Effects of the Putative Transcriptional Regulator IclR on Francisella tularensis Pathogenesis

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.

Download Full-text

Presence of Pili on the Surface of Francisella tularensis

Infection and Immunity ◽

10.1128/iai.72.5.3042-3047.2004 ◽

2004 ◽

Vol 72 (5) ◽

pp. 3042-3047 ◽

Cited By ~ 46

Author(s):

Horacio Gil ◽

Jorge L. Benach ◽

David G. Thanassi

Keyword(s):

Electron Microscopy ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Live Vaccine ◽

Live Vaccine Strain ◽

Negative Bacterium ◽

Gram Negative ◽

Schu S4 ◽

Highly Virulent

ABSTRACT Francisella tularensis is a highly infectious gram-negative bacterium with potential for use as a bioweapon. Analysis of the F. tularensis live vaccine strain (LVS) ultrastructure by electron microscopy revealed the presence of long, thin fibers, similar in appearance to type 4 pili. The highly virulent F. tularensis Schu S4 strain was found to contain type 4 pilus genes, and we confirmed that these genes are present and expressed in the LVS.

Download Full-text

Intranasal Interleukin-12 Treatment for Protection against Respiratory Infection with the Francisella tularensis Live Vaccine Strain

Infection and Immunity ◽

10.1128/iai.73.4.2306-2311.2005 ◽

2005 ◽

Vol 73 (4) ◽

pp. 2306-2311 ◽

Cited By ~ 67

Author(s):

Nathalie S. Duckett ◽

Sofia Olmos ◽

Douglas M. Durrant ◽

Dennis W. Metzger

Keyword(s):

Respiratory Infection ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Live Vaccine ◽

Interleukin 12 ◽

Live Vaccine Strain ◽

Wild Type ◽

Intranasal Infection ◽

Ifn Γ

ABSTRACT Francisella tularensis is a gram-negative intracellular bacterium that can induce lethal respiratory infection in humans and rodents. However, little is known about the role of innate or adaptive immunity in protection from respiratory tularemia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with the live vaccine strain (LVS) of F. tularensis was investigated. It was found that gamma interferon (IFN-γ) and IL-12 were strictly required for protection, since mice deficient in IFN-γ, IL-12 p35, or IL-12 p40 all succumbed to LVS doses that were sublethal for wild-type mice. Furthermore, exogenous IL-12 treatment 24 h before intranasal infection with a lethal dose of LVS (10,000 CFU) significantly decreased bacterial loads in the lungs, livers, and spleens of wild-type BALB/c and C57BL/6 mice and allowed the animals to survive infection; such protection was not observed in IFN-γ-deficient mice. The resistance induced by IL-12 to LVS infection was still observed in NK cell-deficient beige mice but not in CD8−/− mice. These results demonstrate that exogenous IL-12 delivered intranasally can prevent respiratory tularemia through a mechanism that is at least partially dependent upon the expression of IFN-γ and CD8 T cells.

Download Full-text

Bypass of an epigenetic S-phase transcriptional module by convergent nutrient stress signals

10.1101/741363 ◽

2019 ◽

Author(s):

Marie Delaby ◽

Gaël Panis ◽

Coralie Fumeaux ◽

Laurence Degeorges ◽

Patrick H. Viollier

Keyword(s):

Cell Cycle ◽

Transcriptional Activation ◽

Caulobacter Crescentus ◽

Transcriptional Regulator ◽

S Phase ◽

Wild Type ◽

Intracellular Growth ◽

Obligate Intracellular ◽

Stress Signals ◽

Alpha Proteobacteria

AbstractThe signals feeding into bacterial S-phase transcription are poorly understood. Cellular cycling in the alpha-proteobacterium Caulobacter crescentus is driven by a complex circuit of at least three transcriptional modules that direct sequential promoter firing during the G1, early and late S cell cycle phases. In alpha-proteobacteria, the transcriptional regulator GcrA and the CcrM methyltransferase epigenetically activate promoters of cell division and polarity genes that fire in S-phase. By evolving Caulobacter crescentus cells to cycle and differentiate in the absence of the GcrA/CcrM module, we discovered that phosphate deprivation and (p)ppGpp alarmone stress signals converge on S-phase transcriptional activation. The cell cycle oscillations of the CtrA protein, the transcriptional regulator that implements G1 and late S-phase transcription, are essential in our evolved mutants, but not in wild-type cells, showing that the periodicity in CtrA abundance alone can sustain cellular cycling without GcrA/CcrM. While similar nutritional sensing occurs in other alpha-proteobacteria, GcrA and CcrM are not encoded in the reduced genomes of obligate intracellular relatives. We thus propose that the nutritional stress response induced during intracellular growth obviated the need for an S-phase transcriptional regulator.

Download Full-text

Francisella tularensis Live Vaccine Strain Folate Metabolism and Pseudouridine Synthase Gene Mutants Modulate Macrophage Caspase-1 Activation

Infection and Immunity ◽

10.1128/iai.00991-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 201-208 ◽

Cited By ~ 13

Author(s):

Tyler K. Ulland ◽

Ann M. Janowski ◽

Blake W. Buchan ◽

Matthew Faron ◽

Suzanne L. Cassel ◽

...

Keyword(s):

Francisella Tularensis ◽

Vaccine Strain ◽

Live Vaccine ◽

Live Vaccine Strain ◽

Wild Type ◽

Content Type ◽

Pseudouridine Synthase ◽

Caspase 1

Francisella tularensisis a Gram-negative bacterium and the causative agent of the disease tularemia. Escape ofF. tularensisfrom the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses toF. tularensis. We have identified the5-formyltetrahydrofolate cycloligasegene (FTL_0724) as being important forF. tularensislive vaccine strain (LVS) virulence. Infection of micein vivowith aF. tularensisLVSFTL_0724mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. TheFTL_0724mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophagesin vitro. In contrast, infection of macrophages with aF. tularensisLVSrluD pseudouridine synthase(FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophagesin vitrocompared to infection of macrophages with wild-type LVS. In addition, theFTL_0699mutant was not attenuatedin vivo. These findings further illustrate thatF. tularensisLVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogenin vivo.

Download Full-text

Susceptibility to Secondary Francisella tularensis Live Vaccine Strain Infection in B-Cell-Deficient Mice Is Associated with Neutrophilia but Not with Defects in Specific T-Cell-Mediated Immunity

Infection and Immunity ◽

10.1128/iai.69.1.194-203.2001 ◽

2001 ◽

Vol 69 (1) ◽

pp. 194-203 ◽

Cited By ~ 83

Author(s):

Catharine M. Bosio ◽

Karen L. Elkins

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Culture System ◽

Live Vaccine ◽

Cell Mediated Immunity ◽

Live Vaccine Strain ◽

Wild Type

ABSTRACT Previous studies have demonstrated a role for B cells, not associated with antibody production, in protection against lethal secondary infection of mice with Francisella tularensislive vaccine strain (LVS). However, the mechanism by which B cells contribute to this protection is not known. To study the specific role of B cells during secondary LVS infection, we developed an in vitro culture system that mimics many of the same characteristics of in vivo infection. Using this culture system, we showed that B cells do not directly control LVS infection but that control of LVS growth is mediated primarily by LVS-primed T cells. Importantly, B cells were not required for the generation of effective memory T cells since LVS-primed, B-cell-deficient (BKO) mice generated CD4+ and CD8+ T cells that controlled LVS infection similarly to LVS-primed CD4+ and CD8+ T cells from wild-type mice. The control of LVS growth appeared to depend primarily on gamma interferon and nitric oxide and was similar in wild-type and BKO mice. Rather, the inability of BKO mice to survive secondary LVS infection was associated with marked neutrophil influx into the spleen very early after challenge. The neutrophilia was directly associated with B cells, since BKO mice reconstituted with naive B cells prior to a secondary challenge with LVS had decreased bacterial loads and neutrophils in the spleen and survived.

Download Full-text

Respiratory Francisella tularensis Live Vaccine Strain Infection Induces Th17 Cells and Prostaglandin E2, Which Inhibits Generation of Gamma Interferon-Positive T Cells

Infection and Immunity ◽

10.1128/iai.01412-07 ◽

2008 ◽

Vol 76 (6) ◽

pp. 2651-2659 ◽

Cited By ~ 68

Author(s):

Matthew D. Woolard ◽

Lucinda L. Hensley ◽

Thomas H. Kawula ◽

Jeffrey A. Frelinger

Keyword(s):

T Cells ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Th17 Cells ◽

Live Vaccine ◽

Prostaglandin E ◽

Live Vaccine Strain ◽

Intranasal Inoculation ◽

Adaptive Immune ◽

Ifn Γ

ABSTRACT Two key routes of Francisella tularensis infection are through the skin and airway. We wished to understand how the route of inoculation influenced the primary acute adaptive immune response. We show that an intranasal inoculation of the F. tularensis live vaccine strain (LVS) with a 1,000-fold-smaller dose than an intradermal dose results in similar growth kinetics and peak bacterial burdens. In spite of similar bacterial burdens, we demonstrate a difference in the quality, magnitude, and kinetics of the primary acute T-cell response depending on the route of inoculation. Further, we show that prostaglandin E2 secretion in the lung is responsible for the difference in the gamma interferon (IFN-γ) response. Intradermal inoculation led to a large number of IFN-γ+ T cells 7 days after infection in both the spleen and the lung. In contrast, intranasal inoculation induced a lower number of IFN-γ+ T cells in the spleen and lung but an increased number of Th17 cells in the lung. Intranasal infection also led to a significant increase of prostaglandin E2 (PGE2) in the bronchoalveolar lavage fluid. Inhibition of PGE2 production with indomethacin treatment resulted in increased numbers of IFN-γ+ T cells and decreased bacteremia in the lungs of intranasally inoculated mice. This research illuminates critical differences in acute adaptive immune responses between inhalational and dermal infection with F. tularensis LVS mediated by the innate immune system and PGE2.

Download Full-text

Toll-Like Receptor 2-Mediated Signaling Requirements for Francisella tularensis Live Vaccine Strain Infection of Murine Macrophages

Infection and Immunity ◽

10.1128/iai.01868-06 ◽

2007 ◽

Vol 75 (8) ◽

pp. 4127-4137 ◽

Cited By ~ 87

Author(s):

Leah E. Cole ◽

Kari Ann Shirey ◽

Eileen Barry ◽

Araceli Santiago ◽

Prasad Rallabhandi ◽

...

Keyword(s):

Protein Synthesis ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Live Vaccine Strain ◽

Toll Like Receptor ◽

Bacterial Protein ◽

Wild Type ◽

Murine Macrophages ◽

Toll Like Receptor 2 ◽

Bacterial Protein Synthesis

ABSTRACT Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-κB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2−/− macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSΔguaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-κB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication.

Download Full-text

MglA and Igl Proteins Contribute to the Modulation of Francisella tularensis Live Vaccine Strain-Containing Phagosomes in Murine Macrophages

Infection and Immunity ◽

10.1128/iai.00226-08 ◽

2008 ◽

Vol 76 (8) ◽

pp. 3502-3510 ◽

Cited By ~ 80

Author(s):

Linda Bönquist ◽

Helena Lindgren ◽

Igor Golovliov ◽

Tina Guina ◽

Anders Sjöstedt

Keyword(s):

Cell Line ◽

Francisella Tularensis ◽

Vaccine Strain ◽

Live Vaccine ◽

Live Vaccine Strain ◽

Intracellular Replication ◽

Global Regulator ◽

Intracellular Growth ◽

Impaired Ability ◽

Macrophagelike Cell

ABSTRACT The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.

Download Full-text

An In Vitro Model System Used To Study Adherence and Invasion of Francisella tularensis Live Vaccine Strain in Nonphagocytic Cells

Infection and Immunity ◽

10.1128/iai.01811-06 ◽

2007 ◽

Vol 75 (6) ◽

pp. 3178-3182 ◽

Cited By ~ 37

Author(s):

Stephen R. Lindemann ◽

Molly K. McLendon ◽

Michael A. Apicella ◽

Bradley D. Jones

Keyword(s):

Francisella Tularensis ◽

Vaccine Strain ◽

In Vitro Model ◽

Cytochalasin D ◽

Live Vaccine ◽

Live Vaccine Strain ◽

Intracellular Growth ◽

Phagocytic Cells ◽

Vitro Model

ABSTRACT In observing Francisella tularensis interactions with nonphagocytic cell lines in vitro, we noted significant adherence, invasion, and intracellular growth of the bacteria within these cells. F. tularensis live vaccine strain invasion of nonprofessional phagocytic cells is inhibited by cytochalasin D and nocodazole, suggesting that both the actin and microtubule cytoskeletons are important for invasion.

Download Full-text

A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1

Microbiology ◽

10.1099/mic.0.025445-0 ◽

2009 ◽

Vol 155 (5) ◽

pp. 1498-1504 ◽

Cited By ~ 31

Author(s):

Crystal L. Schmerk ◽

Barry N. Duplantis ◽

Perry L. Howard ◽

Francis E. Nano

Keyword(s):

Growth Factors ◽

Francisella Tularensis ◽

Transcriptional Response ◽

Wild Type ◽

Intracellular Replication ◽

Intracellular Growth ◽

Francisella Novicida ◽

Mrna Profile ◽

Growth Phenotype ◽

In Cis

Several genes contained in the Francisella pathogenicity island (FPI) encode proteins needed for intracellular growth and virulence of Francisella tularensis. The pdpA gene is the first cistron in the larger of the two operons found in the FPI. In this work we studied the intracellular growth phenotype of a Francisella novicida mutant in the pdpA gene. The ΔpdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Strains with in cis complementation of the ΔpdpA lesion showed a restoration of intracellular growth to wild-type levels. Infection of macrophages with the ΔpdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild-type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands.

Download Full-text