Document Type


Date of Degree

Spring 2017

Degree Name

PhD (Doctor of Philosophy)

Degree In


First Advisor

Allen, Lee-Ann H.

First Committee Member

Barker, Jason H.

Second Committee Member

Horswill, Alexander R.

Third Committee Member

Houtman, Jon C.D.

Fourth Committee Member

Jones, Bradley D.


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.

Public Abstract

The human immune system defends the body against infection and is comprised of multiple cell types including neutrophils and macrophages. Neutrophils are recruited to sites of inflammation where they rapidly ingest and degrade invading microbes. To prevent the spread of infection, neutrophils commit suicide and are cleared by macrophages. The bacterium Francisella tularensis causes the life-threatening disease tularemia, which is characterized by the deleterious accumulation of neutrophils in the lungs. This microbe’s extreme virulence indicates that the organism possesses robust strategies for evading the host immune response, but many of these tactics remain unidentified. It is well established that Francisella evades neutrophil antibacterial defenses and resides within the host cell, and our lab recently discovered that this bacterium extends neutrophil lifespan to preserve its replicative niche.

The goal of this thesis is to determine the molecular mechanisms by which F. tularensis prevents neutrophil death and to explore the fate of infected neutrophils following engulfment by macrophages. We report that this microbe alters the critical balance of pro-death and pro-survival factors via changes in protein abundance, activity, and location. We also demonstrate that infected cells are resistant to treatment with R-roscovitine, a cell death-inducing drug currently in testing for the treatment of inflammation. Here, we show that macrophages readily consume infected neutrophils, resulting in release of their infectious cargo into the macrophage cytoplasm and substantial bacterial replication. Our data begin to define molecular mechanisms to account for the profound accumulation of neutrophils and tissue damage that occurs during tularemia.


Apoptosis, Efferocytosis, Francisella tularensis, Neutrophils


xv, 180 pages


Includes bibliographical references (pages 158-180).


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Copyright © 2017 Jenna Mae McCracken

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