Adaptation of Francisella tularensis to the Mammalian Environment Is Governed by Cues Which Can Be Mimicked In Vitro

Abstract

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod-or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHIand macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI-and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHBgrown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

Phosphatidylinositol 3-Kinase Activation Attenuates the TLR2-Mediated Macrophage Proinflammatory Cytokine Response toFrancisella tularensisLive Vaccine Strain

“…Berton, unpublished observations). It has recently been shown that the in vitro growth conditions, including the nature of the growth medium, can dramatically affect the virulence and immunostimulatory properties of F. tularensis (85). Thus, multiple factors may account for differences observed among studies of host cell signaling responses by the various Francisella species.…”

Francisella tularensis Antioxidants Harness Reactive Oxygen Species to Restrict Macrophage Signaling and Cytokine Production

“…Recently, Wehrly et al (25) reported an increase in the transcriptional levels of F. tularensis antioxidants following infection of macrophages as early as 1 h post-infection, suggesting a significant role of these antioxidant defenses in intramacrophage survival. Culture conditions that mimic intracellular macrophage growth enhance F. tularensis KatG expression and limit its capacity to stimulate macrophage cytokine production (26). It has been reported that redox-dependent inactivation of signaling components plays an important role in long-term homeostasis of human alveolar macrophages (27).…”

IglG and IglI of the Francisella Pathogenicity Island Are Important Virulence Determinants of Francisella tularensis LVS

“…Stimulation of macrophages with E. coli LPS leads to TNF-␣ production and secretion, an inflammasome-independent event, which is efficiently inhibited upon infection by F. tularensis LVS (73). In contrast, infection with an ⌬iglC mutant or an ⌬mglA mutant resulted in TNF-␣ production that was augmented when cells were activated by E. coli LPS (32,73). Hence, to further characterize the role of the FPI in the inhibition of TNF-␣ production, we infected J774 cells with the ⌬pdpE, ⌬iglG, or ⌬iglI mutant as well as the complemented mutant strains.…”

“…The pathogenicity of F. tularensis is not completely understood, but the primary replication site in humans appears to be macrophages and involves escape from the phagosome prior to lysosomal fusion (19,30), followed by cytosolic replication (68). Upon encounter with a host cell, rapid induction of a proinflammatory response which is completely or partly repressed upon bacterial internalization occurs (32,60,74). Moreover, F. tularensis actively suppresses the ability of both dendritic cells and macrophages to secrete cytokines in response to secondary stimuli (8,21,73).…”