| Lipopolysaccharides (LPS), a major constituent of the outer membrane of Gram-negative bacteria, have been implicated as a contributory factor in many of the pathophysiologic symptoms of Gram-negative infections. The experiments reported in this dissertation were designed to: (i) examine the kinetics and extent of LPS release from intact bacteria after exposure to normal human serum (NHS); (ii) examine the serum components necessary for LPS release; (iii) characterize the physical nature of LPS released from bacteria by the action of NHS; and (iv) compare and contrast the biologic activities of serum released LPS (S-LPS) with phenol-water extracted LPS (PW-LPS). The serum-sensitive, UDP-galactose epimerase deficient mutant strain E. coli J5 was utilized to specifically radiolabel the LPS component of the outer membrane, and in this manner, the fate of LPS following incubation of bacteria in NHS was monitored.;Incubation of E. coli J5 in NHS at 37$spcirc$C results in a rapid loss of bacterial viability, which is accompanied by release of LPS from the bacteria. The extent of LPS released by serum is: (i) growth phase dependent; (ii) operative over a $>$100-fold bacterial concentration range; and (iii) not due solely to the depletion of active serum components. LPS release is finite; maximal LPS release from mid-logarithmic phase bacteria is consistently $sim$30% of the total radiolabel incorporated into the bacteria. Factors influencing the capacity of NHS to mediate LPS release include LPS subunit heterogeneity and the presence or absence of divalent cations. A serum-resistant mutant of E. coli fails to release LPS in response to incubation in NHS. LPS release is complement mediated. Both the alternative and classical pathways of complement may contribute to both bacterial killing and LPS release.;A portion of S-LPS exists in reversible association with serum proteins in a lower molecular weight form than that of PW-LPS, LPS co-elutes with two serum proteins with apparent molecular weights of approximately 68 and 32 kD. The LPS component of S-LPS and the LPS retained on the serum-treated bacteria, do not appear to be significantly different. Thus, it appears that one component of humoral "detoxification" of LPS may involve the diminution of the pathophysiologic effects of LPS, while enhancing the immunomodulatory effects. (Abstract shortened with permission of author.). |
