Siobhán C. Cowley

A Francisella tularensis Pathogenicity Island Required for Intramacrophage Growth

Francisella tularensis is a gram-negative, facultative intracellular pathogen that causes the highly infectious zoonotic disease tularemia. We have discovered a ca. 30-kb pathogenicity island of F. tularensis (FPI) that includes four large open reading frames (ORFs) of 2.5 to 3.9 kb and 13 ORFs of 1.5 kb or smaller. Previously, two small genes located near the center of the FPI were shown to be needed for intramacrophage growth. In this work we show that two of the large ORFs, located toward the ends of the FPI, are needed for virulence. Although most genes in the FPI encode proteins with amino acid sequences that are highly conserved between high- and low-virulence strains, one of the FPI genes is present in highly virulent type A F. tularensis, absent in moderately virulent type B F. tularensis, and altered in F. tularensis subsp. novicida, which is highly virulent for mice but avirulent for humans. The G+C content of a 17.7-kb stretch of the FPI is 26.6%, which is 6.6% below the average G+C content of the F. tularensis genome. This extremely low G+C content suggests that the DNA was imported from a microbe with a very low G+C-containing chromosome.

Innate and Adaptive Immunity to Francisella

Abstract:  Studies of immune responses to Francisella have been conducted for well over 50 years. Here, the basic parameters of innate and adaptive immune responses to Francisella are reviewed, with an emphasis on those that may contribute directly to protection against infection. Although older literature provides a wealth of information on human immune responses to infection and vaccination, most recent information has been derived largely from studies in animals and using animal cells, particularly mice. In experimental animals, activation of macrophages, a major and probably preferred host cell for Francisella, appears central to control of infection. Thus, in animal models and in vitro studies using mouse macrophages, cytokines such as IFN‐γ and TNF‐α, derived first from both nonspecific cells such as natural killer cells and later from Francisella‐specific T cells, collaborate to effect intracellular killing. In mice, these intracellular killing mechanisms include reactive nitrogen and oxygen species, but killing mechanisms remain to be identified in humans. Ultimately both CD4+ and CD8+ T cells develop into Francisella‐specific memory cells and are important for control of primary Francisella infection or vaccination‐induced protection. The effector mechanisms invoked by either CD4+ or CD8+ T cells, beyond production of IFN‐γ and TNF‐α, are the subject of ongoing studies. Both specific antibodies and B cells may contribute to control of primary infection or vaccination‐induced protection in some circumstances, particularly against lower virulence Francisella strains. Thus a number of known proinflammatory and Th‐1 T cell related components of the immune system combat this virulent bacterium; no doubt others remain to be discovered.

In Vivo Clearance of an Intracellular Bacterium, Francisella tularensis LVS, Is Dependent on the p40 Subunit of Interleukin-12 (IL-12) but Not on IL-12 p70

ABSTRACT To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-γ), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4+ and CD8+ T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-γ than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-γ production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.

CD4-CD8- T cells control intracellular bacterial infections both in vitro and in vivo

Memory T cells, including the well-known CD4+ and CD8+ T cells, are central components of the acquired immune system and are the basis for successful vaccination. After infection, CD4+ and CD8+ T cells expand into effector cells, and then differentiate into long-lived memory cells. We show that a rare population of CD4−CD8−CD3+ αβ + γδ −NK1.1− T cells has similar functions. These cells potently and specifically inhibit the growth of the intracellular bacteria Mycobacterium tuberculosis (M. tb.) or Francisella tularensis Live Vaccine Strain (LVS) in macrophages in vitro, promote survival of mice infected with these organisms in vivo, and adoptively transfer immunity to F. tularensis LVS. Furthermore, these cells expand in the spleens of mice infected with M. tb. or F. tularensis LVS, and then acquire a memory cell phenotype. Thus, CD4−CD8− T cells have a role in the control of intracellular infection and may contribute to successful vaccination.

Purified Lipopolysaccharide from Francisella tularensis Live Vaccine Strain (LVS) Induces Protective Immunity against LVS Infection That Requires B Cells and Gamma Interferon

ABSTRACT Previous results have demonstrated that nonspecific protective immunity against lethal Francisella tularensis live vaccine strain (LVS) or Listeria monocytogenes infection can be stimulated either by sublethal infection with bacteria or by treatment with bacterial DNA given 3 days before lethal challenge. Here we characterize the ability of purified lipopolysaccharide (LPS) fromF. tularensis LVS to stimulate similar early protective immunity. Treatment of mice with surprisingly small amounts of LVS LPS resulted in very strong and long-lived protection against lethal LVS challenge within 2 to 3 days. Despite this strong protective response, LPS purified from F. tularensis LVS did not activate murine B cells for proliferation or polyclonal immunoglobulin secretion, nor did it activate murine splenocytes for secretion of interleukin-4 (IL-4), IL-6, IL-12, or gamma interferon (IFN-γ). Immunization of mice with purified LVS LPS induced a weak specific anti-LPS immunoglobulin M (IgM) response and very little IgG; however, infection of mice with LVS bacteria resulted in vigorous IgM and IgG, particularly IgG2a, anti-LPS antibody responses. Studies using various immunodeficient mouse strains, including LPS-hyporesponsive C3H/HeJ mice, μMT− (B-cell-deficient) knockout mice, and IFN-γ-deficient mice, demonstrated that the mechanism of protection does not involve recognition through the Lpsngene product; nonetheless, protection was dependent on B cells as well as IFN-γ.

Multiple T Cell Subsets Control Francisella tularensis LVS Intracellular Growth Without Stimulation Through Macrophage Interferon γ Receptors

A variety of data suggest that in vivo production of interferon (IFN)-γ is necessary, but not sufficient, for expression of secondary protective immunity against intracellular pathogens. To discover specific IFN-γ–independent T cell mediated mechanisms, we took advantage of an in vitro culture system that models in vivo immune responses to the intracellular bacterium Francisella tularensis live vaccine strain (LVS). LVS-immune lymphocytes specifically controlled 99% of the growth of LVS in wild-type murine bone marrow–derived macrophages. Surprisingly, LVS-immune lymphocytes also inhibited LVS intracellular growth by as much as 95% in macrophages derived from IFN-γ receptor knockout (IFNγR KO) mice. CD8+ T cells, and to a lesser degree CD4+ T cells, controlled LVS intracellular growth in both wild-type and IFNγR KO macrophages. Further, a unique population of Thy1+αβ+CD4−CD8− cells that was previously suggested to operate during secondary immunity to LVS in vivo strongly controlled LVS intracellular growth in vitro. A large proportion of the inhibition of LVS intracellular growth in IFNγR KO macrophages by all three T cell subsets could be attributed to tumor necrosis factor (TNF) α. Thus, T cell mechanisms exist that control LVS intracellular growth without acting through the IFN-γ receptor; such control is due in large part to TNF-α, and is partially mediated by a unique double negative T cell subpopulation.

Lung CD4−CD8− Double-Negative T Cells Are Prominent Producers of IL-17A and IFN-γ during Primary Respiratory Murine Infection with Francisella tularensis Live Vaccine Strain

For several intracellular infections, pulmonary vaccination provides measurably better protection against pulmonary challenge. The unique factors that contribute to pulmonary immune responses are not well characterized. In this study, we show that CD4−CD8− double negative (DN) T cells are a major responding T cell subset in the lungs of mice during pulmonary Francisella tularensis live vaccine strain (LVS) infection. DN T cells were a minor (<2%) subset in spleens and lungs of mice during sublethal intradermal infection with LVS. In contrast, they were a major responding T cell subset in lungs during pulmonary LVS infection, producing large quantities of IFN-γ and IL-17A. The numbers of IL-17A+ DN T cells in the lungs exceeded that of CD4+ and CD8+ T cells on day 7 postinfection; by day 14 postinfection, all three IL-17A–producing T cell subsets were present in equivalent numbers. CD4+, CD8+, and DN T cell production of IL-17A was not observed in the spleens of pulmonary-infected mice or the lungs and spleens of intradermally infected mice. Correspondingly, IL-17A knockout mice were more susceptible to respiratory than intradermal LVS infection, with delayed clearance 1–3 wk postinfection. Finally, in vitro treatment of LVS-infected macrophages and alveolar type II epithelial cells with IFN-γ and IL-17A affected significantly greater LVS growth control than treatment with either cytokine alone. The data presented in this study demonstrate that DN cells contribute to production of IL-17A and IFN-γ in the lungs during inhalational Francisella infection and that these cytokines additively activate host cells to control LVS intracellular growth.

Immunity to Francisella.

In recent years, studies on the intracellular pathogen Francisella tularensis have greatly intensified, generating a wealth of new information on the interaction of this organism with the immune system. Here we review the basic elements of the innate and adaptive immune responses that contribute to protective immunity against Francisella species, with special emphasis on new data that has emerged in the last 5 years. Most studies have utilized the mouse model of infection, although there has been an expansion of work on human cells and other new animal models. In mice, basic immune parameters that operate in defense against other intracellular pathogen infections, such as interferon gamma, TNF-α, and reactive nitrogen intermediates, are central for control of Francisella infection. However, new important immune mediators have been revealed, including IL-17A, Toll-like receptor 2, and the inflammasome. Further, a variety of cell types in addition to macrophages are now recognized to support Francisella growth, including epithelial cells and dendritic cells. CD4+ and CD8+ T cells are clearly important for control of primary infection and vaccine-induced protection, but new T cell subpopulations and the mechanisms employed by T cells are only beginning to be defined. A significant role for B cells and specific antibodies has been established, although their contribution varies greatly between bacterial strains of lower and higher virulence. Overall, recent data profile a pathogen that is adept at subverting host immune responses, but susceptible to many elements of the immune systems antimicrobial arsenal.

Transformation and allelic replacement in Francisella spp.

We describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella. Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-Tn10-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.

Differential Requirements by CD4+ and CD8+ T Cells for Soluble and Membrane TNF in Control of Francisella tularensis Live Vaccine Strain Intramacrophage Growth

During primary infection with intracellular bacteria, the membrane-associated form of TNF provides some TNF functions, but the relative contributions during memory responses are not well-characterized. In this study, we determined the role of T cell-derived secreted and membrane-bound TNF (memTNF) during adaptive immunity to Francisella tularensis live vaccine strain (LVS). Although transgenic mice expressing only the memTNF were more susceptible to primary LVS infection than wild-type (WT) mice, LVS-immune WT and memTNF mice both survived maximal lethal secondary Francisella challenge. Generation of CD44high memory T cells and clearance of bacteria were similar, although more IFN-γ and IL-12(p40) were produced by memTNF mice. To examine T cell function, we used an in vitro tissue coculture system that measures control of LVS intramacrophage growth by LVS-immune WT and memTNF-T cells. LVS-immune CD4+ and CD8+ T cells isolated from WT and memTNF mice exhibited comparable control of LVS growth in either normal or TNF-α knockout macrophages. Although the magnitude of CD4+ T cell-induced macrophage NO production clearly depended on TNF, control of LVS growth by both CD4+ and CD8+ T cells did not correlate with levels of nitrite. Importantly, intramacrophage LVS growth control by CD8+ T cells, but not CD4+ T cells, was almost entirely dependent on T cell-expressed TNF, and required stimulation through macrophage TNFRs. Collectively, these data demonstrate that T cell-expressed memTNF is necessary and sufficient for memory T cell responses to this intracellular pathogen, and is particularly important for intramacrophage control of bacterial growth by CD8+ T cells.