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Reactive oxygen and nitrogen in host defence against Francisella tularensis
Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Clinical Microbiology, Infectious Diseases.
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 [en]
Cell and molecular biology, Francisella tularensis, inducible nitric oxide synthase, phagocyte oxidase, macrophages
Keyword [sv]
Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Clinical Bacteriology
Identifiers
URN: urn:nbn:se:umu:diva-474ISBN: 91-7305-851-3 (print)OAI: oai:DiVA.org:umu-474DiVA: diva2:143532
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
List of papers
1. Factors affecting the escape of Francisella tularensis from the phagolysosome.
Open this publication in new window or tab >>Factors affecting the escape of Francisella tularensis from the phagolysosome.
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2004 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, 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.

Identifiers
urn:nbn:se:umu:diva-4613 (URN)10.1099/jmm.0.45685-0 (DOI)15358816 (PubMedID)
Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2009-11-19Bibliographically approved
2. The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages.
Open this publication in new window or tab >>The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages.
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.

Keyword
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
Identifiers
urn:nbn:se:umu:diva-7175 (URN)10.1016/j.micinf.2004.11.020 (DOI)15788155 (PubMedID)
Available from: 2008-01-04 Created: 2008-01-04 Last updated: 2009-11-19Bibliographically approved
3. Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis.
Open this publication in new window or tab >>Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis.
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2004 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 72, no 12, 7172-7182 p.Article in journal (Refereed) Published
Abstract [en]

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are important mediators of the bactericidal host response. We investigated the contribution of these two mediators to the control of infection with the facultative intracellular bacterium Francisella tularensis. When intradermally infected with the live vaccine strain F. tularensis LVS, mice deficient in production of RNS (iNOS(-/-) mice) or in production of ROS by the phagocyte oxidase (p47(phox-/-) mice) showed compromised resistance to infection. The 50% lethal dose (LD(50)) for iNOS(-/-) mice was <20 CFU, and the LD(50) for p47(phox-/-) mice was 4,400 CFU, compared to an LD(50) of >500,000 CFU for wild-type mice. The iNOS(-/-) mice survived for 26.4 +/- 1.8 days, and the p47(phox-/-) mice survived for 10.1 +/- 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-gamma) and interleukin-6 were higher in iNOS(-/-) and p47(phox-/-) mice than in wild-type mice. Histological examination of livers of iNOS(-/-) mice revealed severe liver pathology. Splenocytes obtained 5 weeks after primary infection from antibiotic-treated iNOS(-/-) mice showed an in vitro recall response that was similar in magnitude and greater secretion of IFN-gamma compared to cells obtained from wild-type mice. In summary, mice lacking expression of RNS or ROS showed extreme susceptibility to infection with F. tularensis LVS. The roles of RNS and ROS seemed to be distinct since mice deficient in production of ROS showed dissemination of infection and died during the early phase of infection, whereas RNS deficiency led to severe liver pathology and a contracted course of infection.

Keyword
Animals, Colony Count; Microbial, Disease Susceptibility, Interferon Type II/blood, Liver/microbiology/pathology, Lymphocyte Activation, Mice, Mice; Inbred C57BL, Nitric Oxide Synthase/physiology, Nitric Oxide Synthase Type II, Reactive Nitrogen Species/*physiology, Reactive Oxygen Species/*metabolism, Skin/microbiology, Superoxides/metabolism, Tularemia/*immunology
Identifiers
urn:nbn:se:umu:diva-7172 (URN)10.1128/IAI.72.12.7172-7182.2004 (DOI)15557642 (PubMedID)
Available from: 2008-01-04 Created: 2008-01-04 Last updated: 2011-04-15Bibliographically approved
4. Cu,Zn-superoxide dismutase of Francisella tularensis LVS is a virulence factor
Open this publication in new window or tab >>Cu,Zn-superoxide dismutase of Francisella tularensis LVS is a virulence factor
(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-4427 (URN)
Available from: 2005-03-15 Created: 2005-03-15 Last updated: 2010-01-14Bibliographically approved

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