Thomas H. Kawula

Infected-Host-Cell Repertoire and Cellular Response in the Lung following Inhalation of Francisella tularensis Schu S4, LVS, or U112

ABSTRACT Francisella tularensis causes systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection. F. tularensis is a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation of F. tularensis strains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4- and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4- and LVS- and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11bhigh macrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 infection but not Schu S4 or LVS infection. These results demonstrate directly that F. tularensis is a promiscuous intracellular pathogen in the lung that invades and replicates within cell types ranging from migratory immune cells to structural tissue cells. However, the proportions of cell types infected and the cellular immune response evoked by the human pathogenic strain Schu S4 differ from those of the human avirulent U112.

Genome-Wide Identification of Francisella tularensis Virulence Determinants

ABSTRACT 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 replicates within alveolar type II epithelial cells in vitro and in vivo following inhalation.

ABSTRACT 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.

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

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.

Francisella tularensis Harvests Nutrients Derived via ATG5-Independent Autophagy to Support Intracellular Growth

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.

Francisella tularensis-Infected Macrophages Release Prostaglandin E2 that Blocks T Cell Proliferation and Promotes a Th2-Like Response

Francisella tularensis is a highly infectious bacterial pathogen, and is likely to have evolved strategies to evade and subvert the host immune response. In this study, we show that F. tularensis infection of macrophages alters T cell responses in vitro, by blocking T cell proliferation and promoting a Th2-like response. We demonstrate that a soluble mediator is responsible for this effect and identify it as PGE2. Supernatants from F. tularensis-infected macrophages inhibited IL-2 secretion from both MHC class I and MHC class II-restricted T cell hybridomas, as well as enhanced a Th2-like response by inducing increased production of IL-5. Furthermore, the soluble mediator blocked proliferation of naive MHC class I-restricted T cells when stimulated with cognate tetramer. Indomethacin treatment partially restored T cell proliferation and lowered IL-5 production to wild-type levels. Macrophages produced PGE2 when infected with F. tularensis, and treatment of infected macrophages with indomethacin, a cyclooxygenase-1/cyclooxygenase-2 inhibitor, blocked PGE2 production. To further demonstrate that PGE2 was responsible for skewing of T cell responses, we infected macrophages from membrane PGE synthase 1 knockout mice (mPGES1−/−) that cannot produce PGE2. Supernatants from F. tularensis-infected membrane PGE synthase 1−/− macrophages did not inhibit T cell proliferation. Furthermore, treatment of T cells with PGE2 recreated the effects seen with infected supernatant. From these data, we conclude that F. tularensis can alter host T cell responses by causing macrophages to produce PGE2. This study defines a previously unknown mechanism used by F. tularensis to modulate adaptive immunity.

Deletion of ripA Alleviates Suppression of the Inflammasome and MAPK by Francisella tularensis

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1β and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.

Periplasmic copper–zinc superoxide dismutase protects Haemophilus ducreyi from exogenous superoxide

Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease implicated in increased heterosexual transmission of HIV. As part of an effort to identify H. ducreyi gene products involved in virulence and pathogenesis, we created random TnphoA insertion mutations in an H. ducreyi 35 000 library cloned in Escherichia coli. Inserts encoding exported or secreted PhoA fusion proteins were characterized by DNA sequencing. One such clone encoded a Cu–Zn superoxide dismutase (SOD) enzyme. The Cu–Zn SOD was periplasmic in H. ducreyi and accounted for most of the detectable SOD activity in whole‐cell lysates of H. ducreyi grown in vitro. To investigate the function of the Cu–Zn SOD, we created a Cu–Zn SOD‐deficient H. ducreyi strain by inserting a cat cassette into the sodC gene. The wild‐type and Cu–Zn SOD null mutant strains were equally resistant to excess cytoplasmic superoxide induced by paraquat, demonstrating that the Cu–Zn SOD did not function in the detoxification of cytoplasmic superoxide. However, the Cu–Zn SOD null strain was significantly more susceptible to killing by extracellular superoxide than the wild type. This result suggests that the H. ducreyi Cu–Zn SOD may play a role in bacterial defence against oxidative killing by host immune cells during infection.

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

ABSTRACT 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.

Francisella tularensis Invasion of Lung Epithelial Cells

ABSTRACT 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.