TetR-Based Gene Regulation Systems for Francisella tularensis

ABSTRACT There are a number of genetic tools available for studying Francisella tularensis , the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems for F. tularensis based on the Tet repressor, TetR. For the inducible system, a tet operator sequence was cloned into a modified F. tularensis groESL promoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon, luxCDABE , was cloned under the hybrid Francisella tetracycline-regulated promoter ( FTRp ), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association with tetO. Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml −1 . In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists of FTRp regulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with the lux reporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploring F. tularensis protein function

Identifying Francisella tularensis Genes Required for Growth in Host Cells

Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924 , a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The Δ FTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The Δ FTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence

Francisella tularensis RipA Protein Topology and Identification of Functional Domains

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1β (IL-1β) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function

Francisella tularensis Invasion of Lung Epithelial Cells

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, causes disseminating infections in humans and other mammalian hosts. Macrophages and other monocytes have long been considered the primary site of F. tularensis replication in infected animals. However, recently it was reported that F. tularensis also invades and replicates within alveolar epithelial cells following inhalation in a mouse model of tularemia. TC-1 cells, a mouse lung epithelial cell line, were used to study the process of F. tularensis invasion and intracellular trafficking within nonphagocytic cells. Live and paraformaldehyde-fixed F. tularensis live vaccine strain organisms associated with, and were internalized by, TC-1 cells at similar frequencies and with indistinguishable differences in kinetics. Inhibitors of microfilament and microtubule activity resulted in significantly decreased F. tularensis invasion, as did inhibitors of phosphatidylinositol 3-kinase and tyrosine kinase activity. Collectively, these results suggest that F. tularensis epithelial cell invasion is mediated by a preformed ligand on the bacterial surface and driven entirely by host cell processes. Once internalized, F. tularensis-containing endosomes associated with early endosome antigen 1 (EEA1) followed by lysosome-associated membrane protein 1 (LAMP-1), with peak coassociation frequencies occurring at 30 and 120 min postinoculation, respectively. By 2 h postinoculation, 70.0% (± 5.5%) of intracellular bacteria were accessible to antibody delivered to the cytoplasm, indicating vacuolar breakdown and escape into the cytoplasm

PanG, a New Ketopantoate Reductase Involved in Pantothenate Synthesis

Pantothenate, commonly referred to as vitamin B5, is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG

Identification of Early Interactions between Francisella and the Host

The adaptive immune response to Francisella tularensis is dependent on the route of inoculation. Intradermal inoculation with the F. tularensis live vaccine strain (LVS) results in a robust Th1 response in the lungs, whereas intranasal inoculation produces fewer Th1 cells and instead many Th17 cells. Interestingly, bacterial loads in the lungs are similar early after inoculation by these two routes. We hypothesize that the adaptive immune response is influenced by local events in the lungs, such as the type of cells that are first infected with Francisella. Using fluorescence-activated cell sorting, we identified alveolar macrophages as the first cell type infected in the lungs of mice intranasally inoculated with F. novicida U112, LVS, or F. tularensis Schu S4. Following bacterial dissemination from the skin to the lung, interstitial macrophages or neutrophils are infected. Overall, we identified the early interactions between Francisella and the host following two different routes of inoculation

Measurement invariance of Personal Well-Being Index (PWI-8) across 26 countries

This report examines the measurement invariance of the Personal Well-being Index with 8 items (PWI-8). University students (N = 5731) from 26 countries completed the measure either through paper and pencil or electronic mode. We examined uni-dimensional structure of PWI and performed a Multi-group CFA to assess the measurement invariance across the 26 countries, using conventional approach and the alignment procedure. The findings provide evidence of configural and partial metric invariance, as well as partial scalar invariance across samples. The findings suggest that PWI-8 can be used to examine correlates of life satisfaction across all included countries, however it is impossible to compare raw scores across countries

The Effect of Immune Selection on the Structure of the Meningococcal Opa Protein Repertoire

The opa genes of the Gram negative bacterium Neisseria meningitidis encode Opacity-associated outer membrane proteins whose role is to promote adhesion to the human host tissue during colonisation and invasion. Each meningococcus contains 3–4 opa loci, each of which may be occupied by one of a large number of alleles. We analysed the Opa repertoire structure in a large, well-characterised collection of asymptomatically carried meningococci. Our data show an association between Opa repertoire and meningococcal lineages similar to that observed previously for meningococci isolated from cases of invasive disease. Furthermore, these Opa repertoires exhibit discrete, non-overlapping structure at a population level, and yet low within-repertoire diversity. These data are consistent with the predictions of a mathematical model of strong immune selection upon a system where identical alleles may occupy different loci

Belief in a zero-sum game and subjective well-being across 35 countries

This article presents a short research report on the relationship between perceived antagonism in social relations measured using the Belief in a Zero-Sum Game (BZSG) scale, life satisfaction, and positive and negative affect. Given that individuals who believe that life is like a zero-sum game are likely to perceive their daily interactions with others as unfair, we expected that individuals with high BZSG experience more negative affect and fewer positive one, resulting in a lower satisfaction with life. In addition, we examined whether country-level BZSG may play a moderating role in these associations. Data were collected from student samples (N=7146) in 35 countries. Multilevel modelling revealed that perceived social antagonism in social relations is negatively associated with satisfaction with life and that this relationship is mediated by both positive and negative affect at the individual level. The relation of individual BZSG and negative affect on satisfaction with life were weaker in societies with higher country-level BZSG, suggesting that the effects of BZSG may be less detrimental in these countries. These findings extend previous knowledge about predictors of life satisfaction and suggest that social beliefs might also be an important factor that influences subjective well-being. The contribution of the study is that the separate treatment of life satisfaction and positive and negative affect may be helpful in many research situations, particularly from a cross-cultural perspective

Belief in a zero-sum game and subjective well-being across 35 countries

This article presents a short research report on the relationship between perceived antagonism in social relations measured using the Belief in a Zero-Sum Game (BZSG) scale, life satisfaction, and positive and negative affect. Given that individuals who believe that life is like a zero-sum game are likely to perceive their daily interactions with others as unfair, we expected that individuals with high BZSG experience more negative affect and fewer positive one, resulting in a lower satisfaction with life. In addition, we examined whether country-level BZSG may play a moderating role in these associations. Data were collected from student samples (N = 7146) in 35 countries. Multilevel modelling revealed that perceived social antagonism in social relations is negatively associated with satisfaction with life and that this relationship is mediated by both positive and negative affect at the individual level. The relation of individual BZSG and negative affect on satisfaction with life were weaker in societies with higher country-level BZSG, suggesting that the effects of BZSG may be less detrimental in these countries. These findings extend previous knowledge about predictors of life satisfaction and suggest that social beliefs might also be an important factor that influences subjective well-being. The contribution of the study is that the separate treatment of life satisfaction and positive and negative affect may be helpful in many research situations, particularly from a cross-cultural perspective.info:eu-repo/semantics/publishedVersio