Promoter-specific repression of fimB expression by the Escherichia coli nucleoid-associated protein H-NS.

The H-NS protein is a major component of the Escherichia coli nucleoid. Mutations in hns, the gene encoding H-NS, have pleiotropic effects on the cell altering both the expression of a variety of unlinked genes and the inversion rate of the DNA element containing the fimA promoter. We investigated the interaction between H-NS and fimB, the gene encoding the bidirectional recombinase that catalyzes fimA promoter flipping. In beta-galactosidase assays, we found that fimB expression increased approximately fivefold in an hns2-tetR insertion mutant. In gel mobility shift assays with purified H-NS, we have also shown that H-NS bound directly and cooperatively to the fimB promoter region with greater affinity than for any other known H-NS-regulated gene. Furthermore, this high-affinity interaction resulted in a promoter-specific inhibition of fimB transcription. The addition of purified H-NS to an in vitro transcription system yielded a fivefold or greater reduction in fimB-specific mRNA production. However, the marked increase in cellular FimB levels in the absence of H-NS was not the primary cause of the mutant rapid inversion phenotype. These results are discussed in regard to both H-NS as a transcriptional repressor of fimB expression and its role in regulating type 1 pilus promoter inversion

Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 × 10−8 ± 0.87 × 10−8 per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis

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

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

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 Replicates within Alveolar Type II Epithelial Cells In Vitro and In Vivo following Inhalation

Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell types have not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines. Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated within alveolar type II epithelial cells

Genome-Wide Identification of Francisella tularensis Virulence Determinants

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

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