During infection, membrane components are shed from bacteria in complexes that include LPS and two bacterial membrane lipoproteins: murein lipoprotein (MLP), and peptidoglycans associated lipoprotein (PAL). This renewal proposal seeks to study the physical interactions that each of these bacterial membrane components have with each other and with host lipoproteins, and the relative contribution of each in activation of cellular and systemic inflammatory responses that are mediated through Toll-like receptors (TLRs). Our central hypothesis is that MLP and PAL contribute to the pathogenesis of sepsis, and that their activity in vivo may be altered by interactions with each other, with LPS, and with host proteins and lipoproteins. Whereas most previous work has studied chemically purified or synthetic TLR agonists, this work will study natural forms of MLP and PAL wherever possible. The first specific aim is to study the kinetics of release of MLP and PAL from the bacterial cell wall into bacterial membrane complexes and into low density forms in serum. In vitro studies will utilize human serum. In vivo studies will be performed in two models of infection in mice: peritoneal E. coli infection and cecal ligation and puncture. The physical association of MLP and PAL with subsets of lipoprotein particles containing LPS and apoA 1 and the distribution of MLP and PAL in organs and cells in tissues will be studied over time. The second specific aim is to compare the effects of serum released forms of MLP and PAL with chemically purified forms of each in vitro and in vivo. Effects will be evaluated using both macrophages and endothelial cells because these cells integrate the inflammaory response in sepsis. Bacterial strains deficient in MLP and PAL, and mice deficient in TLR2 and TLR4, will be used to distinguish the contributions of cellular activation by each bacterial lipoprotein and by LPS. Our third specific aim is to study the effect of anti-MLP IgG on the processes above and on survival in the two models. The experiments are designed to evaluate the importance that physiologically relevant forms of MLP and PAL play in the pathophysiology of sepsis during infection.
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