Tularemia is a potentially fatal zoonosis of humans caused by the facultative intracellular bacterium, Francisella tularensis, and inhalation of as few as ten organisms is sufficient to cause severe pneumonic disease, tissue necrosis, and death. Accumulation of neutrophils (polymorphonuclear leukocytes, PMNs) in the lungs is essential for development of severe disease in animals infected with this pathogen, and tissue destruction progresses steadily as more alveoli and bronchioles become clogged with infected PMNs and debris. Direct evidence that neutrophils contribute to tularemia progression and pathogenesis rather than effective host defense is demonstrated by the fact that blockade of PMN influx into the lung is protective, and animals survive what would otherwise be a lethal infection. Nevertheless, most studies of F. tularensis have focused on the fate of this organism in macrophages. Our long-term goal is to define in detail the role of human neutrophils in the pathogenesis of tularemia. To this end we have made several important discoveries. We have shown that natural IgM is required for opsonization of F. tularensis and identified receptors that mediate infection of both neutrophils and macrophages. We discovered that evasion of oxidative host defense is achieved via the ability of F. tularensis to act at multiple levels to disrupt NADPH oxidase assembly and activity, and identified relevant virulence factors. We were also the first to show that F. tularensis can escape the phagosome and replicate in neutrophil cytosol, confirming its ability to successfully infect multiple phagocytes types. Particularly relevant here is our discovery of PMN-specific aspects of virulence. Neutrophils are short-lived cells that are preprogrammed to die by constitutive apoptosis, a process that is typically accelerated by phagocytosis and is essential for resolution of the inflammatory response. In marked contrast, we find that F. tularensis inhibits PMN apoptosis and significantly prolongs cell lifespan, and this is achieved, in part, via effects on neutrophil gene expression. These data are noteworthy as defects in PMN turnover are indicative of an ineffective and dysregulated inflammatory response. Timely clearance of dying PMNs by macrophages is essential to prevent necrosis and tissue damage, and our preliminary data suggest that this process may also be impaired. In view of these data we hypothesize that F. tularensis inhibits PMN apoptosis by affecting expression of a specific subset of anti- and proapoptotic genes and pro-survival factors, and that defects in clearance of infected PMNs by macrophages favors cell necrosis, sustains infection, and prevents the reprogramming of macrophages that is required for termination of the inflammatory response. To test this, we propose the following Specific Aims: 1. To elucidate the molecular mechanisms of apoptosis inhibition by F. tularensis with a focus on the BAX, XIAP and calpastatin. 2. To identify bacterial genes required for PMN apoptosis inhibition. 3. To elucidate the functional consequences of prolonged neutrophil lifespan. We expect that completion of the proposed studies will provide fundamental insight into the molecular mechanisms that account for the dysregulated inflammatory response that is characteristic of tularemia, and that our findings will also inform studies of other diseases that are also characterized by defects in PMN turnover, and affect Veterans more frequently, such as chromic obstructive pulmonary disease.
The high microbicidal capacity and short lifespan of neutrophils are critical for effective antimicrobial defense and regulation of the inflammatory response. Francisella tularensis is a bacterial pathogen that survives inside neutrophils and causes a rapidly progressive and potentially fatal pneumonia when inhaled into the lung. Our recent data indicate that F. tularensis profoundly impairs neutrophil turnover, and we believe that this, in turn, contributes t the profound tissue damage and destruction that characterizes this infection. The objective of this proposal is to define the underlying molecular mechanism. We expect that completion of the proposed studies will provide fundamental new insight into the pathogenesis of tularemia that will also inform studies of other disease states in which neutrophil apoptosis is perturbed, including chronic obstructive pulmonary disease which affects large numbers of people, including Veterans.
