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Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, e50473Article in journal (Refereed) Published
Abstract [en]

Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM beta-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native beta-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species.

Place, publisher, year, edition, pages
2012. Vol. 7, no 11, e50473
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-63027DOI: 10.1371/journal.pone.0050473ISI: 000311535700086PubMedID: 23185631OAI: oai:DiVA.org:umu-63027DiVA: diva2:581120
Available from: 2012-12-28 Created: 2012-12-27 Last updated: 2017-12-06Bibliographically approved
In thesis
1. The Francisella pathogenicity island: its role in type VI secretion and intracellular infection
Open this publication in new window or tab >>The Francisella pathogenicity island: its role in type VI secretion and intracellular infection
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Intracellular bacteria have developed various mechanisms to enter and persist in host cells and, at the same time, to evade the host immune response. One such pathogen is Francisella tularensis, the etiological agent of tularemia. After phagocytosis, this Gram-negative bacterium quickly escapes from the phagocytic compartment and replicates in the host cell cytosol. For this mode of infection, several components of the Francisella pathogenicity island (FPI) are critical. Interestingly, some FPI proteins share homology to components of Type VI Secretion Systems (T6SSs), but their assembly and functionality remains to be shown in Francisella.The thesis focused on the characterization of several of these FPI components; more specifically, how they contribute to the infection cycle as well as their possible role in the putative T6SS. We identified three unique mutants, ΔiglG, ΔiglI and ΔpdpE, which to various degrees were able to escape the phagosomal compartment, replicate in the host cytosol and cause host cell cytotoxicity. In contrast, ΔiglE as well as mutants within the conserved core components of T6SSs, VgrG and DotU, were defective for all of these processes. In the case of IglE, which is a lipoprotein and localized to the outer membrane of the bacterial cell wall, residues within its N-terminus were identified to be important for IglE function. Consistent with a suggested role as a trimeric membrane puncturing device, VgrG was found to form multimers. DotU stabilized the inner membrane protein IcmF, in agreement with its function as a core T6SS component. The functionality of the secretion system was shown by the translocation of several FPI proteins into the cytosol of infected macrophages, among them IglE, IglC and VgrG, of which IglE was the most prominently secreted protein. At the same time, the secretion was dependent on the core components VgrG, DotU but also on IglG. Although we and others have shown the importance of FPI proteins for the escape of F. tularensis, it has been difficult to assess their role in the subsequent replication, since mutants that fail to escape never reach the growth-permissive cytosol. For this reason, selected FPI mutants were microinjected into the cytosol of different cell types and their growth compared to their replication upon normal uptake. Our data suggest that not only the metabolic adaptation to the cytosolic compartment is important for the replication of intracytosolic bacteria, but also the mechanism of their uptake as well as the permissiveness of the cytosolic compartment per se.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 82 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1708
Keyword
Francisella, FPI, Type VI Secretion, Igl, DotU, VgrG, Pdp, microinjection, phagosomal escape, intracellular replication
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-101321 (URN)978-91-7601-246-8 (ISBN)
Public defence
2015-04-24, sal E04, byggnad 6E, NUS, Norrlands universitetssjukhus, Umeå, 10:00 (English)
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Available from: 2015-04-01 Created: 2015-03-27 Last updated: 2015-05-08Bibliographically approved

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Bröms, Jeanette E.Meyer, LenaSun, KunLavander, MoaSjöstedt, Anders
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