scholarly journals Restricted cytosolic growth of Francisella tularensis subsp. tularensis by IFN-γ activation of macrophages

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 327-339 ◽  
Author(s):  
Jessica A. Edwards ◽  
Dedeke Rockx-Brouwer ◽  
Vinod Nair ◽  
Jean Celli
Keyword(s):  
Francisella Tularensis ◽  
Murine Bone Marrow ◽  
Bacterial Proliferation ◽  
Phagocyte Nadph Oxidase ◽  
Human Blood Monocyte ◽  
Oxide Synthase ◽  
Schu S4 ◽  
Intracellular Proliferation

The intracellular bacterium Francisella tularensis ensures its survival and proliferation within phagocytes of the infected host through phagosomal escape and cytosolic replication, to cause the disease tularemia. The cytokine interferon-γ (IFN-γ) is important in controlling primary infections in vivo, and in vitro intracellular proliferation of Francisella in macrophages, but its actual effects on the intracellular cycle of the bacterium are ambiguous. Here, we have performed an extensive analysis of the intracellular fate of the virulent F. tularensis subsp. tularensis strain Schu S4 in primary IFN-γ-activated murine and human macrophages to understand how this cytokine controls Francisella proliferation. In both murine bone marrow-derived macrophages (muBMMs) and human blood monocyte-derived macrophages (MDMs), IFN-γ controlled bacterial proliferation. Schu S4 growth inhibition was not due to a defect in phagosomal escape, since bacteria disrupted their phagosomes with indistinguishable kinetics in both muBMMs and MDMs, regardless of their activation state. Rather, IFN-γ activation restricted cytosolic replication of Schu S4 in a manner independent of reactive oxygen or nitrogen species. Hence, IFN-γ induces phagocyte NADPH oxidase Phox- and inducible nitric oxide synthase (iNOS)-independent cytosolic effector mechanisms that restrict growth of virulent Francisella in macrophages.

scholarly journals Phagocytic Receptors Dictate Phagosomal Escape and Intracellular Proliferation of Francisella tularensis

2011 ◽  
Vol 79 (6) ◽  
pp. 2204-2214 ◽  
Author(s):  
Henriette Geier ◽  
Jean Celli
Keyword(s):  
Francisella Tularensis ◽  
Scavenger Receptor ◽  
Mannose Receptor ◽  
Dependent Manner ◽  
Content Type ◽  
Murine Bone Marrow ◽  
Delayed Maturation ◽  
Schu S4 ◽  
Intracellular Proliferation

ABSTRACTFrancisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Variousin vitromodels ofFrancisella-macrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent ofFrancisellaphagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strainFrancisella tularensissubsp.tularensisSchu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the complement receptor CR3, the scavenger receptor A (SRA), and the Fcγ receptor (FcγR), respectively. CR3-mediated uptake delayed maturation of the earlyFrancisella-containing phagosome (FCP) and restricted phagosomal escape, while FcγR-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate ofFrancisellaby delivering bacteria through phagocytic pathways that restrict phagosomal escape and intracellular proliferation.

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

2011 ◽  
Vol 79 (9) ◽  
pp. 3665-3676 ◽  
Author(s):  
Brian C. Russo ◽  
Joseph Horzempa ◽  
Dawn M. O'Dee ◽  
Deanna M. Schmitt ◽  
Matthew J. Brown ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Febrile Illness ◽  
Host Cells ◽  
High Morbidity ◽  
Wild Type ◽  
Content Type ◽  
Infectious Dose ◽  
Schu S4

ABSTRACTTularemia is a debilitating febrile illness caused by the category A biodefense agentFrancisella 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 whichF. tularensissenses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions ofF. tularensiswith host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis ofF. tularensissubsp.holarcticalive 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 inF. tularensissubsp.tularensisSchu 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 macrophagesin 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 attenuatedin 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 anF. tularensisgene important for virulence and evasion of the host immune response.

scholarly journals A Francisella tularensis SCHU S4 mutant deficient in γ-glutamyltransferase activity induces protective immunity: characterization of an attenuated vaccine candidate

Microbiology ◽  
2011 ◽  
Vol 157 (11) ◽  
pp. 3172-3179 ◽  
Author(s):  
Philip M. Ireland ◽  
Helen LeButt ◽  
Rebecca M. Thomas ◽  
Petra C. F. Oyston
Keyword(s):  
Mutant Strain ◽  
Francisella Tularensis ◽  
Protective Immunity ◽  
Attenuated Vaccine ◽  
Schu S4 ◽  
Glutamyl Transpeptidase

Francisella tularensis is an intracellular pathogen which causes tularaemia. There is no licensed vaccine currently available for prophylaxis. The γ-glutamyl transpeptidase (GGT) encoded by the ggt gene has been shown to be important for the intracellular survival of F. tularensis. In this study we have constructed a ggt deletion mutant in the highly virulent F. tularensis strain SCHU S4. Characterization of the mutant strain confirmed the function of ggt, and confirmed the role of GGT in cysteine acquisition. The mutant strain was highly attenuated both in vitro and in vivo using murine models of infection. Moreover, we have demonstrated that the attenuated mutant is able to induce protective immunity against an F. tularensis SCHU S4 challenge, and thus may be a candidate for the development of an attenuated vaccine.

scholarly journals Resistance of Francisella tularensis Strains against Reactive Nitrogen and Oxygen Species with Special Reference to the Role of KatG

2007 ◽  
Vol 75 (3) ◽  
pp. 1303-1309 ◽  
Author(s):  
Helena Lindgren ◽  
Hua Shen ◽  
Carl Zingmark ◽  
Igor Golovliov ◽  
Wayne Conlan ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Bacterial Pathogen ◽  
Free System ◽  
Virulent Strains ◽  
A Cell ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Schu S4

ABSTRACT Francisella tularensis is a facultative intracellular bacterial pathogen capable of proliferating within host macrophages. The mechanisms that explain the differences in virulence between various strains of the species are not well characterized. In the present study, we show that both attenuated (strain LVS) and virulent (strains FSC200 and SCHU S4) strains of the pathogen replicate at similar rates in resting murine peritoneal exudate cells (PEC). However, when PEC were activated by exposure to gamma interferon (IFN-γ), they killed LVS more rapidly than virulent strains of the pathogen. Addition of N G -monomethyl-l-arginine, an inhibitor of inducible nitric oxide synthase, to IFN-γ-treated PEC, completely inhibited killing of the virulent strains, whereas it only partially blocked the killing of LVS. Similarly, in a cell-free system, SCHU S4 and FSC200 were more resistant to killing by H2O2 and ONOO− than F. tularensis LVS. Catalase encoded by katG is a bacterial factor that can detoxify bactericidal compounds such as H2O2 and ONOO−. To investigate its contribution to the virulence of F. tularensis, katG deletion-containing mutants of SCHU S4 and LVS were generated. Both mutants demonstrated enhanced susceptibility to H2O2 in vitro but replicated as effectively as the parental strains in unstimulated PEC. In mice, LVS-ΔkatG was significantly attenuated compared to LVS whereas SCHU S4-ΔkatG, despite slower replication, killed mice as quickly as SCHU S4. This implies that clinical strains of the pathogen have katG-independent mechanisms to combat the antimicrobial effects exerted by H2O2 and ONOO−, the loss of which could have contributed to the attenuation of LVS.

scholarly journals Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

2015 ◽  
Vol 60 (1) ◽  
pp. 288-295 ◽  
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.

scholarly journals FTT0831c/FTL_0325 Contributes to Francisella tularensis Cell Division, Maintenance of Cell Shape, and Structural Integrity

2014 ◽  
Vol 82 (7) ◽  
pp. 2935-2948 ◽  
Author(s):  
Gregory T. Robertson ◽  
Elizabeth Di Russo Case ◽  
Nicole Dobbs ◽  
Christine Ingle ◽  
Murat Balaban ◽  
...  
Keyword(s):  
Cell Wall ◽  
Francisella Tularensis ◽  
Structural Integrity ◽  
Content Type ◽  
Mueller Hinton ◽  
Host Innate Immune Response ◽  
Altered Surface ◽  
Schu S4

ABSTRACTTheFrancisellaFTT0831c/FTL_0325 gene encodes amino acid motifs to suggest it is a lipoprotein and that it may interact with the bacterial cell wall as a member of the OmpA-like protein family. Previous studies have suggested that FTT0831c is surface exposed and required for virulence ofFrancisella tularensisby subverting the host innate immune response (M. Mahawar et al., J. Biol. Chem. 287:25216–25229, 2012). We also found that FTT0831c is required for murine pathogenesis and intramacrophage growth of Schu S4, but we propose a different model to account for the proinflammatory nature of the resultant mutants. First, inactivation of FTL_0325 from live vaccine strain (LVS) or FTT0831c from Schu S4 resulted in temperature-dependent defects in cell viability and morphology. Loss of FTT0831c was also associated with an unusual defect in lipopolysaccharide O-antigen synthesis, but loss of FTL_0325 was not. Full restoration of these properties was observed in complemented strains expressing FTT0831cin trans, but not in strains lacking the OmpA motif, suggesting that cell wall contact is required. Finally, growth of the LVS FTL_0325 mutant in Mueller-Hinton broth at 37°C resulted in the appearance of membrane blebs at the poles and midpoint, prior to the formation of enlarged round cells that showed evidence of compromised cellular membranes. Taken together, these data are more consistent with the known structural role of OmpA-like proteins in linking the OM to the cell wall and, as such, maintenance of structural integrity preventing altered surface exposure or release of Toll-like receptor 2 agonists during rapid growth ofFrancisellain vitroandin vivo.

scholarly journals Effects of chitosan on nitric oxide production and inducible nitric oxide synthase activity and mRNA expression in weaned piglets

2018 ◽  
Vol 60 (No. 8) ◽  
pp. 359-366
Author(s):  
J. Li ◽  
B. Shi ◽  
S. Yan ◽  
L. Jin ◽  
Y. Guo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mrna Expression ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Weaned Piglets ◽  
Inos Activity ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The effects of chitosan on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity and gene expression in vivo or vitro were investigated in weaned piglets. In vivo, 180 weaned piglets were assigned to five dietary treatments with six replicates. The piglets were fed on a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. In vitro, the peripheral blood mononuclear cells (PBMCs) from a weaned piglet were cultured respectively with 0 (control), 40, 80, 160, and 320 µg chitosan/ml medium. Results showed that serum NO concentrations on days 14 and 28 and iNOS activity on day 28 were quadratically improved with increasing chitosan dose (P < 0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the duodenum on day 28, and were improved quadratically in the jejunum on days 14 and 28 and in the ileum on day 28 (P < 0.01). In vitro, the NO concentrations, iNOS activity, and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentrations and iNOS activity by chitosan were markedly inhibited by N-(3-[aminomethyl] benzyl) acetamidine (1400w) (P < 0.05). Moreover, the increase of NO concentrations, iNOS activity, and mRNA expression in PBMCs induced by lipopolysaccharide (LPS) were suppressed significantly by chitosan (P < 0.05). The results indicated that the NO concentrations, iNOS activity, and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which exerted bidirectional regulatory effects on the NO production via modulated iNOS activity and mRNA expression.

scholarly journals Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge

2016 ◽  
Vol 60 (4) ◽  
pp. 2052-2062 ◽  
Author(s):  
Ky V. Hoang ◽  
Heather Curry ◽  
Michael A. Collier ◽  
Hassan Borteh ◽  
Eric M. Bachelder ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Lethal Challenge ◽  
Content Type ◽  
Human Macrophages ◽  
Gentamicin Treatment ◽  
Significant Toxicity ◽  
Serovar Typhimurium ◽  
Spectrum Activity

ABSTRACTFrancisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis. We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo. No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

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