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Effects of Francisella tularensis infection on macrophage intracellular signaling
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
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: urn:nbn:se:umu:diva-547ISBN: 91-7305-898-3 OAI: oai:DiVA.org:umu-547DiVA: diva2:143787
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
List of papers
1. Francisella tularensis LVS initially activates but subsequently down-regulates intracellular signaling and cytokine secretion in mouse monocytic and human peripheral blood mononuclear cells.
Open this publication in new window or tab >>Francisella tularensis LVS initially activates but subsequently down-regulates intracellular signaling and cytokine secretion in mouse monocytic and human peripheral blood mononuclear cells.
2005 (English)In: Microbial Pathogenesis, ISSN 0882-4010, Vol. 38, no 5-6, 239-247 p.Article in journal (Refereed) Published
Abstract [en]

Monocytic cells constitute an important defense mechanism against invading pathogens by recognizing conserved pathogens components. The recognition leads to activation of intracellular pathways involving nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPK), such as the c-Jun NH2-terminal kinase (JNK), and p38. We show that in vitro infection with Francisella tularensis results in activation of NF-kappaB, phosphorylation of p38 and c-Jun, and secretion of TNF-alpha in adherent mouse peritoneal cells, in the mouse macrophage-like cell line J774A.1, in the human macrophage cell line THP-1, and in human peripheral blood monocytic cells. This occurred after infection with the human live vaccine strain, F. tularensis LVS or a mutant strain denoted deltaiglC, which lacks expression of a 23-kDa protein, or after addition of killed F. tularensis LVS. Addition of purified F. tularensis LPS resulted in no discernible effects on the cells. When the effects were followed up to 5 h, activation persisted in cultures with killed bacteria or infected with the deltaiglC strain. In contrast, the signal transduction activation and secretion of TNF-alpha were down-regulated within the 5h period in mouse peritoneal cells, J774 cells or human peripheral blood mononuclear cells infected with F. tularensis LVS. Together, the results suggest that infection with live F. tularensis LVS bacteria leads to a rapid induction of a proinflammatory response in mouse and human cells but after internalization of bacteria, this response is completely or partly down-regulated in most cell types. This down-regulation does not occur when cells are infected with the mutant deltaiglC.

Identifiers
urn:nbn:se:umu:diva-4612 (URN)10.1016/j.micpath.2005.02.003 (DOI)15925273 (PubMedID)
Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2009-11-30Bibliographically approved
2. 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
3. Francisella tularensis inhibits Toll-like receptor-mediated activation of intracellular signaling and secretion of TNF-alpha and IL-1 in murine macrophages.
Open this publication in new window or tab >>Francisella tularensis inhibits Toll-like receptor-mediated activation of intracellular signaling and secretion of TNF-alpha and IL-1 in murine macrophages.
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2003 (English)In: Cellular Microbiology, ISSN 1462-5814, Vol. 5, no 1, 41-51 p.Article in journal (Refereed) Published
Abstract [en]

Microbial ligands, including lipopolysaccharide (LPS) and bacterial lipoproteins, activate Toll-like receptors (TLR) of mononuclear phagocytes, thereby inducing proinflammatory cytokines and antimicrobial activity. We show that Francisella tularensis, an intracellular pathogen, is capable of inhibiting this macrophage response. Infection with the live vaccine strain F. tularensis LVS rendered cells of the murine macrophage-like cell line J774A.1 incapable of secreting TNF-alpha or IL-1beta and mobilizing an antimicrobial activity in response to bacterial lipopeptide or Escherichia coli-derived LPS. Inhibition of TNF-alpha secretion occurred also when J774 cells were infected with F. tularensis LVS in the presence of chloramphenicol, but not when they were infected with a mutant of F. tularensis LVS defective in expression of a 23 kDa protein that is upregulated during intracellular infection. Purified F. tularensis LPS did not show an agonistic or antagonistic effect on the E. coli LPS-induced activation of the J774 cells. Francisella tularensis LVS suppressed the capability of the cells to respond to LPS or bacterial lipopeptide (BLP) with activation of nuclear factor kappa B (NF-kappaB), and degradation of the in-hibitor of NF-kappaB, IkappaB, was blocked during the infection. Also the LPS- or BLP-induced phosphorylation of the mitogen-activated protein kinase p38 and the transcription factor c-Jun was inhibited by F. tularensis LVS but not by the 23 kDa protein mutant. In conclusion, F. tularensis appears capable of abrogating the TNF-alpha and IL-1 responses of macrophages induced by E. coli LPS or BLP via a mechanism that involves suppression of several intracellular pathways and is dependent on expression of a bacterial 23 kDa protein.

Identifiers
urn:nbn:se:umu:diva-4614 (URN)10.1046/j.1462-5822.2003.00251.x (DOI)12542469 (PubMedID)
Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2009-11-30Bibliographically approved
4. Mechanisms of inhibition of TLR-mediated signaling conferred by Francisella tularensis.
Open this publication in new window or tab >>Mechanisms of inhibition of TLR-mediated signaling conferred by Francisella tularensis.
(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-4615 (URN)
Available from: 2005-05-31 Created: 2005-05-31 Last updated: 2010-01-14Bibliographically approved
5. Drosophila melanogaster as a model for elucidating the pathogenicity of Francisella tularensis
Open this publication in new window or tab >>Drosophila melanogaster as a model for elucidating the pathogenicity of Francisella tularensis
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2008 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 10, no 6, 1327-1338 p.Article in journal (Refereed) Published
Abstract [en]

Drosophila melanogaster is a widely used model organism for research on innate immunity and serves as an experimental model for infectious diseases. The aetiological agent of the zoonotic disease tularaemia, Francisella tularensis, can be transmitted by ticks and mosquitoes and Drosophila might be a useful, genetically amenable model host to elucidate the interactions between the bacterium and its arthropod vectors. We found that the live vaccine strain of F. tularensis was phagocytosed by Drosophila and multiplied in fly haemocytes in vitro and in vivo. Bacteria injected into flies resided both inside haemocytes and extracellularly in the open circulatory system. A continuous activation of the humoral immune response, i.e. production of antimicrobial peptides under control of the imd/Relish signalling pathway, was observed and it may have contributed to the relative resistance to F. tularensis as flies defective in the imd/Relish pathway died rapidly. Importantly, bacterial strains deficient for genes of the F. tularensis intracellular growth locus or the macrophage growth locus were attenuated in D. melanogaster. Our results demonstrate that D. melanogaster is a suitable model for the analysis of interactions between F. tularensis and its arthropod hosts and that it can also be used to identify F. tularensis virulence factors relevant for mammalian hosts.

National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-9517 (URN)10.1111/j.1462-5822.2008.01129.x (DOI)18248629 [PubMed - as supplied by publisher] (PubMedID)
Available from: 2008-04-14 Created: 2008-04-14 Last updated: 2012-09-07

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