Modulation of virulence factors in Francisella tularensis determines human macrophage responses

Carlson, Paul E.; Carroll, James A.; O’Dee, Dawn M.; Nau, Gerard J.

doi:10.1016/j.micpath.2007.02.001

Cited by 40 publications

(58 citation statements)

References 53 publications

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“…LVS bacteria lacking the iglC gene failed to inhibit responsiveness to E. coli LPS following infection of J774.1 macrophages (50). Furthermore, a spontaneous LVS mutant that failed to produce IglC induced both proinflammatory cytokines and was unable to suppress responsiveness of human macrophages to E. coli LPS (11). We detected IglC protein in S4CM.…”

Section: Discussionmentioning

confidence: 79%

Direct and Indirect Impairment of Human Dendritic Cell Function by VirulentFrancisella tularensisSchu S4

2009

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The gram-negative, facultative intracellular bacterium Francisella tularensis causes acute, lethal pneumonic disease following infection with only 10 CFU. The mechanisms used by the bacterium to accomplish this in humans are unknown. Here, we demonstrate that virulent, type A F. tularensis strain Schu S4 efficiently infects and replicates in human myeloid dendritic cells (DCs). Despite exponential replication over time, Schu S4 failed to stimulate transforming growth factor ␤, interleukin-10 (IL-10), IL-6, IL-1␤, IL-12, tumor necrosis factor alpha, alpha interferon (IFN-␣), and IFN-␤ throughout the course of infection. Schu S4 also suppressed the ability of directly infected DCs to respond to different Toll-like receptor agonists. Furthermore, we also observed functional inhibition of uninfected bystander cells. This inhibition was mediated, in part, by a heat-stable bacterial component. Lipopolysaccharide (LPS) from Schu S4 was present in Schu S4-conditioned medium. However, Schu S4 LPS was weakly inflammatory and failed to induce suppression of DCs at concentrations below 10 g/ml, and depletion of Schu S4 LPS did not significantly alleviate the inhibitory effect of Schu S4-conditioned medium in uninfected human DCs. Together, these data show that type A F. tularensis interferes with the ability of a central cell type of the immune system, DCs, to alert the host of infection both intra-and extracellularly. This suggests that immune dysregulation by F. tularensis operates on a broader and more comprehensive scale than previously appreciated.Francisella tularensis is a gram-negative, facultative intracellular bacterium and the causative agent of tularemia. Although the bacteria was identified nearly 100 years ago, the nature of its interaction with host cells and tissues has remained largely undefined until recent times. The unfortunate realization of the potential of F. tularensis as a biological weapon has resulted in a resurgence of research on tularemia. Much of our recent understanding of tularemia pathogenesis has come from manipulation of the mouse model of Francisella infections. While these murine studies have yielded many important advances in our understanding of tularemia pathogenesis, very little is understood about how Francisella, especially the most virulent type A strains such as Schu S4, interacts with human cells.Dendritic cells (DCs) serve as a central cell type in the immune system, bridging the innate and adaptive immune response to effectively eradicate invading pathogens.

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Section: Discussionmentioning

confidence: 79%

Direct and Indirect Impairment of Human Dendritic Cell Function by VirulentFrancisella tularensisSchu S4

2009

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“…Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll gradient centrifugation. Monocytes were then isolated from PBMCs by Optiprep (Sigma-Aldrich) gradient centrifugation as described previously (32). Monocytes were adhered to plastic 60-mm culture dishes in serum-free Dulbecco's modified Eagle's medium (DMEM).…”

Section: Methodsmentioning

confidence: 99%

The Intracellular Environment of Human Macrophages That Produce Nitric Oxide Promotes Growth of Mycobacteria

et al. 2013

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e Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-␥), L-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-␥ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.

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“…Buffy coats from blood donations (Central Blood Bank, Pittsburgh, PA) served as the source of human monocytes that were differentiated into macrophages by in vitro culture as has been detailed previously (9,10,27,29,50). For in vitro infections, primary human macrophages were washed and resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% human serum AB (complement replete; GemCell Gemini Bio-Products), 25 mM HEPES, and 1% GlutaMAX and then plated onto Primaria-coated 96-well culture dishes (Becton, Dickinson and Company) at a density of 5.0 ϫ 10 4 cells per well as we have described previously (27).…”

Section: Methodsmentioning

confidence: 99%

“…As this disease progresses, macrophage numbers decline (26,63), likely due to the induction of caspase-3-dependent apoptosis induced by the virulent type A F. tularensis strain (63). During interactions with host macrophages, F. tularensis can block the activation of these cells, as evidenced by the inhibition of proinflammatory cytokine production (9,60,61). In addition to macrophages and dendritic cells, F. tularensis can invade and replicate in nonphagocytic host cells, such as alveolar epithelial cells, kidney epithelial cells, hepatocytes, and fibroblasts (14,21,(25)(26)(27)47).…”

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confidence: 99%

Francisella tularensisΔpyrFMutants Show that Replication in Nonmacrophages Is Sufficient for PathogenesisIn Vivo

Horzempa¹,

O’Dee²,

Shanks

et al. 2010

Infect Immun

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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 ⌬pyrF 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 ⌬pyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis ⌬pyrF 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 ⌬pyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis ⌬pyrF 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.

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Modulation of virulence factors in Francisella tularensis determines human macrophage responses

Cited by 40 publications

References 53 publications

Direct and Indirect Impairment of Human Dendritic Cell Function by VirulentFrancisella tularensisSchu S4

Direct and Indirect Impairment of Human Dendritic Cell Function by VirulentFrancisella tularensisSchu S4

The Intracellular Environment of Human Macrophages That Produce Nitric Oxide Promotes Growth of Mycobacteria

Francisella tularensisΔpyrFMutants Show that Replication in Nonmacrophages Is Sufficient for PathogenesisIn Vivo

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