scholarly journals Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: Effects of host background and route of immunization

Vaccine ◽  
2010 ◽  
Vol 28 (7) ◽  
pp. 1824-1831 ◽  
Author(s):  
J. Wayne Conlan ◽  
Hua Shen ◽  
Igor Golovliov ◽  
Carl Zingmark ◽  
Petra C.F. Oyston ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Deletion Mutants ◽  
Targeted Deletion ◽  
Differential Ability ◽  
Schu S4

scholarly journals Genome-Wide Identification of Francisella tularensis Virulence Determinants

2007 ◽  
Vol 75 (6) ◽  
pp. 3089-3101 ◽  
Author(s):  
Jingliang Su ◽  
Jun Yang ◽  
Daimin Zhao ◽  
Thomas H. Kawula ◽  
Jeffrey A. Banas ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Stress Responses ◽  
Small Subset ◽  
Virulence Determinants ◽  
Genome Wide ◽  
Genes Encoding ◽  
Capsule Locus ◽  
Life Threatening ◽  
Transposon Mutants ◽  
Schu S4

ABSTRACT Francisella tularensis is a gram-negative pathogen that causes life-threatening infections in humans and has potential for use as a biological weapon. The genetic basis of the F. tularensis virulence is poorly understood. This study screened a total of 3,936 transposon mutants of the live vaccine strain for infection in a mouse model of respiratory tularemia by signature-tagged mutagenesis. We identified 341 mutants attenuated for infection in the lungs. The transposon disruptions were mapped to 95 different genes, virtually all of which are also present in the genomes of other F. tularensis strains, including human pathogenic F. tularensis strain Schu S4. A small subset of these attenuated mutants carried insertions in the genes encoding previously known virulence factors, but the majority of the identified genes have not been previously linked to F. tularensis virulence. Among these are genes encoding putative membrane proteins, proteins associated with stress responses, metabolic proteins, transporter proteins, and proteins with unknown functions. Several attenuated mutants contained disruptions in a putative capsule locus which partially resembles the poly-γ-glutamate capsule biosynthesis locus of Bacillus anthracis, the anthrax agent. Deletional mutation analysis confirmed that this locus is essential for F. tularensis virulence.

scholarly journals Global Analysis of Genes Essential forFrancisella tularensisSchu S4 GrowthIn Vitroand for Fitness during Competitive Infection of Fischer 344 Rats

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Philip M. Ireland ◽  
Helen L. Bullifent ◽  
Nicola J. Senior ◽  
Stephanie J. Southern ◽  
Zheng Rong Yang ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Insertion Site ◽  
Therapeutic Drug ◽  
Content Type ◽  
Fischer 344 ◽  
A Genome ◽  
Fischer 344 Rat ◽  
Schu S4

ABSTRACTThe highly virulent intracellular pathogenFrancisella tularensisis a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis ofFrancisellavirulence in the Fischer 344 rat, we have constructed anF. tularensisSchu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growthin vitro. Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. Thisin vivoselection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-typeF. tularensisSchu S4 strain.IMPORTANCEThe intracellular bacterial pathogenFrancisella tularensiscauses a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches.F. tularensisis one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important forF. tularensisunderin vitroandin vivoconditions, providing candidates that can be evaluated for vaccine or antibacterial development.

scholarly journals Francisella tularensis ΔpyrF Mutants Show that Replication in Nonmacrophages Is Sufficient for Pathogenesis In Vivo

2010 ◽  
Vol 78 (6) ◽  
pp. 2607-2619 ◽  
Author(s):  
Joseph Horzempa ◽  
Dawn M. O'Dee ◽  
Robert M. Q. Shanks ◽  
Gerard J. Nau
Keyword(s):  
Francisella Tularensis ◽  
De Novo ◽  
Human Fibroblasts ◽  
Host Cells ◽  
Hek 293 ◽  
293 Cells ◽  
Animal Infection ◽  
Schu S4

ABSTRACT The pathogenesis of Francisella tularensis has been associated with this bacterium's ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized ΔpyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis ΔpyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis ΔpyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis ΔpyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis ΔpyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.

scholarly journals Monophosphoryl Lipid A Enhances Efficacy of a Francisella tularensis LVS-Catanionic Nanoparticle Subunit Vaccine against F. tularensis Schu S4 Challenge by Augmenting both Humoral and Cellular Immunity

2017 ◽  
Vol 24 (3) ◽  
Author(s):  
Katharina Richard ◽  
Barbara J. Mann ◽  
Aiping Qin ◽  
Eileen M. Barry ◽  
Robert K. Ernst ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Subunit Vaccine ◽  
Lipid A ◽  
Ex Vivo ◽  
The United States ◽  
Content Type ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid ◽  
Ifn Γ ◽  
Schu S4

ABSTRACT Francisella tularensis, a bacterial biothreat agent, has no approved vaccine in the United States. Previously, we showed that incorporating lysates from partially attenuated F. tularensis LVS or fully virulent F. tularensis Schu S4 strains into catanionic surfactant vesicle (V) nanoparticles (LVS-V and Schu S4-V, respectively) protected fully against F. tularensis LVS intraperitoneal (i.p.) challenge in mice. However, we achieved only partial protection against F. tularensis Schu S4 intranasal (i.n.) challenge, even when employing heterologous prime-boost immunization strategies. We now extend these findings to show that both LVS-V and Schu S4-V immunization (i.p./i.p.) elicited similarly high titers of anti-F. tularensis IgG and that the titers could be further increased by adding monophosphoryl lipid A (MPL), a nontoxic Toll-like receptor 4 (TLR4) adjuvant that is included in several U.S. FDA-approved vaccines. LVS-V+MPL immune sera also detected more F. tularensis antigens than LVS-V immune sera and, after passive transfer to naive mice, significantly delayed the time to death against F. tularensis Schu S4 subcutaneous (s.c.) but not i.n. challenge. Active immunization with LVS-V+MPL (i.p./i.p.) also increased the frequency of gamma interferon (IFN-γ)-secreting activated helper T cells, IFN-γ production, and the ability of splenocytes to control intramacrophage F. tularensis LVS replication ex vivo. Active LVS-V+MPL immunization via heterologous routes (i.p./i.n.) significantly elevated IgA and IgG levels in bronchoalveolar lavage fluid and significantly enhanced protection against i.n. F. tularensis Schu S4 challenge (to ∼60%). These data represent a significant step in the development of a subunit vaccine against the highly virulent type A strains.

scholarly journals Francisella tularensis Phagosomal Escape Does Not Require Acidification of the Phagosome

2009 ◽  
Vol 77 (5) ◽  
pp. 1757-1773 ◽  
Author(s):  
Daniel L. Clemens ◽  
Bai-Yu Lee ◽  
Marcus A. Horwitz
Keyword(s):  
Francisella Tularensis ◽  
Membrane Glycoproteins ◽  
Reporter System ◽  
Additional Time ◽  
Virulent Type ◽  
Secondary Lysosomes ◽  
Phagosomal Membrane ◽  
Schu S4 ◽  
The Impact

ABSTRACT Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by “F. tularensis subsp. novicida,” indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.

scholarly journals The 58-Kilodalton Major Virulence Factor of Francisella tularensis Is Required for Efficient Utilization of Iron

2009 ◽  
Vol 77 (10) ◽  
pp. 4429-4436 ◽  
Author(s):  
Helena Lindgren ◽  
Marie Honn ◽  
Igor Golovlev ◽  
Konstantin Kadzhaev ◽  
Wayne Conlan ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Iron Acquisition ◽  
Protein A ◽  
Intracellular Iron ◽  
Major Virulence Factor ◽  
Iron Pool ◽  
Isogenic Mutants ◽  
Schu S4 ◽  
Iron Concentrations

ABSTRACT We investigated the role of the 58-kDa FTT0918 protein in the iron metabolism of Francisella tularensis. The phenotypes of SCHU S4, a prototypic strain of F. tularensis subsp. tularensis, and the ΔFTT0918 and ΔfslA isogenic mutants were analyzed. The gene product missing in the ΔfslA mutant is responsible for synthesis of a siderophore. When grown in broth with various iron concentrations, the two deletion mutants generally reached lower maximal densities than SCHU S4. The ΔFTT0918 mutant, but not the ΔfslA mutant, upregulated the genes of the F. tularensis siderophore locus (fsl) operon even at high iron concentrations. A chrome azurol sulfonate plate assay confirmed siderophore production by all strains except the ΔfslA strain. In a cross-feeding experiment using medium devoid of free iron, SCHU S4 promoted growth of the ΔfslA strain but not of the ΔFTT0918 strain. The sensitivity of SCHU S4 and the ΔFTT0918 and ΔfslA strains to streptonigrin demonstrated that the ΔFTT0918 strain contained a smaller free intracellular iron pool and that the ΔfslA strain contained a larger one than SCHU S4. In contrast to the marked attenuation of the ΔFTT0918 strain, the ΔfslA strain was as virulent as SCHU S4 in a mouse model. Altogether, the data demonstrate that the FTT0918 protein is required for F. tularensis to utilize iron bound to siderophores and that it likely has a role also in siderophore-independent iron acquisition. We suggest that the FTT0918 protein be designated Fe utilization protein A, FupA.

Sign in / Sign up

Export Citation Format

Share Document