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Francisella tularensis infection induces macrophage cell death
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Francisella tularensis, the causative agent of tularemia, is a potent human and animal pathogen. Its principal survival mechanism is rapid intracellular multiplication. The mechanisms that enables it to multiply intracellularly have been ill-defined and the thesis focused on characterizing the outcome of the macrophage-Francisella interaction and also if the interactions differ between the various subspecies of F tularensis.

The nature of host cell death was examined and the correlation of macrophage killing with intramacrophage Francisella growth was investigated by in vitro infection of J774A.1 macrophages with either the live vaccine (LVS) strain of F. tularensis, belonging to subspecies holarctica, or the subspecies novicida strain U112 Macrophage entry was in both cases cytochalasin D-sensitive but the intramacrophage growth of the two Francisella strains led to distinct types of host cell death, i.e., apoptosis vs. necrosis.

The macrophage apoptosis induced by infection with the LVS strain was mediated via the intrinsic pathway with critical involvement of caspase-3 and the mitochondria. The infected and apoptotic macrophages were shrunken, their chromatin was specifically degraded and revealed a typical DNA ladder pattern upon electrophoresis. Moreover, they were TUNEL positive, indicating the occurrence of apoptosis-dependent DNA fragmentation. The necessity of intracellular growth for the apoptosis was shown by the use of an isogenic mutant, denoted iglC, which lacked the ability to multiply intracellularly and this infection did not result in apoptosis.

The F. novicida strain U112, on the other hand, inhibited NF- B activity and ultimately induced macrophage necrosis. The infected and necrotic macrophages were enlarged, their chromatin was randomly degraded which gave a diffuse DNA pattern typical of necrosis. There was no apoptosis-specific caspase activation. By the use of an isogenic mutant, denoted mglA, it was shown that intracellular replication was necessary for the induction of necrosis. A hemolytic protein, novilysin A, was found in the F. novicida strain U112 but lacking in other subspecies of F. tularensis. The protein is a putative virulence factor but most likely not involved in the induction of necrosis. Its significance for the pathogenesis of F. novicida remains to be determined.

The findings of the thesis provide a detailed picture of the interaction between the host cells and various subspecies of F. tularensis. It also shows that the outcome of the interaction is critically dependent on the type of F. tularensis subspecies. The findings also question the use of F. novicida as a model organism for understanding pathogenicity mechanisms of the species in general. The induction of the host cell death is presumably an important mechanism for the survival of F. tularensis since it allows the bacterium to escape from cells deplete of nutrients and subsequently to invade cells with an intact supply of nutrients necessary for its continuous multiplication.

Place, publisher, year, edition, pages
Umeå: Klinisk bakteriologi , 2004. , 89 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 899
Research subject
Clinical Bacteriology
Identifiers
URN: urn:nbn:se:umu:diva-295ISBN: 91-7305-671-5 (print)OAI: oai:DiVA.org:umu-295DiVA: diva2:142975
Public defence
2004-05-27, Sal E 04, By 6 E, Norrlands Universitetssjukhus, 13:00
Opponent
Available from: 2004-06-23 Created: 2004-06-23 Last updated: 2010-08-03Bibliographically approved
List of papers
1. Fancisella tularensis induces cytopathogenicity and apaptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication
Open this publication in new window or tab >>Fancisella tularensis induces cytopathogenicity and apaptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication
2001 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 69, no 7, 4691-4694 p.Article in journal (Refereed) Published
Abstract [en]

The murine macrophage-like cell line J774.A1 ingests and allows intracellular growth of Francisella tularensis. We demonstrate that, after 24 h of infection, a pronounced cytopathogenicity resulted and the J774 cells were undergoing apoptosis. Despite this host cell apoptosis, no decrease in bacterial numbers was observed. When internalization of bacteria was prevented or intracellularly located F. tularensis bacteria were eradicated within 12 h, the progression of host cell cytopathogenicity and apoptosis was prevented.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4023 (URN)10.1128/IAI.69.7.4691-4694.2001 (DOI)11402018 (PubMedID)
Available from: 2004-06-23 Created: 2004-06-23 Last updated: 2017-12-14Bibliographically approved
2. Delineation of the molecular mechanisms of Francisella tularensis-induced apoptosis in murine macrophages
Open this publication in new window or tab >>Delineation of the molecular mechanisms of Francisella tularensis-induced apoptosis in murine macrophages
2003 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 71, no 8, 4642-4646 p.Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. Here we analyzed the pathway leading to apoptosis in the murine macrophage-like cell line J774A.1 after infection with F. tularensis strain LVS (named LVS for live vaccine strain). We obtained evidence that the infection affected the mitochondria of the macrophages, since it induced release of the mitochondrial molecule cytochrome c into the cytosol and changed the potential over the mitochondrial membrane. Moreover, activation of caspase 9 and the executioner caspase 3 was also observed in the LVS-infected J774A.1 macrophages. The activated caspase 3 degraded poly(ADP-ribose) polymerase (PARP). All of these events were observed within 9 to 12 h after the initiation of infection, and maximum degradation of a synthetic caspase 3 substrate occurred at 18 h. The internucleosomal fragmentation and PARP degradation resulting from activation of this apoptotic pathway was prevented by the caspase 3 inhibitor Z-DEVD-fmk. No involvement of caspase 1, caspase 8, Bcl-2, or Bid was observed. Thus, the F. tularensis infection induces macrophage apoptosis through a pathway partly resembling the intrinsic apoptotic pathway.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4024 (URN)10.1128/IAI.71.8.4642-4646.2003 (DOI)12874344 (PubMedID)
Available from: 2004-06-23 Created: 2004-06-23 Last updated: 2017-12-14Bibliographically approved
3. Expression of iglC is mecessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis
Open this publication in new window or tab >>Expression of iglC is mecessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis
2004 (English)In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 37, no 5, 225-230 p.Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. In a previous study, an iglC null mutant of F. tularensis live vaccine strain LVS was generated by allelic replacement and in the current study this iglC mutant was successfully complemented in trans. We characterized the capacity of this iglC mutant and the complemented strain to induce macrophage apoptosis. The iglC mutant did not induce apoptosis in the infected cells. In contrast, the complemented iglC strain was able to multiply in the murine macrophage-like cell line J774A.1 and induced apoptosis similar to that of the wild-type strain. It is the first successful example of complementation in trans of a F. tularensis mutant strain and more importantly this work provides direct evidence that the intracellular growth ability is essential for F. tularensis to induce macrophage apoptosis.

Place, publisher, year, edition, pages
London: Academic P., 2004
Keyword
Intracellular bacterial growth, Francisella, Macrophage apoptosis, Intracellular growth locus C (iglC), Allelic replacement, Trans-complementation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4025 (URN)10.1016/j.micpath.2004.07.002 (DOI)15519043 (PubMedID)
Available from: 2004-06-23 Created: 2004-06-23 Last updated: 2017-12-14Bibliographically approved
4. Infection with Francisella novicida leads to killing of murine macrophages by necrosis
Open this publication in new window or tab >>Infection with Francisella novicida leads to killing of murine macrophages by necrosis
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4026 (URN)
Available from: 2004-06-23 Created: 2004-06-23 Last updated: 2012-06-15Bibliographically approved
5. Francisella strains express hemolysins of distinct characteristics
Open this publication in new window or tab >>Francisella strains express hemolysins of distinct characteristics
2003 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 224, no 1, 91-95 p.Article in journal (Refereed) Published
Abstract [en]

Historically, Francisella strains have been described as nonhemolytic. In this study, we show by use of solid and liquid hemolysis assays that some Francisella strains have hemolytic properties. The Francisella novicida type strain U112 is hemolytic to horse erythrocytes and Francisella philomiragia type strain FSC144 is hemolytic towards both human and horse erythrocytes. The F. novicida strain U112 released a protein (novilysin A) into the culture supernatant which cross-reacted with antiserum against Escherichia coli HlyA whereas there was no similar protein detectable with this cross-reactive property from the supernatant of the F. philomiragia strain.

Keyword
Hemolysin, Novilysin A, Virulence factor, Growth-phase dependence, Francisella
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-4027 (URN)10.1016/S0378-1097(03)00431-2 (DOI)12855173 (PubMedID)
Available from: 2004-06-23 Created: 2004-06-23 Last updated: 2017-12-14Bibliographically approved

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