Francisella tularensis is a facultative intracellular bacterium that infects many cell types including neutrophils. We demonstrated previously that F. tularensis inhibits NADPH oxidase assembly and activity and then escapes the phagosome to the cytosol, but effects on other aspects of neutrophil function are unknown. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis typically accelerates this process. We now demonstrate that F. tularensis significantly inhibited neutrophil apoptosis as indicated by morphological analysis as well as Annexin V and TUNEL staining. Thus, ~80% of infected neutrophils remained viable at 48 h as compared with ~50% of control cells, and ~40% of neutrophils that ingested opsonized zymosan. In keeping with this, processing and activation of procaspases-8, -9 and -3 were markedly diminished and delayed. F. tularensis also significantly impaired apoptosis triggered by Fas crosslinking. Of note, these effects were dose-dependent and could be conferred by either intracellular or extracellular live bacteria, but not by formalin-killed organisms or isolated LPS and capsule, and were not affected by disruption of wbtA2 or FTT1236/FTL0708, genes required for LPS O-antigen and capsule biosynthesis. In summary, we demonstrate for the first time that F. tularensis profoundly impairs constitutive neutrophil apoptosis via effects on the intrinsic and extrinsic pathways, and thereby define a new aspect of innate immune evasion by this organism. As defects in neutrophil turnover prevent resolution of inflammation, our findings also suggest a mechanism that may in part account for the neutrophil accumulation, granuloma formation and severe tissue damage that characterizes lethal pneumonic tularemia.

is a Gram-negative bacterium that is closely related to the highly virulent facultative intracellular pathogen, Data published by us and others demonstrate that virulence correlates directly with its ability to impair constitutive apoptosis and extend human neutrophil lifespan. In contrast, is attenuated in humans, and the mechanisms that account for this are incompletely defined. Our published data demonstrate that binds natural IgG that is present in normal human serum, which in turn, elicits NADPH oxidase activation that does not occur in response to As it is established that phagocytosis and oxidant production markedly accelerate neutrophil death, we predicted that may influence the neutrophil lifespan in an opsonin-dependent manner. To test this hypothesis, we quantified bacterial uptake, phosphatidylserine (PS) externalization, and changes in nuclear morphology, as well as the kinetics of procaspase-3, -8, and -9 processing and activation. To our surprise, we discovered that not only failed to accelerate neutrophil death but also diminished and delayed apoptosis in a dose-dependent, but opsonin-independent, manner. In keeping with this, studies of conditioned media (CM) showed that neutrophil longevity could be uncoupled from phagocytosis and that stimulated neutrophil secretion of CXCL8. Taken together, the results of this study reveal shared and unique aspects of the mechanisms used by species to manipulate neutrophil lifespan and as such, advance understanding of cell death regulation during infection.

Neutrophils are important innate immune effector cells that primarily function during infection by engulfing and killing pathogens using a combination of toxic granule components and reactive oxygen species (ROS) generated by the NADPH oxidase. Francisella tularensis is a Gram-negative bacterium and the causative agent of tularemia, an infectious disease that, in the absence of treatment, results in 30-60% mortality. A closely related species, F. novicida, does not cause human disease but causes a tularemialike illness in mice and productively infects human and murine cells in vitro; thus this organism is often employed as a model. In our previous work, we have shown that virulent and avirulent F. tularensis enters neutrophils without inducing a respiratory burst, as the NADPH oxidase fails to assemble on bacterial phagosomes. Further, this pathogen inhibits enzyme activity upon subsequent neutrophil stimulation despite successful oxidase assembly, indicating that F. tularensis employs multiple mechanisms to inhibit the NADPH oxidase. It remains unknown, however, whether F. novicida retains these mechanisms of oxidase inhibition, or whether its inability to modulate neutrophil function partially accounts for its avirulence in humans. Additional work has suggested a potential role for Francisella acid phosphatases and catalase genes in inhibited production and detoxification of neutrophil-derived ROS, respectively. In the current study, we employ qualitative and quantitative methods to evaluate the magnitude and location of ROS generation during infection with F. tularensis LVS, F. novicida, or F. novicida mutants lacking functional acpA or katG. Our results demonstrate that serumopsonized F. novicida, but not LVS, induced a prominent respiratory burst that coincided with oxidase assembly and intraphagosomal superoxide production in bacterial phagosomes. Furthermore, our data show for the first time that opsonized F. novicida, but not LVS, engaged FcγRIII (CD16) during phagocytosis by neutrophils, suggesting that this receptor may play a role in signaling events that lead to respiratory burst induction. vii Despite its inability to evade burst induction, F. novicida inhibited post-assembly oxidase activity following sequential stimulation of neutrophils, similar to F. tularensis strains. Finally, we conclude that acpA and katG do not play a significant role in F. novicidaneutrophil interactions as these mutants did not induce a stronger respiratory burst during phagocytosis, and their ability to inhibit post-assembly NADPH oxidase activity and survive in neutrophils was indistinguishable from wild-type organisms. Thus, these data strongly suggest that differential opsonization of F. novicida compared to F. tularensis results in engagement of specific receptors that function to activate these cells during infection. Further, the retained ability of F. novicida to inhibit post-assembly oxidase activity confirms that Francisella utilize two independent mechanisms by which they modulate NADPH oxidase function. Finally...