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

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.

Download Full-text

Characterization of the role of Fhit in maintenance of genomic integrity following low dose radiation, in vivo and in vitro

10.2172/990617 ◽

2010 ◽

Author(s):

Ya Wang

Keyword(s):

Low Dose ◽

Genomic Integrity ◽

Low Dose Radiation ◽

Dose Radiation

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00630-18 ◽

2019 ◽

Vol 201 (7) ◽

Cited By ~ 8

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.

Download Full-text

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

Infection and Immunity ◽

10.1128/iai.00134-10 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2607-2619 ◽

Cited By ~ 38

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.

Download Full-text

Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform

Biochemical Journal ◽

10.1042/bcj20190182 ◽

2019 ◽

Vol 476 (10) ◽

pp. 1521-1537 ◽

Cited By ~ 4

Author(s):

Emma J. Goldberg ◽

Katherine A. Buddo ◽

Kelsey L. McLaughlin ◽

Regina F. Fernandez ◽

Andrea S. Pereyra ◽

...

Keyword(s):

Acid Oxidation ◽

Valuable Insight ◽

Keto Acid ◽

Isolated Mitochondria ◽

Mouse Tissues ◽

Branched Chain

Abstract Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism—evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

Download Full-text

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

Microbiology ◽

10.1099/mic.0.031716-0 ◽

2010 ◽

Vol 156 (2) ◽

pp. 327-339 ◽

Cited By ~ 42

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.

Download Full-text

In vivo and in vitro response to octreotide LAR in a TSH-secreting adenoma: characterization of somatostatin receptor expression and role of subtype 5

Pituitary ◽

10.1007/s11102-010-0271-2 ◽

2010 ◽

Vol 14 (2) ◽

pp. 141-147 ◽

Cited By ~ 25

Author(s):

Federico Gatto ◽

Federica Barbieri ◽

Lara Castelletti ◽

Marica Arvigo ◽

Alessandra Pattarozzi ◽

...

Keyword(s):

Somatostatin Receptor ◽

Receptor Expression ◽

Octreotide Lar ◽

In Vitro Response

Download Full-text

Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

Infection and Immunity ◽

10.1128/iai.00140-10 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2370-2376 ◽

Cited By ~ 14

Author(s):

Louise M. Temple ◽

David M. Miyamoto ◽

Manju Mehta ◽

Christian M. Capitini ◽

Stephen Von Stetina ◽

...

Keyword(s):

Whooping Cough ◽

Bioinformatic Analysis ◽

Tracheal Ring ◽

Tracheal Cell ◽

Bordetella Avium ◽

Identification And Characterization

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

Download Full-text

Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in l-Arginine Biosynthesis

Infection and Immunity ◽

10.1128/iai.70.6.3080-3084.2002 ◽

2002 ◽

Vol 70 (6) ◽

pp. 3080-3084 ◽

Cited By ~ 55

Author(s):

Bhavna G. Gordhan ◽

Debbie A. Smith ◽

Heidi Alderton ◽

Ruth A. McAdam ◽

Gregory J. Bancroft ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Auxotrophic Mutant ◽

Phenotypic Characterization ◽

Wild Type ◽

Arginine Biosynthesis ◽

High Concentrations

ABSTRACT A mutant of Mycobacterium tuberculosis defective in the metabolism of l-arginine was constructed by allelic exchange mutagenesis. The argF mutant strain required exogenous l-arginine for growth in vitro, and in the presence of 0.96 mM l-arginine, it achieved a growth rate and cell density in stationary phase comparable to those of the wild type. The mutant strain was also able to grow in the presence of high concentrations of argininosuccinate, but its auxotrophic phenotype could not be rescued by l-citrulline, suggesting that the ΔargF::hyg mutation exerted a polar effect on the downstream argG gene but not on argH. The mutant strain displayed reduced virulence in immunodeficient SCID mice and was highly attenuated in immunocompetent DBA/2 mice, suggesting that l-arginine availability is restricted in vivo.

Download Full-text

NLRP3- and AIM2-autonomy in a mouse model of MSU crystal-induced acute inflammation in vivo suggests imiquimod-dependent targeting of Il-1[beta] expression as relevant therapy for gout patients.

10.1101/772756 ◽

2019 ◽

Author(s):

Alexandre Mariotte ◽

Aurore Decauwer ◽

Chrystelle Po ◽

Cherine Abou-Faycal ◽

Angelique Pichot ◽

...

Keyword(s):

Mouse Model ◽

Acute Inflammation ◽

Joint Inflammation ◽

Inflammatory Action ◽

Recognition Receptor ◽

Msu Crystals

The role of Monosodium Urate (MSU) crystals in gout pathophysiology is well described, as is the major impact of IL-1b in the inflammatory reaction that constitutes the hallmark of the disease. However, despite the discovery of the NLRP3 inflammasome and its role as a Pattern Recognition Receptor linking the detection of a danger signal (MSU) to IL-1b; secretion in vitro, the precise mechanisms leading to joint inflammation in gout patients are still poorly understood. Here, we provide an extensive clinical, biological and molecular characterization of the acute uratic inflammation mouse model induced by subcutaneous injection of MSU crystals, which accurately mimics human gout. Our work reveals several key features of MSU-dependent inflammation and identifies novel therapeutic opportunities, among which the use of topical application of imiquimod to promote interferon-dependent anti-inflammatory action maybe relevant.

Download Full-text

Characterization of AcMNPV with a deletion of ac69 gene

Microbiology Research ◽

10.4081/mr.2011.e4 ◽

2011 ◽

Vol 2 (1) ◽

pp. 4

Author(s):

Jianhao Ke ◽

Jinwen Wang ◽

Riqiang Deng ◽

Lin Lin ◽

Bei Jinlong ◽

...

Keyword(s):

Spodoptera Frugiperda ◽

Trichoplusia Ni ◽

Virus Production ◽

Viral Infectivity ◽

Methyltransferase Activity ◽

Budded Virus

ORF69 (Ac69) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in Spodoptera frugiperda Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an ac69 knockout AcMNPV bacmid mutant and analyses of the influence of ac69 deletion on the viral infectivity in Sf-9 cells and Trichoplusia ni larvae so as to investigate the role of ac69 in the viral life cycle. Results indicated that ac69 deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect AcMNPV infectivity for Trichoplusia ni larvae in LD50 and LT50 bioassay when administered orally. These results suggest that ac69 may be dispensable for viral infectivity both in vitro and in vivo.

Download Full-text