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Characterization of the attenuated Francisella tularensis strain FSC043: with special focus on the gene pdpC
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Francisella tularensis is a highly infective, intracellular bacterium. It is capable of infecting a wide range of mammals and causes the disease tularemia in humans. As a result of its high infectivity there have been a lot of efforts made to create a generally available vaccine against this pathogen. One potential vaccine candidate is the FSC043 strain, a spontaneous mutant that has acquired mutations making it attenuated for replication both in vitro and in the experimental mouse model. However, it was noted that it afforded protection against challenge with a highly virulent F. tularensis strain.

The aim of this thesis has been to delineate the mechanisms of its attenuation to better understand F. tularensis pathogenesis and to obtain a better knowledge about the prerequisites of protective immunity against this potent pathogen. Microarray and whole-genome sequencing revealed four mutations in the attenuated FSC043 strain that were not present in the virulent SCHU S4 isolate. One of these mutations has been described earlier as it results in a fusion protein also found in other attenuated strains. Among the other differences, two mutations were identical nonsense mutations in a duplicated gene region known as the Francisella pathogenicity island (FPI). The affected gene, pdpC, is coding for PdpC (pathogenicity determinant protein C). We found that these mutations resulted in a truncated form of PdpC, and also that the downstream gene was severely downregulated due to these mutations.

Further, our studies revealed that the intracellular phenotype of the FSC043 strain differed from other tested strains in that a small portion of the intracellular bacteria were able to escape the phagosome and multiply within the host, while the majority of intracellular bacteria stayed confined to the phagosome. We wanted to study the specific function of pdpC and therefore deleted both copies of it in the virulent SCHU S4strain as well as the Live Vaccine Strain, an empirically attenuated strain often used as a model for the virulent strains of F. tularensis. The resulting mutants showed an attenuated phenotype; no intracellular growth in murine cells, and no virulence in mice. When studying the intracellular localization of the LVS Δpdpc mutant, we found that it was uniformly located adjacent to phagosomal membrane-like structures but that the membrane was markedly disrupted. Further, this mutant induced an MOI-dependent cytotoxicity, measured by LDH release, and also the release of IL-1β, an inflammatory cytokine not induced by phagosomally contained mutants. Studies on markers for host cell death revealed that the LVS ΔpdpC mutant induced mitochondrial instability, phosphatidylserine (PS) presentation, and TUNEL-specific DNA fragmentation in infected cells, rather similar to the wild-type strain, despite its lack of replication.

This study reveals that the pdpC gene is an important gene required for F. tularensis virulence. We also show that non-replicating intracellular bacteria can induce host cell death, hypothesizing that release of bacterial components in the host cell cytosol is required for this induction. The FSC043 mutant showed a unique phenotype where a small subset of bacteria was able to escape the phagosome and replicate in the host cell. This was also seen in the pdpC deletion mutant of SCHU S4, but not with the LVS ΔpdpC. However, regardless of genetic background, the ΔpdpC mutant had an effect on phagosomal escape; either by affecting the phagosomal membranes in a unique way or by allowing phagosomal escape of a small proportion of the bacteria.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2013. , 39 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1552
Keyword [en]
Francisella tularensis, intracellular bacteria, J774, apoptosis, pyroptosis, PdpC, FSC043
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology; Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-66365ISBN: 978-91-7459-564-2 (print)OAI: oai:DiVA.org:umu-66365DiVA: diva2:606274
Public defence
2013-03-15, Betula, NUS 6M - Laboratoriecentrum, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2013-02-22 Created: 2013-02-18 Last updated: 2013-02-22Bibliographically approved
List of papers
1. Whole-genome sequencing reveals distinct mutational patterns in closely related laboratory and naturally propagated Francisella tularensis strains
Open this publication in new window or tab >>Whole-genome sequencing reveals distinct mutational patterns in closely related laboratory and naturally propagated Francisella tularensis strains
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2010 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 7, e11556- p.Article in journal (Refereed) Published
Abstract [en]

The F. tularensis type A strain FSC198 from Slovakia and a second strain FSC043, which has attenuated virulence, are both considered to be derivatives of the North American F. tularensis type A strain SCHU S4. These strains have been propagated under different conditions: the FSC198 has undergone natural propagation in the environment, while the strain FSC043 has been cultivated on artificial media in laboratories. Here, we have compared the genome sequences of FSC198, FSC043, and SCHU S4 to explore the possibility that the contrasting propagation conditions may have resulted in different mutational patterns. We found four insertion/deletion events (INDELs) in the strain FSC043, as compared to the SCHU S4, while no single nucleotide polymorphisms (SNPs) or variable number of tandem repeats (VNTRs) were identified. This result contrasts with previously reported findings for the strain FSC198, where eight SNPs and three VNTR differences, but no INDELs exist as compared to the SCHU S4 strain. The mutations detected in the laboratory and naturally propagated type A strains, respectively, demonstrate distinct patterns supporting that analysis of mutational spectra might be a useful tool to reveal differences in past growth conditions. Such information may be useful to identify leads in a microbial forensic investigation.

Place, publisher, year, edition, pages
Public library of science, 2010
Keyword
Francisella tularensis, FSC043
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:umu:diva-66131 (URN)10.1371/journal.pone.0011556 (DOI)000280065600004 ()20657845 (PubMedID)
Available from: 2013-02-15 Created: 2013-02-15 Last updated: 2017-12-06Bibliographically approved
2. Characterization of the spontaneous mutant FSC043 of Francisella tularensis subspecies tularensis
Open this publication in new window or tab >>Characterization of the spontaneous mutant FSC043 of Francisella tularensis subspecies tularensis
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(English)Manuscript (preprint) (Other academic)
Keyword
Francisella tularensis, J774 cells, PdpC, FSC043
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-45866 (URN)
Available from: 2011-08-19 Created: 2011-08-19 Last updated: 2013-02-22Bibliographically approved
3. The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection
Open this publication in new window or tab >>The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection
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2013 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, 20Article in journal (Refereed) Published
Abstract [en]

Background: A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system.

Results: We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release.

Conclusions: The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.

Place, publisher, year, edition, pages
BioMed Central, 2013
Keyword
Francisella tularensis, type VI secretion, cytopathogenicity, intracellular replication, PdpC
National Category
Microbiology Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-66129 (URN)10.1186/1471-2180-13-20 (DOI)000314827600002 ()23356941 (PubMedID)
Note

The Francisella tularensis LVS Delta pdpC mutant exhibits a unique phenotype during intracellular infection

Available from: 2013-02-15 Created: 2013-02-15 Last updated: 2017-12-06Bibliographically approved
4. Importance of PdpC, IglC, IglI, and IglG for modulation of a host cell death pathway induced by Francisella tularensis LVS
Open this publication in new window or tab >>Importance of PdpC, IglC, IglI, and IglG for modulation of a host cell death pathway induced by Francisella tularensis LVS
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Modulation of host cell death pathways appears to be a prerequisite for the successful life styles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand how this is achieved, macrophages were infected with the F. tularensis live vaccine strain (LVS) and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes. All of these genes encode components that together with a dozen other proteins form the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ∆iglI or ∆iglC mutant, although the former strain replicated. The ∆iglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ∆pdpC mutant showed no replication, but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to LVS. The unique phenotypes of the mutants provide novel insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection.

Keyword
Francisella tularensis, LVS, apoptosis, PdpC, IglG, IglI
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-66132 (URN)
Available from: 2013-02-18 Created: 2013-02-15 Last updated: 2013-02-22

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