bacterial genome annotation. Further, 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, of particular relevance when profiling host-pathogen interactions. Moreover, in order to comprehensively detect novel, previously unannotated genomic elements, extensive total translatome (ribo-seq), translation initiation (retapamulin assisted ribo-seq) and shotgun proteomics analysis were performed. The conditions used for this riboproteogenomic discovery were based on reported transcriptomic efforts providing a cross section of infection stages and complementary genomic expression patterns. To delineate translated ORFs using ribo-seq data, a new gene discovery pipeline was developed and applied. Moreover, matching proteomics datasets have been interrogated for protein evidence of the novel genes translation further strengthening the confidence of their true nature. Since this way, a large number of new and underexplored genomic elements encoding sORF-encoded polypeptides (SEPs) and N-terminal proteoforms were discovered, we also set out to out to functionally characterize a selection of these at the genome-wide level. Besides (real-time) expression and localization analysis, we developed a novel Ribo-seq method which specifically enables the study of short transmembrane domain containing SEPs, a category frequently found amongst (uncharacterized) SEPs.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 obtained, we also investigated whether computational analysis and state of the art riboproteogenomic approaches can shed light on the challenges faced in the identification of SEPsGiven 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.Our riboproteogenomic workflow also enabled for the first time, to map the genome-wide occurrence and conditional expression of bacterial N-terminal proteoforms. Functional characterization of selected candidates will be performed by applying a recently optimized proximity labelling strategy amongst other. Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts) Novel OMICS workflows, methodologies and data-analysis pipelines were established, the latter of key importance for the successful continuation of the project. 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 establishment 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.Further, by assessing proteome-wide subcellular localization and protein stability, a dynamic view on bacterial protein expression will be provided.By the implementation of the innovative interactomics and proxeome technologies developed, bacterial proteoform 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.