Project Summary/Abstract
Spotted fever group rickettsioses (SFGR) are in aggregate the second most common tick-borne infections in the U.S. and
account for considerable severe disease and death, with case fatality rates over 10% in many regions of the world. Rocky
Mountain spotted fever is the prototypical disease in this group for which the major pathophysiologic adverse consequence
is increased vascular permeability, leading to hypotension, hypoperfusion, and ischemic injury to the lungs, brain and other
organs. SFG rickettsiae infect endothelial cells and parasitize host ATP for growth and spread. Our preliminary studies
showed that regulation of calcium flux in infected endothelial cell barriers using calcium chelators or specific calcium
channel blockers abrogates vascular permeability. We propose that spotted fever rickettsiae utilize a nutritional virulence
strategy to subvert host calcium handling – through an acquired “channelopathy” - that drives increases in access to cellular
metabolic substrates, but that in turn damages key functions of endothelial cells. Our hypothesis is that increasing
competition for cellular ATP by rickettsiae impairs ATP-dependent Ca2+ pumps, which in turn leads to increased
intracellular calcium concentrations, activation of Ca2+ channels, Ca2+-dependent kinases, signaling pathways and
transcriptional regulation that increase cellular content of key nutrients for rickettsial growth. The consequences of improved
rickettsial fitness ultimately leads to reorganization of cellular cytoskeleton, disassembly of inter-endothelial junctions, and
inevitably, increased vascular permeability. The proposal will measure intracellular calcium and ATP concentrations in the
presence or absence of calcium channel-blocking agent, while monitoring for endothelial barrier dysfunction in both early
and late stages of Rickettsia parkeri infection. Evidence of a role for calcium flux in rickettsial vascular permeability
includes localized calcium “puffs” with transient focal cytosolic ATP depletion and local cytoskeletal restructuring as early
post-invasion events. This is predicted to be followed by global increases in intracellular calcium, ATP depletion,
cytoskeleton restructuring, and inter-endothelial junction disassembly with massive rickettsial proliferation, sensitive to
buffering cytosolic calcium concentrations through chelation or blocking or silencing of key calcium channels. Rickettsial
load will also be determined to understand the effect of these manipulations on microbial fitness. This work will identify
key mechanisms that permit changes in vascular permeability with spotted fever rickettsia infection, perhaps driven by
rickettsial effector proteins, and potential targets for host-based pharmacologic intervention to prevent severe and fatal
outcomes of SFGR and potentially other infectious diseases.