Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (Sjöstedt)
Show others and affiliations
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. Vol. 13, 20
Keyword [en]
Francisella tularensis, type VI secretion, cytopathogenicity, intracellular replication, PdpC
National Category
Microbiology Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-66129DOI: 10.1186/1471-2180-13-20ISI: 000314827600002PubMedID: 23356941OAI: oai:DiVA.org:umu-66129DiVA: diva2:605721
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
In thesis
1. Characterization of the attenuated Francisella tularensis strain FSC043: with special focus on the gene pdpC
Open this publication in new window or tab >>Characterization of the attenuated Francisella tularensis strain FSC043: with special focus on the gene pdpC
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
Francisella tularensis, intracellular bacteria, J774, apoptosis, pyroptosis, PdpC, FSC043
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology; Microbiology
Identifiers
urn:nbn:se:umu:diva-66365 (URN)978-91-7459-564-2 (ISBN)
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
2. The Francisella pathogenicity island: its role in type VI secretion and intracellular infection
Open this publication in new window or tab >>The Francisella pathogenicity island: its role in type VI secretion and intracellular infection
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Intracellular bacteria have developed various mechanisms to enter and persist in host cells and, at the same time, to evade the host immune response. One such pathogen is Francisella tularensis, the etiological agent of tularemia. After phagocytosis, this Gram-negative bacterium quickly escapes from the phagocytic compartment and replicates in the host cell cytosol. For this mode of infection, several components of the Francisella pathogenicity island (FPI) are critical. Interestingly, some FPI proteins share homology to components of Type VI Secretion Systems (T6SSs), but their assembly and functionality remains to be shown in Francisella.The thesis focused on the characterization of several of these FPI components; more specifically, how they contribute to the infection cycle as well as their possible role in the putative T6SS. We identified three unique mutants, ΔiglG, ΔiglI and ΔpdpE, which to various degrees were able to escape the phagosomal compartment, replicate in the host cytosol and cause host cell cytotoxicity. In contrast, ΔiglE as well as mutants within the conserved core components of T6SSs, VgrG and DotU, were defective for all of these processes. In the case of IglE, which is a lipoprotein and localized to the outer membrane of the bacterial cell wall, residues within its N-terminus were identified to be important for IglE function. Consistent with a suggested role as a trimeric membrane puncturing device, VgrG was found to form multimers. DotU stabilized the inner membrane protein IcmF, in agreement with its function as a core T6SS component. The functionality of the secretion system was shown by the translocation of several FPI proteins into the cytosol of infected macrophages, among them IglE, IglC and VgrG, of which IglE was the most prominently secreted protein. At the same time, the secretion was dependent on the core components VgrG, DotU but also on IglG. Although we and others have shown the importance of FPI proteins for the escape of F. tularensis, it has been difficult to assess their role in the subsequent replication, since mutants that fail to escape never reach the growth-permissive cytosol. For this reason, selected FPI mutants were microinjected into the cytosol of different cell types and their growth compared to their replication upon normal uptake. Our data suggest that not only the metabolic adaptation to the cytosolic compartment is important for the replication of intracytosolic bacteria, but also the mechanism of their uptake as well as the permissiveness of the cytosolic compartment per se.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 82 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1708
Keyword
Francisella, FPI, Type VI Secretion, Igl, DotU, VgrG, Pdp, microinjection, phagosomal escape, intracellular replication
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
urn:nbn:se:umu:diva-101321 (URN)978-91-7601-246-8 (ISBN)
Public defence
2015-04-24, sal E04, byggnad 6E, NUS, Norrlands universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2015-04-01 Created: 2015-03-27 Last updated: 2015-05-08Bibliographically approved

Open Access in DiVA

The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection(2756 kB)247 downloads
File information
File name FULLTEXT02.pdfFile size 2756 kBChecksum SHA-512
7a308e5b03eb8b7e102324e3132e630d9523e452b764233672d1c882dc9f9624c1467830d8fecfaed8e9be88457086150cc7ba4d78fe0d062dcbeb1ef2a9cdbf
Type fulltextMimetype application/pdf

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Lindgren, MarieBröms, Jeanette E.Meyer, LenaGolovliov, IgorSjöstedt, Anders
By organisation
Clinical BacteriologyMolecular Infection Medicine Sweden (MIMS)
In the same journal
BMC Microbiology
MicrobiologyMicrobiology in the medical area

Search outside of DiVA

GoogleGoogle Scholar
Total: 247 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 152 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf