Rickettsioses as infectious diseases stand out for their historic and continued impact on the global human health. Rocky Mountain spotted fever due to tick-transmitted Rickettsia rickettsii (Rr), a Gram-negative obligate intracellular ?-proteobacterium, is one of the most severe rickettsioses and a notifiable illness in the USA. An important determinant of disease pathogenesis in humans and established laboratory models of infection is disseminated infection of microvascular endothelial cell lining of blood vessels, resulting in ?rickettsial vasculitis? defined by inflammation of the vasculature and compromised vascular permeability leading to tissue fluid imbalance and edema of vital organs. Rr-induced endothelial responses include acquisition of pro-adhesive, pro-coagulant, and pro-inflammatory phenotypes and activation of master transcription regulator nuclear factor- ?B, yet the mechanisms underlying determination of innate immune responses and host cell fate represent a neglected area of fundamental relevance. Notably, potential roles of autophagy as a ?double-edged? host defense mechanism capable of supporting intracellular pathogens, anti-inflammatory endothelial responses to minimize vascular damage and dysfunction, and the balance of pro- vis--vis anti-inflammatory signaling as the biological basis of rickettsial virulence represent unexplored arenas of scientific enquiry. We present evidence suggesting increased phosphorylation of signal transducer and activator of transcription protein STAT3, a response coinciding with the onset of autophagy and activation of mechanistic target of rapamycin (mTOR) complexes C1 and C2 during Rr infection of human endothelial cells. Our intriguing preliminary findings and extensive published work on Rr-induced transcriptional activation serve as the basis of our hypothesis that mTOR functions as a central regulatory determinant of the balance of pro-inflammatory/pro-adhesive vis-a-vis anti-inflammatory innate immune responses of Rr-infected endothelium. Considering that mTOR signaling involves two structurally and functionally distinct complexes, Aim 1 will address the fundamentally important definition of the activation status of mTORC1 and mTORC2 during endothelial infection with Rr strains of varying virulence and determine the effects of their inhibition on host cell autophagy and rickettsial replication.
Aim 2 will then decipher potentially differential roles of mTORC1 and C2 in infection-induced inflammatory signaling. We will strategically employ contemporary, cutting-edge, and interdisciplinary approaches of cell and molecular biology to gain new and unique insights into host mTORC1/C2 activation in correlation with rickettsial virulence and their roles in governing innate immune responses via regulation of autophagy and pro- /anti-inflammatory signaling using in vitro and in vivo models of infection. The proposed analysis of pathogen interactions with novel signaling checkpoints capable of regulating beneficial and/or detrimental host responses will guide innovative therapeutic strategies to combat the sequelae associated with (re)emerging rickettsioses.
Mechanistic target of rapamycin (mTOR) is a key intracellular hub for the convergence of a multitude of signaling pathways regulating critical molecular processes including autophagy, innate immunity, and inflammation. The proposed work will pave the way to significant new knowledge of the regulation of mTOR complexes C1 (Raptor) and C2 (Rictor) during host cell interactions with virulent and avirulent strains of Rickettsia rickettsii. Since mTOR represents a stable pharmacologic target due to inherent resistance to mutation(s), manipulation of cellular responses under its control using newly developed modulators of mTOR offers an innovative stratagem for their use as novel therapeutics against intracellular pathogens.