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Importance of PdpC, IglC, IglI, and IglG for modulation of a host cell death pathway induced by Francisella tularensis LVS
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
(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 [en]
Francisella tularensis, LVS, apoptosis, PdpC, IglG, IglI
National Category
URN: urn:nbn:se:umu:diva-66132OAI: diva2:606132
Available from: 2013-02-18 Created: 2013-02-15 Last updated: 2013-02-22
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.
Umeå University medical dissertations, ISSN 0346-6612 ; 1552
Francisella tularensis, intracellular bacteria, J774, apoptosis, pyroptosis, PdpC, FSC043
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology; Microbiology
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)
Available from: 2013-02-22 Created: 2013-02-18 Last updated: 2013-02-22Bibliographically approved

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