Hagmann, Cristina Amparo: Cytosolic recognition of pathogenic nucleic acids. - Bonn, 2011. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-25709
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-25709,
author = {{Cristina Amparo Hagmann}},
title = {Cytosolic recognition of pathogenic nucleic acids},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2011,
month = jul,

note = {The innate immune system senses pathogens by pattern recognition receptors (PRR) located in different cell compartments. Components of the bacterial cell wall and conserved proteins are detected by Toll-like receptors (TLRs) on the cell surface, and by NODs or NOD like receptors (NLRs) in the cytosol of cells. Viruses are recognized via their nucleic acids by TLRs in the endosome and RIG-I like helicases in the cytosol. Even though viral and bacterial DNA possess, with TLR9, a PRR in the endosome, the cytosolic type I inducing DNA receptor (DNAR) has as yet remained elusive. In the first part of this thesis, the minimal recognition motif for the cytosolic DNAR will be investigated. We dissected recognition motifs of short double stranded DNA oligonucleotides (dsODN) in primary monocytes and cell lines and identified two different types of immune stimulatory ODN: In addition to recognition of long dsDNA, including plasmid DNA, human monocytes detected concatamerized short dsODN. Surprisingly, monocytes were also able to recognize short dsODN when harboring mismatched terminal G extensions at 5 and 3 ends (Gn-dsODN). As determined by gel electrophoresis and circular dichroism (CD) spectroscopy under physiological salt and temperature conditions, the G extensions of stimulatory G3-dsODN did not form G quartet structures, which excluded a polymerization-dependentrecognitioneffect.FluorescenceandFRE T -basedanalysis techniques revealed no considerable difference in uptake and degradation between G- ended and C-ended dsODNs. ShRNA-mediated knock-down experiments revealed involvement of the adaptor molecule MITA downstream of the DNAR in dsODN-mediated signaling. Our results define a new sequence-dependent recognition motif of short cytosolic dsDNA in human immune cells by an as yet unknown DNA receptor.
Like the genetic material of viruses, nucleic acids of bacteria are recognized by TLRs in the endosome. In contrast to exclusively extracellular bacteria, intracellular bacteria have evolved mechanisms to escape the endosome and penetrate into the cytosol, where they are accessible to cytosolic PRRs. Listeria monocytogenes infection has been linked to cytosolic nucleic acid sensor-dependent type I IFN induction. Current knowledge indicates that immune cell type I IFN response to Listeria is processed via the bacterial DNA. However, it is tempting to speculate that like DNA, RNA can enter into the cytosol. Using a novel, sensitive labeling method, we were able to show cytosolic access of Listeria RNA during infection. Furthermore, transfection of bacterial RNA into the cytosol of human monocytes or epithelial cells resulted in a type I IFN response, which was dependent on RNA phosphorylation. In contrast to monocytes, epithelial cells were not triggered by bacterial DNA, providing a non-redundant nucleic acid recognition pathway. Notably, knock-down of RIG-I in epithelial cells showed a diminished IFN response to both Listeria RNA and infection. This characteristic was only minimally evident in monocytic cells. We conclude that RIG-I mediated recognition of Listeria monocytogenes plays a role in infection of non- immune cells lacking a mechanism for a direct response to bacterial DNA.},

url = {http://hdl.handle.net/20.500.11811/4997}

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