Development and characterization of protein and derivative peptide endotoxin antagonists.

摘要

Patients with serious gram-negative bacterial infections frequently develop sepsis syndrome with attendant harmful sequelae that include multiple organ failure and death. Systemic elaboration of endotoxin (lipopolysaccharide, LPS), an integral component of the outer membrane of gram-negative bacteria, has been causally implicated in the development of sepsis syndrome. Despite critical care improvements such as aggressive resuscitation, early nutritional support, and judicious use of antibiotics, sepsis has an overall 40% mortality rate that has not appreciably changed over the past several decades. Bactericidal permeability-increasing protein (BPI) is a mammalian host defense protein found in the azurophilic granules of polymorphonuclear leukocytes that exhibits potent gram-negative bactericidal and LPS-neutralizing biological activity. However, the efficacy of BPI in vitro is limited by (1) an inability to access lipid A imbedded within the outer membrane of gram-negative bacteria, and (2) the presence of competing serum proteins that also bind LPS. Bactericidal and LPS-neutralizing biological activity of BPI, known to reside within its amino terminus, has been further localized to a distinct amino acid domain. While BPI is mammalian in origin, Limulus anti-LPS Factor (LALF) is a primitive LPS-binding protein derived from the horseshoe crab Limulus polyphemus that also exhibits gram-negative bactericidal and LPS-neutralizing biological activity. Interestingly, BPI and LALF share the following features within their active domains: (1) alternating hydrophobic and cationic amino acid residues and (2) β-turn secondary structure.; In this thesis small peptides representing minimal functional amino acid domains derived from naturally occurring LPS-binding proteins BPI and LALF are characterized in vitro for (1) the capacity to abrogate TNF-α secretion from LPS-stimulated macrophages, (2) bactericidal activity for P. aeruginosa. In vitro characterization examines LPS-neutralizing capacity of these same peptides through TNF-α abrogation and bacterial clearance during P. aeruginosa endotoxemia and bacteremia as well as protective capacity after lethal challenge with P. aeruginosa .