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The mechanisms behind CEACAM3-mediated recognition and uptake of pathogenic bacteria

The mechanisms behind CEACAM3-mediated recognition and uptake of pathogenic bacteria

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ADRIAN, Jonas, 2019. The mechanisms behind CEACAM3-mediated recognition and uptake of pathogenic bacteria [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Adrian2019mecha-47297, title={The mechanisms behind CEACAM3-mediated recognition and uptake of pathogenic bacteria}, year={2019}, author={Adrian, Jonas}, address={Konstanz}, school={Universität Konstanz} }

<rdf:RDF xmlns:dcterms="http://purl.org/dc/terms/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#" xmlns:foaf="http://xmlns.com/foaf/0.1/" xmlns:void="http://rdfs.org/ns/void#" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/47297"> <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/47297"/> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/47297/3/Adrian_2-4r7g46sk16fk4.pdf"/> <dc:contributor>Adrian, Jonas</dc:contributor> <dcterms:abstract xml:lang="eng">The selective pressure exerted by infectious agents has always been a major driving force in the evolution of humans and other mammals. Positive selection of traits leading to superior survival and reproduction rates of adapted hosts promote the development of extensive innate and adaptive defense systems. In a similar fashion, microbial pathogens are selected for pathogenic traits that allow them to suppress, evade or subvert the immune systems. This concept of a constant molecular arms race between pathogens and their hosts is in line with the Red Queen hypothesis. Several gram-negative pathogens, including Neisseria gonorrhoeae, Haemophilus influenzae, Helicobacter pylori, and Moraxella catarrhalis, have independently evolved adhesins that target epithelial surface proteins of the human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family on mucosal cells lining the nasopharynx, the intestine, and the urogenital tract. Interaction with these epithelial CEACAMs is highly advantageous to the pathogens, as they facilitate colonization of the epithelium, penetration into deeper tissues and suppression of innate and adaptive immune functions. Remarkably, the relative recently evolved granulocyte specific CEACAM family member CEACAM3 is able to turn this advantage against the invading pathogens. While its extracellular domain shares high sequence similarity with the bacterial-targeted regions of epithelial CEACAMs, the intracellular region of CEACAM3 contains a unique hemi-immunoreceptor tyrosine-based activation motif (HemITAM). Binding of bacteria to the extracellular domain triggers rapid tyrosine phosphorylation within in the HemITAM-sequence, which activates antimicrobial effector functions, such as pathogen phagocytosis, and clearance by generation of reactive oxygen species. While several positive regulators involved in CEACAM3-mediated intracellular signaling have already been described, we strived to fill in the gaps by establishing a comprehensive genome-wide CRISPR knockout (GeCKO) screening system for the assessment of phagocytosis in a myeloid cellular background. Generation and validation of a CEACAM3 expressing myeloid cell line were followed by extensive screen optimization to assure adequate lentiviral transduction efficiency, assay sensitivity, and reliability. Expectedly, screening the GeCKO lentiviral library revealed several proteins to influence CEACAM3 mediated phagocytosis. For some hits, like the Rac-guanine nucleotide exchange factor (GEF) Vav or the C-terminal Src kinase (CSK), CEACAM3 related functions have already been described. Among the newly identified are CYRI, a negative regulator of Rac-signaling, as well as NEDD4, an E3 ubiquitin-ligase targeting receptor-type protein tyrosine phosphatases (PTPRs). Both, CYRI and PTPRs, might play an important role in the negative regulation of CEACAM3. To investigate the influence of PTPRs on CEACAM3 signaling, individual knockout cells were produced for all PTPRs encoded in the human genome. Indeed, loss of multiple granulocyte-expressed PTPRs enhanced CEACAM3-mediated phagocytosis. Conversely, co-expression of CEACAM3 with PTPR-variants revealed that PTPRC and PTPRJ are able to suppress CEACAM3-mediated phagocytosis, by dephosphorylation of its HemITAM-contained tyrosine residues when paired with phosphatase constructs lacking their large ectodomains. Indeed, expression of full-length phosphatases failed to affect phagocytosis negatively. Derived from these results a phagocytic synapse–based activation model was deduced for CEACAM3-mediated pathogen phagocytosis. Briefly, CEACAM3 receptors are clustered underneath adhesin-bearing pathogens while PTPRC and PTPRJ are passively excluded from the site of tight engagement due to their large ectodomains. In this CEACAM3-clustered region, Src-family kinases are no longer counteracted by the phosphatase action of PTPRs and rapidly phosphorylate CEACAM3 HemITAM residues, triggering localized activation of actin-polymerization, which leads to pathogen engulfment. Interestingly, the granulocyte CEACAM3 as well as the primary targets of pathogen adhesins, epithelial CEACAMs, are among the fastest evolving human genes. We identify CEACAM3 orthologues in higher primates and confirm that its extracellular domain evolved with even stronger rapidity, while its intracellular signaling platform appears to be conserved. Analysis of the binding affinity of primate CEACAM3 variants to human CEACAM3-binding pathogens revealed a reduction in the recognized spectrum with increasing phylogenetic distance. This demonstrates that amino acid alterations occurred, at least in part, in sites that define binding specificity. Indeed, we could identify a single amino acid exchange in gorilla CEACAM3 being sufficient to reestablish receptor recognition of Haemophilus aegyptius. These results demonstrate that CEACAM3 is under extraordinary positive selection, presumably to keep up with bacterial adaptations. Accordingly, several pathogens, such as Neisseria gonorrhoeae and Haemophilus influenzae, have evolved adhesins which evade recognition by human CEACAM3 but retain binding to epithelial CEACAM1. We report a single amino acid divergence between these receptors that is utilized by all CEACAM1 specific neisserial adhesins to avoid CEACAM3 binding, while all other sequence differences did not affect adhesin specificity. An additional mutation at a secondary site also reestablished interaction with the CEACAM1-binding adhesin of Haemophilus influenzae. Remarkably, a human CEACAM3 variant harboring these combined alterations is present in modern African populations with approximately 30% of the population possessing at least one such allele. These CEACAM3 variants in the human gene pool might well demonstrate the ongoing struggle between pathogen adaptation and host counter-adaptation.</dcterms:abstract> <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/> <dc:language>eng</dc:language> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2019-10-23T12:03:37Z</dc:date> <dc:creator>Adrian, Jonas</dc:creator> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2019-10-23T12:03:37Z</dcterms:available> <dcterms:issued>2019</dcterms:issued> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/47297/3/Adrian_2-4r7g46sk16fk4.pdf"/> <dcterms:title>The mechanisms behind CEACAM3-mediated recognition and uptake of pathogenic bacteria</dcterms:title> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <foaf:homepage rdf:resource="http://localhost:8080/jspui"/> <dc:rights>terms-of-use</dc:rights> </rdf:Description> </rdf:RDF>

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