Faculty Bibliography

We have shown previously that a group IIA phospholipase A2 (PLA2) is responsible for the potent bactericidal activity of inflammatory fluids against many Gram-positive bacteria. To exert its antibacterial activity, this PLA2 must first bind and traverse the bacterial cell wall to produce the extensive degradation of membrane phospholipids (PL) required for bacterial killing. In this study, we have examined the properties of the cell-wall that may determine the potency of group IIA PLA2 action. Inhibition of bacterial growth by nutrient deprivation or a bacteriostatic antibiotic reversibly increased bacterial resistance to PLA2-triggered PL degradation and killing. Conversely, pretreatment of Staphylococcus aureus or Enterococcus faecium with subinhibitory doses of beta-lactam antibiotics increased the rate and extent of PL degradation and/or bacterial killing after addition of PLA2. Isogenic wild-type (lyt+) and autolysis-deficient (lyt-) strains of S. aureus were equally sensitive to the phospholipolytic action of PLA2, but killing and lysis was much greater in the lyt+ strain. Thus, changes in cell-wall cross-linking and/or autolytic activity can modulate PLA2 action either by affecting enzyme access to membrane PL or by the coupling of massive PL degradation to autolysin-dependent killing and bacterial lysis or both. Taken together, these findings suggest that the bacterial envelope sites engaged in cell growth may represent preferential sites for the action and cytotoxic consequences of group IIA PLA2 attack against Gram-positive bacteria

The bactericidal/permeability-increasing protein (BPI) is a 456-residue cationic protein produced only by precursors of polymorphonuclear leukocytes (PMN) and is stored in the primary granules of these cells. The potent (nM) cytotoxicity of BPI is limited to gram-negative bacteria (GNB), reflecting the high affinity (<10 nM) of BPI for bacterial lipopolysaccharides (LPS). The biological effects of isolated BPI are linked to complex formation with LPS. Binding of BPI to live bacteria via LPS causes immediate growth arrest. Actual killing coincides with later damage to the inner membrane. Complex formation of BPI with cell-associated or cell-free LPS inhibits all LPS-induced host cell responses. BPI-blocking antibodies abolish the potent activity of whole PMN lysates and inflammatory fluids against BPI-sensitive GNB. The antibacterial and the anti-endotoxin activities of BPI are fully expressed by the amino terminal half of the molecule. These properties of BPI have prompted preclinical and subsequent clinical testing of recombinant amino-terminal fragments of BPI. In animals, human BPI protein products protect against lethal injections of isolated LPS and inocula of GNB. Phase I trials in healthy human volunteers and multiple Phase I/II clinical trials have been completed or are in progress (severe pediatric meningococcemia, hemorrhagic trauma, partial hepatectomy, severe peritoneal infections, and cystic fibrosis) and two phase III trials (meningococcemia and hemorrhagic trauma) have been initiated. In none of >900 normal and severely ill individuals have issues of safety or immunogenicity been encountered. Preliminary evidence points to overall benefit in BPI-treated patients. These results suggest that BPI may have a place in the treatment of life-threatening infections and conditions associated with bacteremia and endotoxemia

The inability to reduce the high mortality due to overwhelming bacterial infection and sepsis has prompted a search for new therapeutic agents. Among these may be a wide range of endogenous antibiotic polypeptides that are prominent components of effective antimicrobial host defences. One of these polypeptide antibiotics is the bactericidal/permeability-increasing protein (BPI), a protein of approximately 55kD which is present in human and other mammalian neutrophils. BPI is toxic for Gram-negative bacteria and binds to endotoxin, resulting in its clearance and neutralisation. A recombinant 21kD N-terminal BPI fragment is at least as active as holo-BPI and protects both animals and humans against the effects of Gram-negative infections and their complications. Phase II/III clinical trials in fulminant paediatric meningococcaemia, haemorrhagic trauma, hepatectomy and severe peritoneal infections are in progress

Much has been learned recently about the structure and function of 55 kDa bactericidal/permeability-increasing protein (BPI), a member of a genomically conserved lipid-interactive protein family. Analysis of BPI fragments and the crystal structure of human BPI have established that BPI consists of two functionally distinct domains: a potently antibacterial and anti-endotoxin amino-terminal domain (approximately 20 kDa) and a carboxy-terminal portion that imparts opsonic activity to BPI. A recombinant amino-terminal fragment (rBPI21) protects animals against the effects of Gram-negative bacteria and endotoxin. In man, rBPI21 is nontoxic and non-immunogenic and is in Phase II/III clinical trials with apparent therapeutic benefit

The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain of Escherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria

Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are closely related LPS-binding proteins whose binding to LPS has markedly different functional consequences. To gain better insight into the possible basis of these functional differences, the physical properties of LBP-LPS and BPI-LPS complexes have been compared in this study by sedimentation, light scattering, and fluorescence analyses. These studies reveal dramatic differences in the physical properties of LPS complexed to LBP versus BPI. They suggest that of the two proteins, only LBP can disperse LPS aggegates. However, BPI can enhance both the sedimentation velocity and apparent size of LPS aggregates while inhibiting LPS-LBP binding even at very low (1:40 to 1:20) BPI:LPS molar ratios

The bactericidal potency toward complement-resistant Escherichia coli of bactericidal/permeability-increasing protein (BPI) released from polymorphonuclear leukocytes (PMNs) in glycogen-induced inflammatory peritoneal exudates of rabbits is dependent on synergy with extracellular p15s. This synergy depends on the high molar ratio of p15s to BPI in the extracellular fluid (approximately 50:1), which greatly exceeds the intracellular ratio (approximately 5:1). To explore the possible basis of the greater accumulation of p15s in inflammatory fluid, we examined the subcellular localization of BPI and p15 in PMNs. Immunogold electron microscopy confirmed the storage of BPI in primary granules and showed that p15s are stored in secondary granules. Reverse-transcription polymerase chain reaction of density-fractionated rabbit bone marrow cells verified that p15s are expressed later than BPI during myeloid differentiation. As the inflammatory response evolves, p15 mRNA appears earlier in blood and exudate cells than mRNA for BPI, consistent with release of progressively less mature precursors from bone marrow. Finally, Ca(2+)-ionophore-mediated exocytosis of p15s occurs more readily than release of BPI. We therefore propose that localization of a synergistic partner of BPI (p15s) in more readily released secondary granules allows the neutrophil to mobilize potent BPI-dependent antibacterial activity extracellularly without significant depletion of intracellular BPI stores

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells

The cell-free fluid (ascitic fluid, AF) of a sterile inflammatory peritoneal exudate elicited in rabbits is potently bactericidal for complement-resistant gram-negative as well as gram-positive bacterial species. This activity is absent in plasma. We now show that essentially all activity in AF against Staphylococcus aureus is attributable to a group II 14-kD phospholipase A2 (PLA2), previously purified from AF in this laboratory. Antistaphylococcal activity of purified PLA2 and of whole AF containing a corresponding amount of PLA2 was comparable and blocked by anti-AF-PLA2 serum. At concentrations present in AF (approximately 10 nM), AF PLA2 kills > 2 logs of 10(6) S. aureus/ml, including methicillin-resistant clinical isolates, and other species of gram-positive bacteria. Human group II PLA2 displays similar bactericidal activity toward S. aureus (LD90 approximately 1-5 nM), whereas 14-kD PLA2 from pig pancreas and snake venom are inactive even at micromolar doses. Bacterial killing by PLA2 requires Ca2+ and catalytic activity and is accompanied by bacterial phospholipolysis and disruption of the bacterial cell membrane and cell wall. These findings reveal that group II extracellular PLA2, the function of which at inflammatory sites has been unclear, is an extraordinarily potent endogenous antibiotic against S. aureus and other gram-positive bacteria