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Translational control in infection biology: riboproteogenomics of bacterial pathogens

Periodic Reporting for period 1 - PROPHECY (Translational control in infection biology: riboproteogenomics of bacterial pathogens)

Reporting period: 2018-11-01 to 2020-04-30

The aim of our research is to better our understanding of bacterial infection biology. Deep sequencing has enabled the study of gene expression at the transcript level. However, 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 proteogenomics approaches that can enable protein translation studies of bacterial pathogens in a host context.
Recent findings of our research team revealed translation of numerous previously unidentified (small) open reading frames and expression of alternative protein isoforms when studying bacterial translation. Therefore, we are exploring the repertoire of bacterial proteins employed to establish a successful interaction with its host cell. For this, the research team develops and applies a complementary cutting-edge riboproteogenomic toolset which will enable, for the first time, targeted systematic genome- and proteome-wide surveys of bacterial transcriptional and translational activity during actual host cell infection. By exploiting this 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. Further, the identification of new pathogen virulence factors will contribute to the development of innovative therapeutics and diagnostics for multiple models of infectious diseases.
The project started effectively on November 1th, 2018. During this first reporting period significant progress was already 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. The PROPHECY project is organized in 7 WPs, each subdivided in specific (complementary) tasks.
Regarding the major achievements during this first reporting period, an optimized proteogenomic workflow was established that uses bacterial ribo-seq and proteomic data using Salmonella Typhimurium as test-case. Overall, this workflow aids the identification of unannotated proteins (including small ORF encoded polypeptides or SEPs) and alternative protein forms raised upon alternative translation initiation (i.e. N-terminal proteoforms) and demonstrated its great promise to assist in experimentally-based bacterial genome annotation. This work is already available as preprint at https://doi.org/10.1101/2019.12.18.881375. Further, complementary to dual Ribo-seq, 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. When compared to searching data-dependent acquisition (DDA) experiment-specific libraries only, the use of hybrid libraries significantly improved peptide detection of relevance when profiling host-pathogen interactions. This work is currently available as preprint at WP5, WP6. Besides, DIA data in the context of infection was obtained, the sorting of subpopulations of infected cells by means of fluorescence dilution and FACS optimized, and scarefree homologues recombineering strategies implemented, all feeding into the PROPHECY project.
As a significant fraction of newly identified ORFs have been predicted to encode transmembrane proteins, we set out to out to functionally characterize these at the genome-wide level. For this, in collaboration with the Bernd Bukau lab (Heidelberg, Germany), a novel method to study co-translational membrane engagement of ribosome-nascent chain complexes (RNCs) was developed. Further, by the optimized Proximity-dependent Biotin Labelling Approaches at hand (https://doi.org/10.1101/701425) bacterial proteoform interaction mapping will be enabled. Finally, by optimizing a strategy based on subcellular fractionation, TMT labelling and machine learning, proof-of-concept data and unique information on the subcellular localization of newly discovered bacterial proteoforms, SEPs and uncharacterized proteins was obtained.
Novel OMICS workflows and methodologies were established, the latter of key importance for the successful continuation of the project. 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 in the context of infected hosts. Further, proteome-wide subcellular localization studies using the recently optimized strategy based on subcellular fractionation, TMT labelling and machine learning will enable to provide a dynamic spatiotemporal view on bacterial protein expression. By the implementation and development of innovative interactomics and proxeome technologies, bacterial proteoform interaction maps of newly discovered proteoforms or putative bacterial effector host proteins will be created. The identification of new pathogen virulence factors will contribute to the development of therapeutics and diagnostics for multiple models of infectious diseases.
HeLa cells infected with GFP-expressing Salmonella Thyphimurium at MOI100 and 24 hpi.