Deacylation of Lipopolysaccharide in Whole Escherichia coli during Destruction by Cellular and Extracellular Components of a Rabbit Peritoneal Inflammatory Exudate

Abstract

Deacylation of purified lipopolysaccharides (LPS)markedly reduces its toxicity toward mammals. However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established. We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS. We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting. Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone. Isolated mononuclear cells and neutrophils removed 50% and 20 -30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria. When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred. In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h. Deacylation of LPS was always restricted to the secondary acyl chains. Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria. This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.The prominent role ascribed to LPS 1 in evoking beneficial as well as harmful mammalian host responses to invading Gramnegative bacteria has prompted intense scrutiny of host recognition, detoxification, and elimination of this bacterial product. Underacylated and underphosphorylated biosynthetic precursors and chemically synthesized analogs of lipid A (the endotoxic part of LPS) possess greatly diminished endotoxic activity and can act as antagonists in human cell bioassays (1, 2). Mammalian enzyme systems capable of removing acyl and phosphate groups from lipid A may therefore play an important role in host defense against Gram-negative bacteria and their LPS. Thus far, only one mammalian enzyme, acyloxyacyl hydrolase, has been shown to deacylate LPS (3, 4).We recently showed that both the cells and extracellular fluid of a sterile inflammatory exudate actively deacylate purified LPS ex vivo in an acyloxyacyl hydrolase-like fashion (5). It has been much more difficult to analyze LPS breakdown products when the LPS is presented in intact bacteria...

The Carboxyl-terminal Domain of Closely Related Endotoxin-binding Proteins Determines the Target of Protein-Lipopolysaccharide Complexes

“…The targeting of BPI-coated bacteria and cell-free LPS to different cells should benefit host defense. Neutrophils are best equipped to quickly eliminate rapidly multiplying and disseminating organisms, whereas monocyte-like cells are better endowed with a digestive apparatus possibly important in detoxification or antigen presentation (42,67). It should be noted, however, that the extent of cellular uptake of BPI:LPS agg , even at relatively low LPS concentrations (Fig.…”

The bactericidal/permeability-increasing protein (BPI) in infection and inflammatory disease

“…Acyloxyacyl hydrolase converts hexa-acylated endotoxins that are potent TLR4 agonists to tetra-acylated species that are TLR4 antagonists [3,105,106]. Endotoxin associated with intact bacteria and bacterial remnants is also partially deacylated in the extracellular inflammatory fluid [88] but at a much slower rate. Thus, a more rapid detoxification mechanism may be needed in inflammatory fluids and could be mediated by locally mobilized endotoxin binding proteins, such as BPI [86].…”

“…During and after phagocytosis, the lipid A region within bacterial endotoxin is partially deacylated by the host enzyme acyloxyacyl hydrolase [88,105]. Acyloxyacyl hydrolase converts hexa-acylated endotoxins that are potent TLR4 agonists to tetra-acylated species that are TLR4 antagonists [3,105,106].…”

“…In addition, recruited and activated neutrophils release some of their granule contents into the surrounding inflammatory fluid [83][84][85][86]. The released proteins may contribute to containment and killing of extracellular bacteria that have exceeded or evaded/resisted the phagocytic capacity of the neutrophils and also to elimination of bioactive bacterial remnants [87,88].…”

Endotoxin-binding Proteins Modulate the Susceptibility of Bacterial Endotoxin to Deacylation by Acyloxyacyl Hydrolase

“…Tissue invasion by even minute quantities of many Gramnegative bacteria (GNB) 2 initiates rapid mobilization of the innate immune responses of the host. In these circumstances, both GNB recognition and many responses depend upon activation of the exquisitely sensitive Toll-like receptor 4 (TLR4) by endotoxins, structurally unique and abundant glycolipids that occupy much of the outer leaflet of the GNB outer membrane (3).…”