HU protein is involved in intracellular growth and full virulence of Francisella tularensis

ABSTRACT Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53). In this study, we investigated the contribution of IclR to the intracellular growth, virulence, and gene regulation of human pathogenic F. tularensis subspecies. Deletion of iclR from the live vaccine strain (LVS) and SchuS4 strain of F. tularensis subsp. holarctica and F. tularensis subsp. tularensis, respectively, did not affect their abilities to replicate within macrophages or epithelial cells. In contrast to F. tularensis subsp. novicida iclR mutants, LVS and SchuS4 ΔiclR strains were as virulent as their wild-type parental strains in intranasal inoculation mouse models of tularemia. Furthermore, wild-type LVS and LVSΔiclR were equally cytotoxic and induced equivalent levels of interleukin-1β expression by infected bone marrow-derived macrophages. Microarray analysis revealed that the relative expression of a limited number of genes differed significantly between LVS wild-type and ΔiclR strains. Interestingly, many of the identified genes were disrupted in LVS and SchuS4 but not in their corresponding F. tularensis subsp. novicida U112 homologs. Thus, despite the impact of iclR deletion on gene expression, and in contrast to the effects of iclR deletion on F. tularensis subsp. novicida virulence, IclR does not contribute significantly to the virulence or pathogenesis of F. tularensis LVS or SchuS4.

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In Vivo Clearance of an Intracellular Bacterium, Francisella tularensis LVS, Is Dependent on the p40 Subunit of Interleukin-12 (IL-12) but Not on IL-12 p70

Infection and Immunity ◽

10.1128/iai.70.4.1936-1948.2002 ◽

2002 ◽

Vol 70 (4) ◽

pp. 1936-1948 ◽

Cited By ~ 95

Author(s):

Karen L. Elkins ◽

Allison Cooper ◽

Susan M. Colombini ◽

Siobhán C. Cowley ◽

Tara L. Kieffer

Keyword(s):

T Cells ◽

Francisella Tularensis ◽

Intracellular Bacterium ◽

Interleukin 12 ◽

Intracellular Growth ◽

Lethal Challenge ◽

Immune Lymphocytes ◽

Ifn Γ

ABSTRACT To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-γ), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4+ and CD8+ T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-γ than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-γ production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.

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The capBCA Locus is Required for Intracellular Growth of Francisella tularensis LVS

Frontiers in Microbiology ◽

10.3389/fmicb.2011.00083 ◽

2011 ◽

Vol 2 ◽

Cited By ~ 10

Author(s):

Jingliang Su ◽

Rexford Asare ◽

Jun Yang ◽

Manoj Kumar Mohan Nair ◽

Joseph E. Mazurkiewicz ◽

...

Keyword(s):

Francisella Tularensis ◽

Intracellular Growth

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Virulence of Francisella tularensis Subspecies holarctica Biovar japonica and Phenotypic Change during Serial Passages on Artificial Media

Microorganisms ◽

10.3390/microorganisms8121881 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1881

Author(s):

Akitoyo Hotta ◽

Neekun Sharma ◽

Osamu Fujita ◽

Akihiko Uda ◽

Kiyoshi Tanabayashi ◽

...

Keyword(s):

Francisella Tularensis ◽

Defined Medium ◽

Biological Properties ◽

Phenotypic Change ◽

Chemically Defined Medium ◽

Intracellular Growth ◽

Phenotypic Stability ◽

Phenotypic Differences ◽

Time To Death ◽

Colony Forming Units

Francisella tularensis (F. tularensis) is the etiological agent of the zoonotic disease tularemia. F. tularensis subspecies holarctica biovar japonica has rarely been isolated in Japan and is considered to have moderate virulence, although the biological properties of fresh isolates have not been analyzed in detail. Here, we analyzed the virulence of two strains of F. tularensis subspecies holarctica biovar japonica (NVF1 and KU-1) and their phenotypic stability during serial passages in Eugon chocolate agar (ECA) and Chamberlain’s chemically defined medium (CDM) based agar (CDMA). C57BL/6 mice intradermally inoculated with 101 colony-forming units of NVF1 or KU-1 died within 9 days, with a median time to death of 7.5 and 7 days, respectively. Both NVF1 and KU-1 strains passaged on ECA 10 times had comparable virulence prior to passaging, whereas strains passaged on ECA 20 times and on CDMA 50 times were attenuated. Attenuated strains had decreased viability in 0.01% H2O2 and lower intracellular growth rates, suggesting both properties are important for F. tularensis virulence. Additionally, passage on ECA of the KU-1 strains altered lipopolysaccharide antigenicity and bacterial susceptibility to β-lactam antibiotics. Our data demonstrate F. tularensis strain virulence in Japan and contribute to understanding phenotypic differences between natural and laboratory environments.

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Identifying Francisella tularensis Genes Required for Growth in Host Cells

Infection and Immunity ◽

10.1128/iai.00004-15 ◽

2015 ◽

Vol 83 (8) ◽

pp. 3015-3025 ◽

Cited By ~ 8

Author(s):

J. Brunton ◽

S. Steele ◽

C. Miller ◽

E. Lovullo ◽

S. Taft-Benz ◽

...

Keyword(s):

Epithelial Cell ◽

Cell Lines ◽

Francisella Tularensis ◽

Host Cells ◽

Hypertonic Solution ◽

Hypotonic Solution ◽

Intracellular Growth ◽

Content Type ◽

Epithelial Cell Lines ◽

Schu S4

Francisella tularensisis a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark ofF. tularensisvirulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes.FTT_0924, a gene of unknown function but highly conserved amongFrancisellaspecies, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines.FTT_0924was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating thatFTT_0924is required for resisting osmotic stress. The ΔFTT_0924mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest thatFTT_0924is required for maintaining peptidoglycan integrity and virulence.

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Faculty Opinions recommendation of Francisella tularensis harvests nutrients derived via ATG5-independent autophagy to support intracellular growth.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718083115.793484221 ◽

2013 ◽

Author(s):

Olivia Steele-Mortimer ◽

Jose Antonio Ibarra

Keyword(s):

Francisella Tularensis ◽

Intracellular Growth

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Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01240-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 288-295 ◽

Cited By ~ 4

Author(s):

Helena Lindgren ◽

Anders Sjöstedt

Keyword(s):

Francisella Tularensis ◽

Iron Uptake ◽

Combined Treatment ◽

Bactericidal Effect ◽

Intracellular Growth ◽

Content Type ◽

Intimate Link ◽

Schu S4

ABSTRACTThe reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacteriumFrancisella tularensisstrain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslAmutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoBmutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment ofF. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicinin vitroandin vivo.

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