scholarly journals Disruption of Francisella tularensis Schu S4iglI,iglJ, andpdpCGenes Results in Attenuation for Growth in Human Macrophages andIn VivoVirulence in Mice and Reveals a Unique Phenotype forpdpC

2012 ◽  
Vol 81 (3) ◽  
pp. 850-861 ◽  
Author(s):  
Matthew E. Long ◽  
Stephen R. Lindemann ◽  
Jed A. Rasmussen ◽  
Bradley D. Jones ◽  
Lee-Ann H. Allen
Keyword(s):  
Francisella Tularensis ◽  
Bacterial Pathogen ◽  
Human Monocyte ◽  
Pathogenicity Island ◽  
Intermediate Phenotype ◽  
Small Subset ◽  
Content Type ◽  
Isolation And Characterization ◽  
Schu S4

ABSTRACTFrancisella tularensisis a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization ofFrancisella tularensissubsp.tularensisstrain Schu S4 mutants that lack functionaliglI,iglJ, orpdpC, three genes of theFrancisellapathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. TheiglIandiglJmutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, thepdpCmutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed bytranscomplementation.In vivovirulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 108CFUiglJ-orpdpC-deficient bacteria. Nevertheless, thepdpCmutant disseminated to the liver and spleen before being eliminated, whereas theiglJmutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential forF. tularensisSchu S4 virulence and further suggest thatpdpCmay play a unique role in this process, as indicated by its distinct intermediate phenotype.

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 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 Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4

2014 ◽  
Vol 82 (9) ◽  
pp. 3622-3635 ◽  
Author(s):  
Marie Lindgren ◽  
Linda Tancred ◽  
Igor Golovliov ◽  
Wayne Conlan ◽  
Susan M. Twine ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Virulent Strain ◽  
Small Subset ◽  
Intracellular Replication ◽  
Content Type ◽  
Infected Cells ◽  
Essential Contribution ◽  
Respiratory Challenge ◽  
Schu S4 ◽  
Highly Virulent

ABSTRACTPreviously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 ofFrancisella tularensissubsp.tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that thepdpEgene and most of thepdpCgene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fusedfupAandfupBgenes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the ΔfupAmutant were <10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the ΔpdpCand ΔpdpCΔpdpEmutants. Complementation of FSC043 with the intactfupAandfupBgenes did not affect the phenotype, whereas complementation with thepdpCandpdpEgenes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in thepdpCgene makes an essential contribution to the phenotype.

scholarly journals Targeting the “Rising DAMP” during a Francisella tularensis Infection

2013 ◽  
Vol 57 (9) ◽  
pp. 4222-4228 ◽  
Author(s):  
Riccardo V. D'Elia ◽  
Thomas R. Laws ◽  
Alun Carter ◽  
Roman Lukaszewski ◽  
Graeme C. Clark
Keyword(s):  
Francisella Tularensis ◽  
Time Course ◽  
Bacterial Pathogen ◽  
Potential Therapeutic Target ◽  
Content Type ◽  
Molecular Pattern ◽  
Significant Survival ◽  
Time Course Experiment ◽  
Schu S4 ◽  
Antibiotic Efficacy

ABSTRACTAntibiotic efficacy is greatly enhanced the earlier it is administered following infection with a bacterial pathogen. However, in a clinical setting antibiotic treatment usually commences following the onset of symptoms, which in some cases (e.g., biothreat agents) may be too late. In a BALB/c murine intranasal model of infection forFrancisella tularensisSCHU S4 infection, we demonstrate during a time course experiment that proinflammatory cytokines and the damage-associated molecular pattern HMGB1 were not significantly elevated above naive levels in tissue or sera until 72 h postinfection. HMGB1 was identified as a potential therapeutic target that could extend the window of opportunity for the treatment of tularemia with antibiotics. Antibodies to HMGB1 were administered in conjunction with a delayed/suboptimal levofloxacin treatment ofF. tularensis. We found in the intranasal model of infection that treatment with anti-HMGB1 antibody, compared to an isotype IgY control antibody, conferred a significant survival benefit and decreased bacterial loads in the spleen and liver but not the lung (primary loci of infection) 4 days into infection. We also observed an increase in the production of gamma interferon in all tested organs. These data demonstrate that treatment with anti-HMGB1 antibody is beneficial in enhancing the effectiveness of current antibiotics in treating tularemia. Strategies of this type, involving antibiotics in combination with immunomodulatory drugs, are likely to be essential for the development of a postexposure therapeutic for intracellular pathogens.

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 The Francisella tularensis migR,trmE, andcphAGenes Contribute to F. tularensis Pathogenicity Island Gene Regulation and Intracellular Growth by Modulation of the Stress Alarmone ppGpp

2013 ◽  
Vol 81 (8) ◽  
pp. 2800-2811 ◽  
Author(s):  
Matthew Faron ◽  
Joshua R. Fletcher ◽  
Jed A. Rasmussen ◽  
Matthew E. Long ◽  
Lee-Ann H. Allen ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Mammalian Cells ◽  
Bacterial Pathogen ◽  
Pathogenicity Island ◽  
Cellular Stress Response ◽  
Intracellular Growth ◽  
Content Type ◽  
New Information ◽  
Cellular Stresses ◽  
Transcriptional Complex

ABSTRACTTheFrancisella tularensispathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within themigR,trmE, andcphAgenes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions inFrancisella. Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations inmigR,trmE, orcphAsignificantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction iniglAreporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that themigR,trmE, andcphAgenes are likely parts of different cellular stress response pathways inFrancisella. These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand howFrancisellaregulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen.

scholarly journals Complement C3 as a Prompt for Human Macrophage Death during Infection with Francisella tularensis Strain SCHU S4

2017 ◽  
Vol 85 (10) ◽  
Author(s):  
Susan R. Brock ◽  
Michael J. Parmely
Keyword(s):  
Cell Death ◽  
Francisella Tularensis ◽  
Single Cell Analysis ◽  
Bacterial Pathogen ◽  
Complement C3 ◽  
Human Macrophage ◽  
Bacterial Cells ◽  
Content Type ◽  
Progression Of Disease ◽  
Schu S4

ABSTRACT Tularemia is caused by the Gram-negative bacterial pathogen Francisella tularensis. Infection of macrophages and their subsequent death are believed to play important roles in the progression of disease. Because complement is a particularly effective opsonin for Francisella, we asked whether complement-dependent uptake of F. tularensis strain SCHU S4 affects the survival of primary human macrophages during infection. Complement component C3 was found to be an essential opsonin in human serum not only for greatly increased uptake of SCHU S4 but also for the induction of macrophage death. Single-cell analysis also revealed that macrophage death did not require a high intracellular bacterial burden. In the presence of C3, macrophage death was observed at 24 h postinfection in a quarter of the macrophages that contained only 1 to 5 bacterial cells. Macrophages infected in the absence of C3 rarely underwent cell death, even when they contained large numbers of bacteria. The need for C3, but not extensive replication of the pathogen, was confirmed by infections with SCHU S4 ΔpurMCD, a mutant capable of phagosome escape but of only limited cytosolic replication. C3-dependent Francisella uptake alone was insufficient to induce macrophage death, as evidenced by the failure of the phagosome escape-deficient mutant SCHU S4 ΔfevR to induce cell death despite opsonization with C3. Together, these findings indicate that recognition of C3-opsonized F. tularensis, but not extensive cytosolic replication, plays an important role in regulating macrophage viability during intracellular infections with type A F. tularensis.

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.

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.

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