<i>Francisella tularensis</i>Invasion of Lung Epithelial Cells

Craven, Robin R.; Hall, Joshua D.; Fuller, James R.; Taft-Benz, Sharon; Kawula, Thomas H.

doi:10.1128/iai.00043-08

Cited by 54 publications

(73 citation statements)

References 57 publications

(73 reference statements)

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“…It is established that Francisella bacteria replicate in the cytosol of primary macrophages and macrophagelike cell lines (18,56,66), and recent data indicate that these organisms also parasitize primary alveolar epithelial cells and epithelial cell lines (21,35,46,57,61). In this regard, it is noteworthy that disruption of pyrB in SchuS4 slows bacterial growth in HepG2 epithelial cells (63).…”

Section: Identification Of Lvs Tn5 Mutants That No Longer Prevent Neumentioning

confidence: 99%

Francisella tularensisGenes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS

Schulert

McCaffrey

Buchan³

et al. 2009

Infect Immun

111

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Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. We have shown that F. tularensis subspecies holarctica strain LVS prevents NADPH oxidase assembly and activation in human neutrophils, but how this is achieved is unclear. Herein, we used random transposon mutagenesis to identify LVS genes that affect neutrophil activation. Our initial screen identified carA, carB, and pyrB, which encode the small and large subunits of carbamoylphosphate synthase and aspartate carbamoyl transferase, respectively. These strains are uracil auxotrophs, and their growth was attenuated on cysteine heart agar augmented with sheep blood (CHAB) or in modified Mueller-Hinton broth. Phagocytosis of the uracil auxotrophic mutants triggered a respiratory burst in neutrophils, and ingested bacteria were killed and fragmented in phagosomes that contained superoxide. Conversely, phagocytosis did not trigger a respiratory burst in blood monocytes or monocyte-derived macrophages (MDM), and phagosomes containing wild-type or mutant bacteria lacked NADPH oxidase subunits. Nevertheless, the viability of mutant bacteria declined in MDM, and ultrastructural analysis revealed that phagosome egress was significantly inhibited despite synthesis of the virulence factor IglC. Other aspects of infection, such as interleukin-1␤ (IL-1␤) and IL-8 secretion, were unaffected. The cultivation of carA, carB, or pyrB on uracil-supplemented CHAB was sufficient to prevent neutrophil activation and intramacrophage killing and supported escape from MDM phagosomes, but intracellular growth was not restored unless uracil was added to the tissue culture medium. Finally, all mutants tested grew normally in both HepG2 and J774A.1 cells. Collectively, our data demonstrate that uracil auxotrophy has cell type-specific effects on the fate of Francisella bacteria.

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Section: Identification Of Lvs Tn5 Mutants That No Longer Prevent Neumentioning

confidence: 99%

Francisella tularensisGenes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS

Schulert

McCaffrey

Buchan³

et al. 2009

Infect Immun

111

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“…Intracellular bacteria accessible to cytoplasmically delivered antibodies were enumerated by the methods described previously (15). The F. tularensis LVS ⌬ripA mutant's accessibility to the cytoplasm was compared to that of the wild type at 20 min, 60 min, and 180 min postinvasion into J774A.1 cells in triplicate assays per time point.…”

Section: Methodsmentioning

confidence: 99%

RipA, a Cytoplasmic Membrane Protein Conserved amongFrancisellaSpecies, Is Required for Intracellular Survival

et al. 2008

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Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS ⌬ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS ⌬ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS ⌬ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.

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“…Phagocytes, particularly macrophages, are thought to be an important replicative niche for F. tularensis in vivo. F. tularensis is detectable within host macrophages and dendritic cells within 1 h after infection (4,14,25). The proinflammatory response within these cells is blocked by F. tularensis, which prevents the production of cytokines and chemokines (4,6,59).…”

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confidence: 99%

A Francisella tularensis Locus Required for Spermine Responsiveness Is Necessary for Virulence

et al. 2011

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Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.

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Francisella tularensisInvasion of Lung Epithelial Cells

Cited by 54 publications

References 57 publications

Francisella tularensisGenes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS

Francisella tularensisGenes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS

RipA, a Cytoplasmic Membrane Protein Conserved amongFrancisellaSpecies, Is Required for Intracellular Survival

A Francisella tularensis Locus Required for Spermine Responsiveness Is Necessary for Virulence

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