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 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 T Cells from Lungs and Livers of Francisella tularensis-Immune Mice Control the Growth of Intracellular Bacteria

2009 ◽  
Vol 77 (5) ◽  
pp. 2010-2021 ◽  
Author(s):  
Carmen M. Collazo ◽  
Anda I. Meierovics ◽  
Roberto De Pascalis ◽  
Terry H. Wu ◽  
C. Rick Lyons ◽  
...  
Keyword(s):  
T Cells ◽  
Francisella Tularensis ◽  
Necrosis Factor Alpha ◽  
Immunological Responses ◽  
Factor Alpha ◽  
Ifn Γ ◽  
Aerosol Infection ◽  
Oxide Synthase ◽  
Intranasal Instillation

ABSTRACT Parenteral and respiratory vaccinations with the intracellular bacterium Francisella tularensis have been studied using the live vaccine strain (LVS) in a mouse model, and spleen cells from immune mice are often used for immunological studies. However, mechanisms of host immunological responses may be different in nonlymphoid organs that are important sites of infection, such as lung and liver. Using parenteral (intradermal) or respiratory (cloud aerosol) vaccination, here we examine the functions of resulting LVS-immune liver or lung cells, respectively. Surprisingly, LVS was considerably more virulent when administered by cloud aerosol than by intranasal instillation, suggesting method-dependent differences in initial localization and/or dissemination patterns. Only low doses were sublethal, and resolution of sublethal cloud aerosol infection was dependent on gamma interferon (IFN-γ), tumor necrosis factor alpha, and inducible nitric oxide synthase. Nonetheless, survival of cloud aerosol or parenteral infection resulted in the development of a protective immune response against lethal LVS intraperitoneal or aerosol challenge, reflecting development of systemic secondary immunity in both cases. Such immunity was further detected by directly examining the functions of LVS-immune lung or liver lymphocytes in vitro. Lung lymphocytes primed by respiratory infection, as well as liver lymphocytes primed by parenteral infection, clearly controlled in vitro intracellular bacterial growth primarily via mechanisms that were not dependent on IFN-γ activity. Thus, our results indicate functional similarities between immune T cells residing in spleens, livers, and lungs of LVS-immune mice.

scholarly journals Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro

Science ◽  
2020 ◽  
Vol 370 (6513) ◽  
pp. eabc8420 ◽  
Author(s):  
Devin E. Christensen ◽  
Barbie K. Ganser-Pornillos ◽  
Jarrod S. Johnson ◽  
Owen Pornillos ◽  
Wesley I. Sundquist
Keyword(s):  
Cell Extract ◽  
Free System ◽  
Double Stranded Dna ◽  
A Cell ◽  
Dna Ends ◽  
Hiv 1 ◽  
Target Plasmid

During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells. We therefore reconstituted and imaged the early stages of HIV-1 replication in a cell-free system. HIV-1 cores released from permeabilized virions supported efficient, capsid-dependent endogenous reverse transcription to produce double-stranded DNA genomes, which sometimes looped out from ruptured capsid walls. Concerted integration of both viral DNA ends into a target plasmid then proceeded in a cell extract–dependent reaction. This reconstituted system uncovers the role of the capsid in templating replication.

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 Clearance of Shigella flexneri Infection Occurs through a Nitric Oxide-Independent Mechanism

1998 ◽  
Vol 66 (6) ◽  
pp. 3012-3016 ◽  
Author(s):  
Sing Sing Way ◽  
Marcia B. Goldberg
Keyword(s):  
Nitric Oxide ◽  
Shigella Flexneri ◽  
Intracellular Pathogens ◽  
Free System ◽  
Innate Resistance ◽  
Cell Free System ◽  
Independent Mechanism ◽  
A Cell ◽  
Ifn Γ

ABSTRACT Nitric oxide (NO) generated by gamma interferon (IFN-γ) activation of macrophages mediates the killing of many intracellular pathogens. IFN-γ is essential to innate resistance to Shigella flexneri infection. We demonstrate that NO is produced followingS. flexneri infection both in mice and in activated cells in vitro and that while it is able to kill S. flexneri in a cell-free system, it is not required for clearance of S. flexneri in either infected mice or in activated cells in vitro.

scholarly journals Host innate recognition of an intestinal bacterial pathogen induces TRIF-dependent protective immunity

2011 ◽  
Vol 208 (13) ◽  
pp. 2705-2716 ◽  
Author(s):  
John Sotolongo ◽  
Cecilia España ◽  
Andrea Echeverry ◽  
David Siefker ◽  
Norman Altman ◽  
...  
Keyword(s):  
Protective Immunity ◽  
Nk Cell ◽  
Interleukin 1 ◽  
Bacterial Pathogen ◽  
Mesenteric Lymph Nodes ◽  
Gram Negative ◽  
Enteric Infections ◽  
Ifn Γ

Toll-like receptor 4 (TLR4), which signals through the adapter molecules myeloid differentiation factor 88 (MyD88) and toll/interleukin 1 receptor domain-containing adapter inducing IFN-β (TRIF), is required for protection against Gram-negative bacteria. TRIF is known to be important in TLR3-mediated antiviral signaling, but the role of TRIF signaling against Gram-negative enteropathogens is currently unknown. We show that TRIF signaling is indispensable for establishing innate protective immunity against Gram-negative Yersinia enterocolitica. Infection of wild-type mice rapidly induced both IFN-β and IFN-γ in the mesenteric lymph nodes. In contrast, TRIF-deficient mice were defective in these IFN responses and showed impaired phagocytosis in regional macrophages, resulting in greater bacterial dissemination and mortality. TRIF signaling may be universally important for protection against Gram-negative pathogens, as TRIF-deficient macrophages were also impaired in killing both Salmonella and Escherichia coli in vitro. The mechanism of TRIF-mediated protective immunity appears to be orchestrated by macrophage-induced IFN-β and NK cell production of IFN-γ. Sequential induction of IFN-β and IFN-γ leads to amplification of macrophage bactericidal activity sufficient to eliminate the invading pathogens at the intestinal interface. Our results demonstrate a previously unknown role of TRIF in host resistance to Gram-negative enteropathogens, which may lead to effective strategies for combating enteric infections.

scholarly journals PapRIV, a BV-2 microglial cell activating quorum sensing peptide

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yorick Janssens ◽  
Nathan Debunne ◽  
Anton De Spiegeleer ◽  
Evelien Wynendaele ◽  
Marta Planas ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Cell Model ◽  
Dependent Manner ◽  
Communication Signals ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Gastro Intestinal Tract

AbstractQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

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

2005 ◽  
Vol 73 (4) ◽  
pp. 2306-2311 ◽  
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.

Modulation of vascular tone in renal microcirculation by erythrocytes: role of EDRF

1993 ◽  
Vol 264 (1) ◽  
pp. H190-H195 ◽  
Author(s):  
J. D. Imig ◽  
D. Gebremedhin ◽  
D. R. Harder ◽  
R. J. Roman
Keyword(s):  
Vascular Tone ◽  
Physiological Salt Solution ◽  
Nitric Oxide Synthase Inhibitor ◽  
Renal Microcirculation ◽  
Vasoconstrictor Response ◽  
A Cell ◽  
Afferent Arterioles ◽  
Synthase Inhibitor

The effect of erythrocytes (red blood cells, RBC) on vascular tone in the renal microcirculation was examined using the juxtamedullary nephron microvascular preparation perfused in vitro with a physiological salt solution containing 5% albumin. The basal diameters of the arcuate, interlobular, proximal, and distal afferent arterioles averaged 444 +/- 24, 74 +/- 3, 29 +/- 1, and 19 +/- 1 micron, respectively, when perfused with a cell-free solution at a pressure of 80 mmHg. The diameters of the arcuate and interlobular arteries increased by 14 +/- 4 and 13 +/- 4%, respectively, whereas the diameter of the proximal and distal portions of the afferent arterioles decreased by 7 +/- 2% when perfusion pressure was elevated from 80 to 160 mmHg. The addition of RBC to the perfusate reduced the basal diameters of interlobular and afferent arterioles by 11 +/- 4 and 15 +/- 3%, respectively. The maximal vasoconstrictor response was seen after the addition of only 1% RBC to the perfusate. Removal of platelets did not block the vasoconstrictor response to addition of RBC to the perfusate. The role of endothelium-derived relaxing factor (EDRF) in the vasoconstrictor response to RBC was studied by addition of nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NNA, 100 microM) to the perfusate. L-NNA reduced the basal diameters of interlobular and afferent arterioles by 7 +/- 3 and 9 +/- 3%, respectively, and abolished the vasoconstrictor response to RBC. L-NNA had no effect on the pressure-diameter relationships of the preglomerular vasculature when added to perfusates already containing RBC.(ABSTRACT TRUNCATED AT 250 WORDS)

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