RESUMO
Much of the very extensive literature describing the (bio)chemistry and biology of bacterial lipopolysaccharides (LPS, endotoxin) has dealt with the properties of these molecules as potent triggers of host responses. This brief review will focus on what has been learned recently about mechanisms by which the host can dispose of LPS and counter its often excessive stimulatory effects.
Assuntos
Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Infecções por Bactérias Gram-Negativas/patologia , Humanos , FagocitoseRESUMO
Killing of gram-positive bacteria by mammalian group IIA phospholipases A2 (PLA2) requires the catalytic activity of the enzyme. However, nearly complete degradation of the phospholipids can occur with little effect on bacterial viability, suggesting that PLA2-treated bacteria can biosynthetically replace phospholipids that are lost due to PLA2 action. In the presence of albumin, phospholipid degradation products are quantitatively sequestered extracellularly. In the absence of albumin, the bacteria retain and substantially reutilize the phospholipid breakdown products and survive an otherwise lethal dose of PLA2. PLA2-treated bacteria also continue to incorporate sodium [2-(14)C]acetate into phospholipids, suggesting that the bacteria are attempting to repair the damaged membranes by de novo synthesis of phospholipids. To determine whether PLA2 action also triggers activation of bacterial lipolytic enzymes, the effects of nisin and PLA2 on the degradation of S. aureus lipids were compared. In contrast to nisin treatment, PLA2 treatment does not stimulate endogenous phospholipase activity in S. aureus. These findings show that S. aureus responds to PLA2 attack by continued phospholipid (re)synthesis by both de novo and salvage pathways. The fate of PLA2-treated S. aureus therefore appears to depend on the relative rates of phospholipid degradation and synthesis.
Assuntos
Lipídeos de Membrana/biossíntese , Fosfolipases A/farmacologia , Fosfolipídeos/biossíntese , Staphylococcus aureus/efeitos dos fármacos , Acetatos/metabolismo , Albuminas/farmacologia , Animais , Lisofosfolipídeos/biossíntese , Fosfolipases A2 , Coelhos , Staphylococcus aureus/metabolismoAssuntos
Imunidade Inata , Plantas/imunologia , Animais , Humanos , Insetos/imunologia , Mamíferos/imunologia , Plantas/virologiaRESUMO
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.
Assuntos
Líquido Ascítico/imunologia , Líquido Ascítico/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Acilação , Animais , Escherichia coli , Lipopolissacarídeos/imunologia , Doenças Peritoneais/imunologia , Doenças Peritoneais/metabolismo , CoelhosRESUMO
The extent to which the mammalian host is capable of enzymatic degradation and detoxification of bacterial lipopolysaccharides (LPS) is still unknown. Partial deacylation of LPS by the enzyme acyloxyacyl hydrolase (AOAH) provides such a mechanism, but its participation in the disposal of LPS under physiological conditions has not been established. In this study, deacylation of isolated radiolabeled LPS by both cellular and extracellular components of a sterile inflammatory peritoneal exudate elicited in rabbits was examined ex vivo. AOAH-like activity, tested under artificial conditions (pH 5.4, 0.1% Triton X-100), was evident in all components of the exudate (mononuclear cells [MNC] > polymorphonuclear leukocytes [PMN] > inflammatory [ascitic] fluid [AF]). Under more physiological conditions, in a defined medium containing purified LPS-binding protein, the LPS-deacylating activity of MNC greatly exceeded that of PMN. In AF, MNC (but not PMN) also produced rapid and extensive CD14-dependent LPS deacylation. Under these conditions, almost all MNC-associated LPS underwent deacylation within 1 h, a rate greatly exceeding that previously found in any cell type. The remaining extracellular LPS was more slowly subject to CD14-independent deacylation in AF. Quantitative analysis showed a comparable release of laurate and myristate but no release of 3-hydroxymyristate, consistent with an AOAH-like activity. These findings suggest a major role for CD14(+) MNC and a secondary role for AF in the deacylation of cell-free LPS at extravascular inflammatory sites.
Assuntos
Líquido Ascítico/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Cavidade Peritoneal , Acilação , Animais , Biodegradação Ambiental , Cavidade Peritoneal/patologia , Cavidade Peritoneal/fisiopatologia , CoelhosRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Fosfolipases A/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Interações Medicamentosas , Lactamas , Fosfolipases A2RESUMO
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.
Assuntos
Proteínas Sanguíneas/fisiologia , Proteínas Sanguíneas/uso terapêutico , Proteínas de Membrana , Animais , Peptídeos Catiônicos Antimicrobianos , Atividade Bactericida do Sangue , Infecções por Bactérias Gram-Negativas/prevenção & controle , HumanosRESUMO
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.
Assuntos
Antibacterianos/imunologia , Proteínas Sanguíneas/imunologia , Proteínas de Membrana , Animais , Antibacterianos/química , Antibacterianos/classificação , Peptídeos Catiônicos Antimicrobianos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/classificação , Líquidos Corporais/imunologia , Cristalografia por Raios X , Humanos , Inflamação , Lipopolissacarídeos/imunologia , Fagocitose , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
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.
Assuntos
Atividade Bactericida do Sangue , Proteínas Sanguíneas/metabolismo , Proteínas de Membrana , Neutrófilos/metabolismo , Proteínas Opsonizantes/metabolismo , Peptídeos Catiônicos Antimicrobianos , Proteínas Sanguíneas/química , Escherichia coli/imunologia , Humanos , Microscopia Eletrônica , Ativação de Neutrófilo , FagocitoseRESUMO
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.
Assuntos
Proteínas de Fase Aguda , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana , Peptídeos Catiônicos Antimicrobianos , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Centrifugação com Gradiente de Concentração , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Luz , Substâncias Macromoleculares , Ligação Proteica , Espalhamento de Radiação , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Anti-Infecciosos/análise , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/farmacologia , Grânulos Citoplasmáticos/metabolismo , Granulócitos/metabolismo , Inflamação/metabolismo , Proteínas de Membrana , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Líquidos Corporais/metabolismo , Sinergismo Farmacológico , Granulócitos/ultraestrutura , Reação em Cadeia da Polimerase , CoelhosRESUMO
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.
Assuntos
Proteínas de Fase Aguda , Proteínas de Transporte/fisiologia , Escherichia coli/imunologia , Receptores de Lipopolissacarídeos/fisiologia , Lipopolissacarídeos/imunologia , Glicoproteínas de Membrana , Proteínas de Membrana , Neutrófilos/imunologia , Peptídeos Catiônicos Antimicrobianos , Aderência Bacteriana , Sequência de Bases , Atividade Bactericida do Sangue , Proteínas Sanguíneas/fisiologia , Células Cultivadas , Primers do DNA/química , Escherichia coli/patogenicidade , Humanos , Medições Luminescentes , Dados de Sequência Molecular , Antígenos O/imunologia , Fosfolipases A/metabolismo , Explosão Respiratória , Transdução de SinaisRESUMO
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.
Assuntos
Líquido Ascítico/enzimologia , Fosfolipases A/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Bactérias/efeitos dos fármacos , Atividade Bactericida do Sangue , Cálcio/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Humanos , Soros Imunes , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Peso Molecular , Testes de Neutralização , Fosfatidilgliceróis/metabolismo , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , CoelhosRESUMO
The bactericidal/permeability-increasing protein (BPI), a potent cytotoxin specific for Gram-negative bacteria and an endotoxin-neutralizing agent, is a major component of the antimicrobial arsenal of mammalian polymorphonuclear leukocytes. The antibacterial and endotoxin-neutralizing activities of the N-terminal portion (approximately 25 kDa) of BPI are at least equal to those of the holoprotein (approximately 50 kDa). Recombinant N-terminal fragments of BPI are antibacterial and inhibit host cell responses to endotoxin in whole blood ex vivo and in animal experiments. BPI administered to both animals and man is apparently nontoxic and nonimmunogenic and acts synergistically with some antibiotics. Thus, the prospects for the therapeutic use of bioactive BPI fragments in serious Gram-negative bacterial infections are highly encouraging.
Assuntos
Antibacterianos/uso terapêutico , Proteínas Sanguíneas/uso terapêutico , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Proteínas de Membrana , Animais , Peptídeos Catiônicos Antimicrobianos , HumanosRESUMO
Bactericidal/permeability-increasing protein (BPI), antibacterial 15-kDa protein isoforms (p15s), and defensins (neutrophil peptides or NPs) are granule-associated antibacterial proteins of polymorphonuclear leukocytes (PMN) that have both direct and synergistic growth inhibitory activity against Gram-negative bacteria. In this study, we have compared in vitro the abilities of these antibacterial proteins, alone and in combination, to inhibit the endotoxic activity of isolated LPS and whole bacteria. All three proteins blocked endotoxin activity in: 1) the Limulus amoebocyte lysate assay, 2) priming of PMN for enhanced arachidonate release, and 3) stimulating leukocyte oxidase activity in 1% blood. However, the proteins differ markedly in both relative potency (BPI >> p15s = NP1) in the presence of the plasma LPS-binding protein and in the range of LPS chemotypes that can be inhibited. BPI potently neutralizes LPS of any chemotype, but p15s and defensins are less active against long-chain (S-type) LPS. In whole blood ex vivo, the p15s and NP1 are approximately 1000-fold less potent than BPI, but at subinhibitory doses act in synergy with BPI to inhibit the TNF-inducing activity of a serum-resistant encapsulated strain of Escherichia coli (K1/r). The anti-endotoxic effects of p15 and NP1 against E. coli K1/r in whole blood appear secondary to growth arrest, because, in marked contrast to BPI, they are not evident against nonviable bacteria (pretreated with antibiotic) nor isolated LPS. Thus, BPI stands out for its ability to inhibit isolated or bacterial LPS under physiologic conditions. However, p15s and defensins may also contribute to suppression of endotoxic signaling by Gram-negative bacteria via synergistic (with BPI) growth inhibition upon extracellular release of these proteins from PMN during inflammation.
Assuntos
Proteínas Sanguíneas/fisiologia , Escherichia coli/imunologia , Lipopolissacarídeos/imunologia , Proteínas de Membrana , Neutrófilos/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos , Defensinas , Humanos , Teste do Limulus , Medições Luminescentes , Coelhos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
To what extent the host defense role of granule-associated antibacterial proteins and peptides of PMN includes extracellular action has not been established. To address this question, we have analyzed the antibacterial activity of cell-free (ascitic) fluid (AF) obtained from glycogen-induced sterile inflammatory rabbit peritoneal exudates in which > 95% of the accumulating cells are PMN. AF, but not plasma collected in parallel, exhibits potent activity toward serum-resistant Gram-negative and Gram-positive bacteria. Total and specific antibacterial activity of AF increases during the first 12 h after injection of glycogen in parallel with the influx of PMN. At maximum, > 99% of 10(7) encapsulated Escherichia coli and Staphylococcus aureus are killed in 30 min/ml of AF. Neutralizing antibodies against the bactericidal/permeability-increasing protein (BPI) of PMN abolishes activity of AF toward encapsulated E. coli but has no effect on activity vs staphylococci. However, BPI alone (approximately 1 microgram/ml in AF) can only account for < or = 20% of AF activity toward E. coli. AF also contains 15 kD PMN proteins (p15s) that act in synergy with BPI. Purified BPI and p15s, in amounts present in AF, reconstitute the growth-inhibitory activity of AF toward encapsulated E. coli. These findings show for the first time an extracellular function of endogenous BPI, providing, together with the p15s, a potent microbicidal system toward Gram-negative bacteria resistant to plasma-derived proteins and phagocytes in inflammatory exudates.
Assuntos
Antibacterianos/isolamento & purificação , Líquido Ascítico/química , Atividade Bactericida do Sangue , Proteínas Sanguíneas/isolamento & purificação , Proteínas de Membrana , Neutrófilos/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos , Líquido Ascítico/citologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inflamação/metabolismo , Testes de Sensibilidade Microbiana , CoelhosRESUMO
Extracellular 14-kDa phospholipases A2 (PLA2) in inflammatory exudates can contribute to bacterial phospholipid (PL) degradation during phagocytosis of Escherichia coli by polymorphonuclear leukocytes (PMN) and are highly active toward E. coli treated with the bactericidal/permeability-increasing protein (BPI) purified from PMN. PLA2 activity toward BPI-treated E. coli varies greatly among members of this conserved family of enzymes and apparently depends on a cluster of basic residues in a variable surface region near the NH2 terminus for recognition of this biological target (Weiss, J., Wright, G.W., Bekkers, A.C.A.P.A., van den Bergh, C.J., and Verheij, H.M. (1991) J. Biol. Chem. 266, 4162-4167). We have examined by site-specific mutagenesis of a recombinant PLA2 that is identical to an enzyme in human synovial fluid (containing His-6, Arg-7, Lys-10, and Lys-15 and a global net charge of +15) the role of basic residues in this region in PLA2 action against PLA-deficient (pldA-) E. coli. Substitution of Ser for Arg-7 +/- Gln for Lys-15 caused, respectively, about a 10- and 25-fold reduction in BPI-dependent PLA2 binding and activity to E. coli, but had no effect on hydrolysis of PL of autoclaved E. coli or dispersions of purified PL. PL degradation during phagocytosis was increased after pretreatment of E. coli (or PMN) with wild-type PLA2 followed by removal of unbound PLA2. Thus, the PLA2 binds to cells before phagocytosis followed by internalization of the enzyme along with E. coli and intracellular action. Mutant (e.g. R7S +/- K15Q) PLA2 show the same BPI-independent binding to E. coli as the wild-type enzyme but 10-30-fold reduced activity during phagocytosis, reflecting lower intracellular activity of these enzymes. Thus, structural determinants first implicated in PLA2 action toward E. coli treated with purified BPI apparently are also important in the intracellular action of PLA2 during phagocytosis by PMN.
Assuntos
Atividade Bactericida do Sangue , Proteínas Sanguíneas/fisiologia , Escherichia coli/efeitos dos fármacos , Inflamação/enzimologia , Proteínas de Membrana , Neutrófilos/fisiologia , Fosfolipases A/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos , Escherichia coli/imunologia , Humanos , Mutagênese Sítio-Dirigida , Fagocitose , Fosfolipases A/química , Fosfolipases A2 , Coelhos , Relação Estrutura-AtividadeRESUMO
Our understanding of the complex and integrated host-defense systems against microbial infection has progressed rapidly with the characterization of individual components. However, the various factors must be studied not only in isolation, but also in a closer approximation to the in vivo situation, where these factors interact. This is well illustrated in recent studies of the role of the bactericidal/permeability-increasing protein.