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Functional characterization of Bartonella effector protein C (BepC) in the context of infection

Marlaire, Simon. Functional characterization of Bartonella effector protein C (BepC) in the context of infection. 2018, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_13291

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Abstract

A wide variety of bacterial pathogens evolved a panel of virulence factors in order to subvert cellular processes and achieve a successful infection. Bacteria of the genus Bartonella translocate a cocktail of effector proteins (Beps) via a type IV secretion system (T4SS) into mammalian cells. BepC, one of the most conserved effectors in the Bartonella species of the lineage 4, has been previously shown to be involved in the internalization of bacterial aggregates and migration defect in vitro.
In this work, we show that the effector BepC localizes at cell-to-cell contact and triggers strong actin rearrangements as well as the formation of bacterial aggregates during infection of human cells. The actin phenotype is induced by BepC from different Bartonella species, indicating an important role of this effector during pathogenesis.
BepC pull-down from infected cells led to the identification of two interacting partners, GEF-H1 and MRCKα, which are two host proteins involved in the RhoA and Cdc42 pathways, respectively. We demonstrate that the ability of BepC to bind GEF-H1 and MRCKα highly correlates with its ability to trigger actin rearrangements. Accordingly, infected cells show an increase of GTP-bound RhoA and phosphorylated myosin light chain while both RhoA and its downstream effector ROCK are required for actin rearrangements mediated by BepC. Thus, our results indicate that BepC activates the RhoA pathway by interacting with GEF-H1 and thereby inducing actin rearrangements although MRCKα might also be involved.
The majority of Beps, including BepC, carries an enzymatic FIC domain that is usually involved in posttranslational modifications. Most Fic proteins carry a canonical FIC motif that is essential for ATP binding and the transfer of AMP onto the target protein (AMPylation). By contrast, BepC is characterized by a non-canonical FIC motif and only displays a weak AMPylation and phosphorylation activity, independently from its conserved motif. Nevertheless, structural analysis and binding assays demonstrate that ATP binds to the FIC domain of BepC and is critical for its thermal stability.
In absence of FIC domain, BepC loses its ability to localize at cell junctions, to interact with GEF-H1 and MRCKα, and to trigger actin rearrangement, suggesting a central role for this domain in the effector function. However, a conserved FIC motif is not necessary to trigger actin rearrangements, which indicates that BepC acts by protein-protein interaction rather than by posttranslational modification. Thus, we propose that BepC is recruited to cell contacts where it triggers the activation of the RhoA pathway by interacting with GEF-H1 and eventually leads to actin rearrangements, possibly with the help of MRCKα.
Ultimately, the subversion of RhoA signaling by BepC could help Bartonella to interfere with the immune response by preventing phagocytosis or impair cell migration. Furthermore, it could play an important role in the disruption of the endothelial barrier in order to reach the blood and establish a long-lasting bacteremia inside the host.
Advisors:Dehio, Christoph and Jenal, Urs
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Infection Biology > Molecular Microbiology (Dehio)
UniBasel Contributors:Dehio, Christoph and Jenal, Urs
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13291
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (iv, 141 Seiten)
Language:English
Identification Number:
Last Modified:08 Feb 2020 15:11
Deposited On:13 Nov 2019 14:32

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