edoc

Innate immune recognition of Salmonella and Francisella : two model intracellular bacterial pathogens

Dreier, Roland F.. Innate immune recognition of Salmonella and Francisella : two model intracellular bacterial pathogens. 2017, Doctoral Thesis, University of Basel, Faculty of Science.

[img] PDF
Restricted to Repository staff only until 1 January 2020.
Available under License CC BY-NC (Attribution-NonCommercial).

48Mb

Official URL: http://edoc.unibas.ch/diss/DissB_12436

Downloads: Statistics Overview

Abstract

The innate immune system is the first line of host defense against invading pathogens. In multicellular organisms, specialized innate immune cells recognize conserved pathogen-associated molecular patters (PAMPs) with germ-line encoded pattern recognition receptors (PRR). Thereby, the organism discriminates between self and non-self and engages mechanisms to eliminate the invader. Beside PAMPs, PRRs recognize mislocalized self-molecules, so called danger-associated molecular patterns (DAMPs), which are indicators of tissue or cellular damage.
Upon PAMP or DAMP recognition, PRRs induce innate immune signaling pathways leading to the activation of pro-inflammatory genes and interferon production, which are important mediators of inflammation. Therefore the recognition of invading pathogen and thereby activation of innate immune signaling pathways determines the success of the immune system to eliminate the potential threat.
Innate immune signaling pathways largely depend on phosphorylation cascades. Today, global phosphorylation changes are analyzed by mass spectrometry, however the number of detected phosphopeptides remains unchanged despite technical improvements. Therefore, we investigated the issue of phosphopeptide detection in mass spectrometry.
The analyses of phosphopeptide-enriched samples have revealed lower signal intensities in MS1 spectra compared to total cell lysate samples, which results in poor phosphopeptide detection with mass spectrometry. Based on these observations, we hypothesized that the phosphate groups of phosphopeptides account for this poor detection. Indeed, we significantly increase the signal intensities in MS1 spectra after enzymatic removal of phosphate groups from phosphopeptides, and consequently we detect three-times more peptides in phosphatase-treated samples. Validation experiments elucidate that most of the newly detected peptides have been initially phosphorylated. Moreover, the newly detected peptides enlarge the activated signaling network upon Salmonella infection. Importantly, we identify known innate immune signaling pathways, which were missing in the analyses of phospho-enriched samples.
Taken together, the phosphate groups of phosphopeptides globally suppress peptide ionization efficacy and therefore account for the low phosphopeptide detection rate by mass spectrometry. By removing the phosphate groups, we identify three times more peptides after phosphatase treatment. The newly detected peptides enlarge the network of activated innate immune signaling pathways upon Salmonella infection and include signaling pathways that are important but have not been detected in phospho-enriched samples. Therefore our findings improve the analyses of innate immune signaling pathways by mass spectrometry and consequently the understanding of innate immunity.
One of the main mechanisms to eliminate invading microbes is by phagocytosis and degradation within phago-lysosomes. However, professional pathogens have developed various defense mechanisms to resist intracellular killing and can even use innate immune cells as replicative niches. For example, the bacterial pathogen Francisella tularensis causes a severe and life-threatening disease called tularemia in humans, because Francisella can survive and replicate in macrophages and dendritic cells. Critical for Francisella pathogenicity is the ability of the phagocytosed bacteria to escape from the phagosome to the host cytosol. Even though we know that genes encoded on the Francisella pathogenicity island (FPI) are essential for escaping from the phagosome, the mechanism is unknown. Homology analyses have suggested that the FPI encodes a type 6 secretion system (T6SS). However experimental evidence is missing, which show that the FPI encode a functional T6SS. Therefore, we investigated whether the FPI encodes a functional T6SS and what impact a functional T6SS has on Francisella virulence in vitro and in vivo.
We show that the FPI of Francisella novicida (F. novicida) encodes a functional T6SS that assembles exclusively at bacterial poles. T6SS function depends on the unfoldase ClpB, which specifically recognizes contracted T6SS sheaths leading to their disassembly. Furthermore we have characterized FPI genes that show no homology with known T6SSs. We have identified IglF, IglG, IglI and IglJ as structural components of the T6SS and PdpC, PdpD, PdpE and AnmK as potential T6SS effector proteins. Whereas PdpE and AnmK are dispensable for phagosomal escape, AIM2 inflammasome activation and virulence in mice, pdpC- and pdpD-deficient bacteria are impaired in all aforementioned analyses. This suggests that PdpC and PdpD are bacterial effector proteins involved in phagosomal escape and thereby in the establishment of a F. novicida infection.
Taken together, F. novicida uses its T6SS to deliver the effector proteins PdpC and PdpD into host cells. PdpC and PdpD are involved in phagosomal rupture and consequently in bacterial escape to the cytosol. These findings are a major breakthrough in the understanding of Francisella pathogenicity and could lead to new vaccination strategies to eradicate the life-threatening human disease Tularemia.
Advisors:Broz, Petr and Bumann, Dirk
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Infection Biology (Broz)
UniBasel Contributors:Broz, Petr and Bumann, Dirk
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12436
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (281 Seiten)
Language:English
Identification Number:
Last Modified:05 Apr 2018 17:36
Deposited On:31 Jan 2018 15:33

Repository Staff Only: item control page