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Factors affecting the escape of Francisella tularensis from the phagolysosome.
Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
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2004 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 53, no 10, 953-958 p.Article in journal (Refereed) Published
Abstract [en]

The highly virulent bacterium Francisella tularensis is well adapted to the intracellular habitat but the mechanisms behind its intracellular survival have been elusive. Recently, it was shown that the bacterium is capable of escaping from the phagosome of human and mouse monocytic cells. Here it is shown that this escape is affected by gamma interferon (IFN-gamma) treatment of mouse peritoneal exudate cells since in treated cells the proportion that escaped was significantly lower (80%) than in untreated cells (97%) as determined by transmission electron microscopy. By contrast, < 1% of mutant bacteria lacking expression of a 23 kDa protein denoted IglC were able to escape from the phagosome. Infection with the DeltaiglC strain complemented with the iglC gene resulted in 60% of the bacteria escaping from the phagosome. Whereas IFN-gamma treatment conferred a static effect on intracellular wild-type bacteria, the treatment had a bactericidal effect on the DeltaiglC strain. The results show that the activation status of infected cells affects the escape of F. tularensis from the phagosome. An even more profound effect on this escape is related to expression of IglC by F. tularensis. Its absence rendered the mutant bacteria incapable of escaping from the phagosome and of multiplying intracellularly.

Place, publisher, year, edition, pages
2004. Vol. 53, no 10, 953-958 p.
Identifiers
URN: urn:nbn:se:umu:diva-4613DOI: 10.1099/jmm.0.45685-0PubMedID: 15358816OAI: oai:DiVA.org:umu-4613DiVA: diva2:143783
Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Reactive oxygen and nitrogen in host defence against Francisella tularensis
Open this publication in new window or tab >>Reactive oxygen and nitrogen in host defence against Francisella tularensis
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Francisella tularensis, the causative agent of tularemia, is a potent human and animal pathogen. Initially upon infection of the host, intramacrophage proliferation of F. tularensis occurs but after activation of the acquired host immunity, the phagocytes become activated to kill the bacterium. In my thesis, I focused on mechanisms utilized by F. tularensis to survive intracellularly and on host mechanisms responsible for macrophage-mediated killing and control of infection.

The F. tularensis-specific protein IglC has been previously shown to be essential to the intramacrophage proliferation and virulence of the bacterium in mice. By electron microscopy of macrophages infected with either the live vaccine strain of F. tularensis or an isogenic mutant, denoted ∆iglC, expression of IglC was found to be necessary for the bacterium to escape from the phagosome. IFN-g-activated macrophages significantly inhibited the escape of the live vaccine strain of F. tularensis from the phagosome.

iNOS and phox generate NO and O2-, respectively. These molecules and their reaction products possess both bactericidal and immunoregulatory properties. We investigated the capability of IFN-g-activated peritoneal exudate cells from gene deficient iNOS-/- or p47phox-/- mice to control an intracellular F. tularensis LVS infection. iNOS was found to contribute significantly to the IFN-g induced killing, while phox contributed only to a minor extent. Unexpectedly, bacteria were eradicated even in the absence of both a functional phox and an active iNOS. The eradication was found to depend on ONOO-, the reaction product of NO and O2-, because addition of a decomposition catalyst of ONOO- completely inhibited the killing.

Studies on iNOS-/- or p47phox-/- mice infected with F. tularensis LVS showed phox to be important during the first days of infection, a stage when iNOS seemed dispensable. Eventually, iNOS-/- mice died of the infection, suggesting a role of iNOS later in the course of infection. iNOS-/- mice exhibited elevated IFN-g serum levels and severe liver damage suggesting that the outcome of infection was at least in part related to an uncontrolled immune response.

Several pathogenic bacteria express Cu,Zn-SOD, which in combination with other enzymes detoxifies reactive oxygen species produced by the host. A deletion mutant of F. tularensis LVS lacking the gene encoding Cu,Zn-SOD was attenuated at least 100-fold compared to LVS in mice. In peritoneal exudate cells from mice, Cu,Zn-SOD was found to be required for effective intramacrophage proliferation and, in mice, important for bacterial replication at the very early phase of infection.

In summary, the most conspicuous findings were a capability of IFN-g activated macrophages to retain F. tularensis LVS in the phagosome, an essential role of ONOO- in intracellular killing of F. tularensis, and an importance of Cu,Zn-SOD to the virulence of F. tularensis LVS.

Place, publisher, year, edition, pages
Umeå: Klinisk mikrobiologi, 2005. 55 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 954
Keyword
Cell and molecular biology, Francisella tularensis, inducible nitric oxide synthase, phagocyte oxidase, macrophages, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-474 (URN)91-7305-851-3 (ISBN)
Public defence
2005-04-15, E04, 6E, Norrlands Universitetssjukhus i Umeå, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2005-03-15 Created: 2005-03-15 Last updated: 2009-11-19Bibliographically approved
2. Effects of Francisella tularensis infection on macrophage intracellular signaling
Open this publication in new window or tab >>Effects of Francisella tularensis infection on macrophage intracellular signaling
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microbes contain a number of structural components, also known as pathogen associated molecular patterns (PAMP), which can be recognized by the host defense system. The PAMP serve as ligands for Toll-like receptors (TLR) expressed on phagocytic cells. Binding of PAMP to TLR leads to activation of a number of intracellular transduction pathways including NF-�B and mitogen-activated protein kinase pathways (MAPK). Activation of those pathways in turn leads to activation of proinflammatory immune responses and antimicrobial defense mechanisms. In this study we show that the intracellular pathogen F. tularensis LVS can induce a proinflammatory response during internalization in both human and mouse macrophages. However, after internalization, F. tularensis LVS effectively blocks all these pathways resulting in no secretion of proinflammatory cytokines in mouse macrophages. In human macrophages, a downregulation was observed, however, there was not a complete block as in the murine cells. Furthermore, the infected cells do not become activated by stimulation with TLR agonists such as Escherichia coli LPS or bacterial lipoprotein. This phenomenon is observed only when the cells were infected with the LVS strain, but not with killed bacteria or specific mutants such as iglC, or mglA mutants, suggesting that not only their presence but also rapid regulation of the Igl proteins are needed. Thus, Igl/Mgl system plays a very important role in mediating these inhibitory effects. To further investigate the role of the Igl/Mgl proteins in the virulence of F. tularensis, we established a novel model for tularemia, infection of Drosophila melanogaster. Using the fly model as a way to mimic the F. tularensis infection in arthropod vectors, which are important for its life cycle, we observed that bacterial mutants in each of the IglB, IglC, IglD, or MglA proteins all showed less efficient killing of the flies than did the parental LVS strain. Nitric oxide, superoxide, and the reaction product of the two mediators, peroxynitrite, all are important for regulation of eukaryotic cell signaling and effectuating bactericidal mechanisms. To this end, we investigated the role of these mediators for the F. tularensismediated TLR-signaling inhibition. We observed that whereas peroxynitrite had little effect on the inhibition, addition of SNAP, a donor of nitric oxide, partially restored the ability of the cells to respond to LPS stimulation and secrete proinflammatory cytokine such as TNF-�. However, since FeTTPS reversed the SNAP-mediated effect, the results indicate that NO forms peroxynitrite intracellular and that the latter molecule is the effector. Together these results indicate that F. tularensis contains PAMP that can activate proinflammatory immune response while the bacteria are located outside of phagocytic cell and during internalization, however, the intracellular bacteria have means to inhibit the activation of anti-bacterial systems of macrophages and block inflammatory responses completely in mouse cells and partially in human cells. This suppression may lead to disruption of synthesis of bactericidal effector molecules, allowing F. tularensis to survive and rapidly proliferate inside phagocytic cells. The ability to suppress the inflammatory responses is dependent on expression of the Igl/Mgl proteins. The Igl/Mgl system is also critical for the ability of F. tularensis to replicate in mammalian cells and in D. melanogaster.

Place, publisher, year, edition, pages
Umeå: Klinisk mikrobiologi, 2005. 55 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 960
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-547 (URN)91-7305-898-3 (ISBN)
Public defence
2005-05-31, Sal E04, NUS, By 6E, Umeå, 09:00 (English)
Opponent
Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2012-06-28Bibliographically approved

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