Francisella tularensis is the causative agent of the life-threatening disease tularemia. The Centers for Disease Control considers F. tularensis among the most likely agents of biowarfare due to its high mortality rate, ease of aerosol transmission, and low infectious dose. A fundamental aspect of tularemia pathogenesis is the overwhelming accumulation of neutrophils in the lung that are incapable of bacterial clearance and furthermore injurious to the host tissue, as neutrophilia exacerbates disease and blockade of neutrophil influx into the lungs favors host survival. We hypothesized that the pathologic accretion of neutrophils may be the result of decreased neutrophil death and/or decreased clearance by macrophages.
Our lab recently demonstrated that F. tularensis delays neutrophil apoptosis by at least 48 hours to preserve its replicative niche, but the mechanism by which this occurs was poorly defined. Here, we investigate alterations in neutrophil apoptosis and survival signaling at the molecular level and find that, in addition to effects on neutrophil transcription, F. tularensis also modulates protein abundance, activity, and subcellular localization. Specifically, we report that F. tularensis preserves mitochondrial integrity by inhibiting the pro-apoptotic proteins Bid and Bax as well as maintaining expression of the pro-survival factors XIAP and calpastatin. Moreover, we found that infection diminishes the ability of R-roscovitine to induce apoptosis, suggesting bacterial modulation of CDK-mediated survival signaling.
Following apoptosis, effete neutrophils are rapidly cleared by macrophages in a process termed efferocytosis to avoid neutrophil progression to secondary necrosis and consequent host tissue damage. We demonstrate for the first time that neutrophils laden with F. tularensis are readily consumed by macrophages and release their infectious cargo into the macrophage cytoplasm. The engulfing cell is unable to eradicate the infection and extensive bacterial replication ensues. Intriguingly, we found that unlike other pathogens, covert infection of macrophages by F. tularensis triggers an inflammatory cytokine response that is highly similar to that of directly infected cells, suggesting that efferocytosis is not an essential virulence mechanism for this bacterium. Together, these studies significantly advance our understanding of fundamental F. tularensis virulence mechanisms and disease pathophysiology as well as shed light on other inflammatory disorders characterized by dysregulated neutrophil turnover and clearance.