Ursache und Wirkung: der Beitrag des Wirtszell-Mikrotubuli-Cytoskeletts zur Chlamydia pneumoniae Infektion
Zellbiologie
Zusammenfassung der Projektergebnisse
Intracellular pathogenic bacteria need to use and manipulate host cellular pathways for their survival and growth. The Gram-negative obligate intracellular Chlamydiae, which are able to infect a wide range of organisms that encompass protozoans to humans and can have serious consequences for human health, massively rely on host cell functions due to their reduced genome size. The initiating process of infection is adherence of infectious elementary bodies (EBs) to host cells leading to the injection of so-called effector proteins into the host cytosol. A prime target for bacterial effectors in general is the host cytoskeleton scaffold, and chlamydial invasion is dependent on the interaction of effector proteins with the host actin cytoskeleton. This requires the binding of actin and actin-modulating proteins by effector proteins such as the conserved chlamydial TarP family, which leads to remodelling of the actin cytoskeleton. The function of the second member of the dynamic host cytoskeletal network, the microtubule (MT) cytoskeleton in the infection process of intracellular bacteria is understood poorly. Given the finding that this network cross talks with the actin cytoskeleton and its importance in essential cellular processes such as motor-driven intracellular transport, we decided to study the impact of this cytoskeletal structure on the chlamydial infection process. Our approach was twofold: (i) a general analysis to determine the importance of the MT cytoskeleton on the early infection of human cells by Chlamydia pneumoniae, and (ii) identification and characterization of chlamydial proteins that modulate MT function. (i) Manipulation of the MT cytoskeleton by MT-destabilizing drugs prior to EB infection resulted in a significant decrease of C. pneumoniae infected cells compared to control cells. Using an experimental set-up that generated a MT-rich subcellular compartment with an ordered array of dynamic MTs, we scored the vast majority of invading EBs to be associated with this part of the cell and on/in the vicinity of the MTs present. Thus, the MT cytoskeleton plays a vital part in the efficient C. pneumoniae early infection process. (ii) To identify C. pneumoniae proteins hijacking the host MT cytoskeleton, we turned to the fission yeast Schizosaccharomyces pombe, as C. pneumoniae is not amenable to phenotypic screening. The interphase MT cytoskeleton of S. pombe and the regulators of its dynamic behaviour have been studied in detail and the structural components of the human and yeast MT cytoskeletons and their main regulators are evolutionary conserved. Unexpectantly, our analysis in yeast identified 14 chlamydial proteins that modulated yeast MT dynamics in various ways leading either to more stable or unstable MTs. As these 14 proteins represented 12% of all C. pneumoniae proteins tested and 8/14 were homologous to proteins from other Chlamydiae, we conclude that the impact of the MT cytoskeleton for chlamydial infection is extraordinary. We studied the function of two of the newly identified proteins in detail: CPn0443, which has a putative C. trachomatis homolog, binds to mammalian MTs in vivo and in vitro and appears to bundle them. Analysis in S. pombe pointed to a possible role in altering MT dynamics by indirectly titrating the conserved EB1/Mal3 MT stabilizing protein. Binding to the mammalian MT cytoskeleton and altering its dynamic behaviour and function was also demonstrated for the second analysed chlamydial protein, Cpn0572. We showed that Cpn0572 is a member of the chlamydial TarP family of early secreted actin regulators. Our extensive analysis revealed that CPn0527 is a multi-domain protein that regulates actin dynamics directly (via monomeric and filamentous actin binding domains) and indirectly (via Vinculin) and that CPn0572 harbours a MT binding domain separate from the actin binding domains. The latter feature allowed us to demonstrate that the Cpn0572 MT function is infection relevant. Furthermore, human cells infected with C. trachomatis ectopically expressing CPn0572 show a co-localization of this C. pneumoniae protein with the interphase MT cytoskeleton. Lastly, CPn0572 also bound to and modulated the MTs of the mitotic spindle, leading to massively aberrant chromosome transmission. As a C. pneumoniae infection has been implicated as a contributor to lung cancer, a hallmark of which is genome instability, we speculate that C. pneumoniae hijacking of MTs might give rise to aneuploidy in the infected cell.
Projektbezogene Publikationen (Auswahl)
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The Chlamydia pneumoniae Tarp Ortholog CPn0572 Stabilizes Host F-Actin by Displacement of Cofilin. Front Cell Infect Microbiol. 2017 Dec 12;7:511
Zrieq R, Braun C, Hegemann JH
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CPn0572, the C. pneumoniae ortholog of TarP, reorganizes the actin cytoskeleton via a newly identified F-actin binding domain and recruitment of vinculin. PLoS One. 2019 Jan 10;14(1):e0210403
Braun C, Alcázar-Román AR, Laska A, Mölleken K, Fleig U, Hegemann JH
