Host responses to lipopolysaccharides (LPS) or lipooligosaccharides (LOS) of Gram-negative bacteria (GNB) are believed to play a major role in defense against many invasive GNB infections. These normally protective responses can also cause life-threatening systematic inflammatory disorders when inadequately controlled. This is dramatically illustrated in fulminant meningococcal septicemia, an extreme acute disease that appears linked to the accumulation of extraordinary high levels of LOS. The broad long-term objectives of this project are to better understand the molecular determinants of endotoxin mobilization and of its interaction with defined host targets. A major focus will be on the biogenesis and biological properties of released bacterial membrane blebs, believed to represent an important of disseminated endotoxin in vivo and in a particularly prominent feature of growing N. meningitidis in vitro.
Our specific aims are to: I) Determine the effect of interaction of N. meningitidis with components of intravascular host defenses on the synthesis and mobilization of meningococcal LOS; II) Characterize the molecular determinants of delivery of purified LOS, membrane blebs and intact bacteria to polymorphonuclear leukocytes (PMN) and endothelial cells; and III) Compare the susceptibility of LOS presented in the forms of aggregates of purified LOS, membrane blebs and intact bacteria to host-mediated clearance and degradations. We will employ several experimental approaches to quantitatively analyze the metabolism and interactions of meningococcal LOS including define bacterial mutants to permit selective radiolabeling of bacterial (glyco)lipids, TLC/image analysis, HPLC, GC-MS and MALTI-TOF, purified endotoxin-binding proteins and neutralizing antibodies, and affinity purification to search for novel mediators of bacterial membrane bled-host interactions. We will draw heavily on collaborating investigators in this program who provide expertise and tools to study in an unusually systematic and controlled fashion the dynamics of meningococcal (endotoxin)-host interactions. These studies are likely to provide new insights concerning two fundamental questions: 1) What controls the mobilization of endo during the mobilization of endotoxin during bacterial interaction with the host? 2) How do the unique physical characteristics meningococcal LOS and of cell-free LOS-containing membrane from fragments affect the interaction of endotoxin with host machinery that mediate either pro- or anti- inflammatory responses?
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