Characterization of the neutrophil respiratory burst during infection with Francisella novicida

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 tularemia-like 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 subjective and objective techniques to evaluate the magnitude and location of ROS generation during infection with F. tularensis LVS, F. novicida, or F. novicida mutants acpA or katG. Our results demonstrate that serum-opsonized 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 Fc-gammaRIII (CD16) during phagocytosis by neutrophils suggesting that this receptor may play a role in signaling events that lead to respiratory burst induction. 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. novicida-neutrophil 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, our conclusions that acpA and katG are disposable for these interactions with neutrophils suggest that F. novicida encodes other important genes that enable them to productively infect these cells.