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The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages.
Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Clinical Microbiology, Infectious Diseases.
Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Clinical Microbiology, Infectious Diseases.
Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology.
2005 (English)In: Microbes and infection, ISSN 1286-4579, Vol. 7, no 3, 467-475 p.Article in journal (Refereed) Published
Abstract [en]

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.

Place, publisher, year, edition, pages
2005. Vol. 7, no 3, 467-475 p.
Keyword [en]
Animals, Francisella tularensis/*immunology, Macrophage Activation/physiology, Macrophages/*physiology, Mice, Mice; Inbred C57BL, Mice; Knockout, Nitric Oxide Synthase/genetics/physiology, Nitric Oxide Synthase Type II, Oxidoreductases/genetics/physiology, Reactive Nitrogen Species/*physiology, Reactive Oxygen Species/*metabolism
URN: urn:nbn:se:umu:diva-7175DOI: 10.1016/j.micinf.2004.11.020PubMedID: 15788155OAI: diva2:146846
Available from: 2008-01-04 Created: 2008-01-04 Last updated: 2009-11-19Bibliographically 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.
Umeå University medical dissertations, ISSN 0346-6612 ; 954
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
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)
Available from: 2005-03-15 Created: 2005-03-15 Last updated: 2009-11-19Bibliographically approved

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