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Host-pathogen interactions between Francisella tularensis and Drosophila melanogaster
Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Francisella tularensis is a highly virulent Gram-negative bacterium causing the zoonotic disease tularemia. Arthropod-borne transmission plays an important role in transferring the disease to humans. F. tularensis induces very low amounts of pro-inflammatory cytokines during infection, due to inhibition of immune signaling pathways and an unusual structure of its lipopolysaccharide (LPS). To date, there is no vaccine available that is approved for public use, although an attenuated live vaccine strain (LVS) is commonly used as a model of the more infectious Francisella strains. To produce an effective vaccine it is important to understand the lifecycle of F. tularensis, including the interaction with the arthropod hosts. Drosophila melanogaster is a widely used model organism, which is increasingly being used in host-pathogen interaction studies as the immune pathways in flies are evolutionary conserved to the immune pathways in humans. An important part of the immune defense of D. melanogaster as well as of arthropods in general is the production of antimicrobial peptides. These peptides primarily target the bacterial membrane, inhibiting bacterial proliferation or directly killing the bacteria.

The aim of this thesis was to establish D. melanogaster as a model for F. tularensis infection and as a model for arthropod vectors of F. tularensis. Also, to use D. melanogaster to further study the interaction between F. tularensis and arthropod vectors, with specific regard to the host immune signaling and arthropod antimicrobial peptides.

F. tularensis LVS infects and kills D. melanogaster in a dose-dependent manner. During an infection, bacteria are found inside fly hemocytes, phagocytic blood cells, similar as in human infections. In mammals genes of the intracellular growth locus (igl) are important for virulence. In this work it is shown that the igl genes are also important for virulence in flies. These results demonstrate that D. melanogaster can be used as a model to study F. tularensis-host interactions.

LVS induces a prolonged activation of several immune signaling pathways in the fly, but seem to interfere with the JNK signaling pathway, similarly as in mammals. Overexpression of the JNK pathway in flies has a protective effect on fly survival.

Relish mutant flies, essentially lacking a production of antimicrobial peptides, succumb quickly to a F. tularensis infection, however, F. tularensis is relatively resistant to individual D. melanogaster antimicrobial peptides. Overexpressing antimicrobial peptide genes in wildtype flies has a protective effect on F. tularensis infection, suggesting that a combination of several antimicrobial peptides is necessary to control F. tularensis. The production of numerous antimicrobial peptides might be why D. melanogaster survives relatively long after infection. An intact structure of the lipid A and of the Kdo core of Francisella LPS is necessary for resistance to antimicrobial peptides and full virulence in flies. These results are similar to previous studies in mammals. In contrast to studies in mammals, genes affecting the O-antigen of F. tularensis LPS are not necessary for virulence in flies.

In conclusion, this thesis work shows that D. melanogaster can be used as a model for studying F. tularensis-host interactions. LVS activates several immune pathways during infection, but interfere with the JNK pathway. Overexpressing the JNK pathway results in increased survival of flies infected with LVS. Despite rather high resistance to individual antimicrobial peptides, exposure to a combination of several D. melanogaster antimicrobial peptides reduces the virulence of F. tularensis.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2012. , 45 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1504
Keyword [en]
arthropod vector, JNK, lipopolysaccharide, antimicrobial peptides
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology; Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-54604ISBN: 978-91-7459-432-4 (print)OAI: oai:DiVA.org:umu-54604DiVA: diva2:524388
Public defence
2012-05-25, E04, by 6E, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-05-04 Created: 2012-05-02 Last updated: 2012-05-04Bibliographically approved
List of papers
1. 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
2. The role of JNK signaling in Francisella tularensis infection of Drosophila melanogaster
Open this publication in new window or tab >>The role of JNK signaling in Francisella tularensis infection of Drosophila melanogaster
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The causative agent of tularemia, Francisella tularensis, inhibits immune signaling pathways during infection and thus reduces the production of proinflammatory cytokines. NF-kB and MAPK pathways are initially activated, but subsequently down-regulated in macrophages. The mechanism(s) of inhibition, or which host proteins that F. tularensis interacts with are not known. Here, we have used Drosophila melanogaster as a genetically amendable model host to study the interaction between F. tularensis LVS and host immune signaling. We found that similar as in mammalian macrophages, LVS inhibits the JNK pathway in flies. LVS interferes with the JNK pathway upstream or at the same level as the JNKK Hemipterous. Expression of an activated Hemipterous in fly macrophage-like cells had a protective effect on fly survival, while inhibiting the JNK pathway in the fly fat body, an important immune tissue, had no effect on fly survival.

Keyword
Francisella tularensis, LVS, Drosophila melanogaster, JNK, Hemipterous
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-54411 (URN)
Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2012-05-04Bibliographically approved
3. Francisella is sensitive to insect antimicrobial peptides
Open this publication in new window or tab >>Francisella is sensitive to insect antimicrobial peptides
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2013 (English)In: Journal of Innate Immunity, ISSN 1662-811X, E-ISSN 1662-8128, Vol. 5, no 1, 50-59 p.Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis causes the zoonotic disease tularemia. Arthropod vectors are important transmission routes for the disease, although it is not known how Francisella survives the efficient arthropod immune response. Here, we used Drosophila melanogaster as a model host for Francisella infections and investigated whether the bacteria are resistant to insect humoral immune responses, in particular to the antimicrobial peptides (AMPs) secreted into the insect hemolymph. Moreover, we asked to which extent such resistance might depend on LPS structure and surface characteristics of the bacteria. We analyzed F. novicida mutant strains in genes, directly or indirectly involved in specific steps of LPS biosynthesis, for virulence in wildtype and Relish E20 immune deficient flies, and tested selected mutants for sensitivity to AMPs in vitro. We demonstrate that Francisella is sensitive to specific fly AMPs, i.e. Attacin, Cecropin, Drosocin and Drosomycin. Furthermore, six bacterial genes, kpsF, manB, lpxF, slt, tolA and pal, were found to be required for resistance to Relish-dependent immune responses, illustrating the importance of structural details of Francisella lipid A and Kdo core for interactions with AMPs. Interestingly, a more negative surface charge and lack of O-antigen did not render mutant bacteria more sensitive to cationic AMPs and attenuated virulence in flies.

Place, publisher, year, edition, pages
Basel: Karger, 2013
Keyword
Francisella tularensis, Drosophila melanogaster, Antimicrobial peptides, Lipopolysaccharide, Host-pathogen interactions
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-54412 (URN)10.1159/000342468 (DOI)23037919 (PubMedID)
Note

Originally published in thesis in manuscript form.

Available from: 2012-04-26 Created: 2012-04-26 Last updated: 2017-12-07Bibliographically approved

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