A gut commensal microbiome-host protein network map reveals bacterial modulation of human immune signaling

Young, Veronika (2024): A gut commensal microbiome-host protein network map reveals bacterial modulation of human immune signaling. Dissertation, LMU München: Fakultät für Biologie

Abstract

Complex diseases such as cardiovascular illnesses, cancer, respiratory diseases, and diabetes have been on the rise worldwide contributing to 70% of all deaths. While these illnesses are superficially not associated with microbial organisms accumulating research links bacteria of the gut microbiome to a diverse range of complex diseases. Especially Pseudomonadota, the third most abundant phylum in the gut, has been associated with several of those illnesses e.g., metabolic sicknesses and cancer. However, the underlying mechanisms mediating the bacterial impact on host health remain largely unknown. Pathogenic representatives of the Pseudomonadota phylum are well-known for employing a type three secretion system (T3SS) to manipulate host cells and thereby mediate infectious diseases. Prominent examples are Salmonella, Shigella, enterohemorrhagic Escherichia coli (EHEC), and enteropathogenic Escherichia coli (EPEC) as well as Yersinia pestis responsible for wiping out one-third of the European population during the plague. Yet, T3SSs have also been detected in plant mutualists e.g., rhizobia strains of the rhizobia-legume symbiosis. Furthermore, bacteria that do not exhibit a pathogenic or mutualistic lifestyle also seem to encode for T3SSs. Work outside of this thesis as part of the same project detected T3SSs also in commensal Pseudomonadota of the human gut microbiome. However, the impact of the T3SS effectors on the host cell and subsequently host health is not known. Therefore, this thesis aimed to elucidate the impact of T3SS effectors expressed by commensal gut Pseudomonadota on host functions in the context of human health and disease. To assess the impact of bacterial effectors on the human host a network map of protein-protein interactions (PPIs) between gut commensal bacterial effectors and human proteins was generated. To this end, an ORFeome collection of bacterial effectors was established by cloning 959 T3SS effectors from known, culturable strains as well as from metagenomic data of the human gut. Testing these bacterial effectors against the human ORFeome v9.1 collection consisting of 17,408 protein-coding genes with a systematic, high-throughput yeast two-hybrid (Y2H) pipeline gave rise to the human-microbiome meta-interactome map (HuMMI). The network consists of 1,263 interactions mediated by 289 effectors and 430 human proteins. HuMMI was subjected to a detailed quality control to assess the reliability of the used pipeline and the quality of the interactions. For this purpose, reference sets were assembled to benchmark the Y2H and an orthogonal assay, which was employed to re-test a subset of HuMMI. In addition, the saturation of HuMMI i.e., the percentage of discovered interactions compared to all detectable interactions, was assessed by a Y2H repeat screen. After demonstrating the reliability of the employed Y2H pipeline and the comparability of HuMMI to well-documented, literature-curated-interactions, validation experiments in vitro were conducted based on the functional analysis of the effector targets. As the bacterial effectors targeted human proteins involved in immune signaling their impact on the transcription factor nuclear factor kappa B (NF-κB) was assessed. A cell-based reporter assay was employed testing the ability of the effectors to modulate NF-κB activity. Five effectors significantly activated NF-κB, while three effectors seemed to inhibit the transcription factor significantly. Further impacts on human immune signaling by T3SS effectors were shown by collaborators reporting increased ICAM1 expression as well as up- and downregulation of pro-inflammatory cytokine secretion from a colon cell line upon effector transfection. The opposing effects of bacterial effectors on immune signaling pathways suggest different influences of gut commensal T3SS effectors on the human host. In conclusion, this study introduced a novel mechanism by which gut commensals might impact the human host. T3SS effectors potentially affect human immune signaling locally and systemically via cytokine secretion potentially affecting the risk of complex disease. Thereby, this works launches the investigation into gut commensal T3SS effectors and their impacts on host health and disease.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 11. Juni 2024
• 1. Bericht­erstatter:in: Falter-Braun, Pascal