Complement-mediated killing of Escherichia coli: dissipation of membrane potential by a C9-derived peptide

The molecular mechanism of complement-mediated killing of Gram-negative bacteria has yet to be resolved, but it is generally accepted that assembly of the membrane attack complex (MAC) of complement on the outer bacterial membrane is a required step. We have now investigated the effect of the MAC and its precursor complex, C5b-8, on the membrane potential (delta Em) across the inner bacterial membrane. Delta Em of whole cells was measured directly by using a lipophilic cation (tetraphenylphosphonium) that equilibrates with the potential or indirectly by measuring transport of solutes (proline and galactoside), which is dependent on delta Em. Our results indicate that the C5b-8 complex caused a transient collapse of delta Em in the absence of cell killing. Addition of C9 to allow formation of the MAC dissipated delta Em irreversibly, and the cells were killed. Since delta Em is generated across the inner membrane in Gram-negative bacteria, inner membrane vesicles were prepared and membrane potentials were generated either by adding D-lactate to energize the electron-transport chain or by creating a K+ diffusion potential with valinomycin. C9 added in the absence of earlier acting complement proteins had no effect on delta Em of isolated, actively respiring vesicles or on K+ diffusion potentials. In contrast, its C-terminal thrombin fragment (C9b), which has been shown earlier to contain the membrane-active domain of C9, efficiently collapsed delta Em in such vesicles. C9b did not require a specific receptor since it was effective on "right-side-out" and "inside-out" vesicles. These results are interpreted to indicate that a C9-derived fragment deenergizes cells and may be the causative agent for cell death.     

Transfer of preformed terminal C5b-9 complement complexes into the outer membrane of viable gram-negative bacteria: effect on viability and integrity

An efficient fusion system between Gram-negative bacteria and liposomes incorporating detergent-extracted C5b-9 complexes has been developed that allows delivery of preformed terminal complexes to the cell envelope (Tomlinson et al., 1989b). Fusion of Salmonella minnesota Re595 and Escherichia coli 17 with C5b-9-incorporated liposomes resulted in the transfer of 1900 C5b-9 complexes to each target bacterial cell. No loss in viability of bacteria was observed following fusion, even though the deposotion of 900 complexes onto the envelope following exposure to lysozyme-free serum effected a greater than 99% loss of viability. Increased sensitivity to antibiotics normally excluded from the cell by an integral outer membrane (OM), as well as the ability of the chromogenic substrate PADAC to gain access to periplasmically located beta-lactamase, indicated that transferred C5b-9 complexes functioned as water-filled channels through the OM. A similar conclusion was drawn from measurements demonstrating the uptake by cells of the lipophilic cation tetraphenylphosphonium (bromide), a result further indicating that the membrane potential across the cytoplasmic membrane was maintained following C5b-9 transfer to the OM. Examination of S. minnesota Re595 by electron microscopy revealed no obvious difference between cells exposed to lethal concentrations of lysozyme-free serum and cells following fusion with C5b-9-incorporated liposomes. These data suggest either that there are critical sites in the OM to which liposome-delivered C5b-9 complexes are unable to gain access or that bacterial cell death is related to events occurring during polymerization of C9 on the cell surface.     

Serum-resistant strains of Borrelia burgdorferi evade complement-mediated killing by expressing a CD59-like complement inhibitory molecule

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab')(2) anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8 beta.     

Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing

We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.     

Studies on the mechanism of bacterial resistance to complement-mediated killing. VI. IgG increases the bactericidal efficiency of C5b-9 for E. coli 0111B4 by acting at a step before C5 cleavage

Our previous experiments showed that immune IgG and F(ab')2, but not Fab', mediated serum killing of Escherichia coli 0111B4, strain 12015 (12015), without significantly increasing the extent of terminal complement (C) component attachment to the bacterial surface. We concluded that bactericidal antibody must change either the site or the nature of C5b-9 bacterial attachment. To pursue this possibility, conditions necessary for elution of C5b-9 from the bacterial surface were examined. Forty-two to 44% of 125I-C9 was released from the serum-resistant nonpresensitized 12015 by 1 M NaCl or 0.1% trypsin, compared with the 21 to 24% release from the serum-sensitive presensitized isolate under the same condition. When strain 12015 bearing 125I-C9 was lysed in a French pressure cell, 73.1% of 125I-C9 was released with the capsular fraction if the organisms had not been presensitized. In contrast, on presensitized 12015, 70.2% of 125I-C9 remained associated with the outer membrane after such lysis. These results suggested that C5b-9 was trapped within or underneath the capsule of 12015 in the absence of bactericidal antibody, but that addition of antibody led to C5b-9 insertion into the outer membrane with bacterial killing. The requirement of C components preceding C5 for bacterial killing was next examined. Minimal killing of presensitized 12015 occurred when a terminal C complex was formed by acid activation from purified C5, C6, C7, C8, and C9 in the absence of C3 or earlier components. In contrast, between 1.2 and 3 log killing of nonpresensitized rough Salmonella minnesota and rough E. coli was observed in the same system. Killing of 12015 was examined with bacteria incubated in C5-deficient serum (C5D), followed by washing and the addition of purified C5, C6, C7, C8, and C9 to permit C5b-9 formation. Antibody was added before or after incubation in C5D serum, or after the addition of purified C5-C9. Under conditions of equivalent C3 and C9 binding, significant killing occurred only when antibody was added before incubation in C5D serum. These results show that antibody must be present at or before the time of C5 convertase formation to mediate killing of 12015 by C5b-9. Therefore, antibody is unlikely to be functioning primarily to alter the bacterial surface to expose sites for C5b-9 insertion, nor is the effect of antibody simply to increase C3 and terminal component binding. We postulate that antibody mediates killing of 12015 by localizing C5b-9 around antibody-clustered sites of C3 and C5 convertase formation.     

Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.     

Activation of glomerular mesangial cells by the terminal membrane attack complex of complement

Treatment of cultured renal glomerular mesangial cells (MC) with nonlytic concentrations of the purified components (C5b-9) of the terminal membrane attack complex (MAC) of complement induced significant functional alterations characteristic of cellular activation. C5b-9-treated MC released large quantities of primarily vasodilatory prostaglandins. In addition, the secretion of an MC-derived auto-growth factor (MC interleukin 1) was greatly enhanced. Examination of the action of C5b-9 on MC phospholipid metabolism indicated that complement induced the activation of phospholipases, leading to quantitative changes in the fatty acid profile of MC membrane phospholipids. These findings demonstrate that cultured MC are highly responsive to nonlytic concentrations of the C5b-9 complex, and suggest that the mesangial deposition of the MAC in many forms of glomerular disease, with resultant cellular activation, may play a major role in the hemodynamic and cellular proliferative events characteristic of these disorders.     

Complement-mediated killing of the Lyme disease spirochete Borrelia burgdorferi. Role of antibody in formation of an effective membrane attack complex

Lyme disease is a multisystemic illness caused by the spirochete Borrelia burgdorferi. In the absence of specific antibody, the spirochete is resistant to the bactericidal activity of C, despite the capacity of B. burgdorferi to activate both C pathways. We examined the mechanism of serum resistance by measuring the deposition of C3 and terminal C components on B. burgdorferi in the presence and absence of immune IgG. In normal human serum antibody-sensitized borreliae bound similar amounts of C3, and similar or increased amounts of C8 and C9, in comparison to unsensitized bacteria. However, at comparable levels of C3, C8, or C9 uptake, only sensitized bacteria were killed. The requirement of antibody for killing could not be explained by differences in the rate of C deposition or by differences in the C9 to C8 ratio in the membrane attack complex (MAC). We found that bacteria incubated in C5-depleted human serum, but not in C6-depleted serum, were killed when this treatment was followed by antibody and the missing C components. Bacteria were also killed by reactive lysis (C5b-9) provided that antibody was present. Therefore, the effect of bactericidal IgG occurred at the stage of C5b binding to the bacterial surface. Elution studies of bound C9 indicated that the MAC was stably bound to the outer membrane of B. burgdorferi, whether or not the bacteria were treated with antibody. However, treatment with 0.1% trypsin released 48% of 125I-C9 from the surface of unsensitized borreliae and 24% from IgG-sensitized cells, demonstrating that the presence of the antibody changed the accessibility to trypsin of C9 in the MAC. These results indicate that the effect of antibody in the killing process is not to enhance the rate or extent of initial or terminal component binding, but rather to alter the bacterial outer membrane to allow effective MAC formation.     

Formation of ion-conducting channels by the membrane attack complex proteins of complement.

The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different.     

Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.     

Assembly of the membrane attack complex of complement on small unilamellar phospholipid vesicles

Light-scattering intensity was shown to be a reliable, direct, and quantitative technique for monitoring the assembly of the membrane attack complex of complement (proteins C5b-6, C7, C8, and C9) on small unilamellar phosphatidylcholine vesicles. The assembly on vesicles occurred in a simple fashion; complexes of C5b-7 bound noncooperatively to the vesicles, and final assembly of C5b-9 did not induce vesicle aggregation or fragmentation. When C5b-6 and C7 were mixed in the presence of vesicles but at molar protein/vesicle ratios of less than 1, there was quantitative binding of C5b-7 to the vesicles with no concomitant aggregation of C5b-7. If C7 was added at a slower rate, quantitative binding was obtained at molar C5b-7/vesicle ratios of up to 5. The latter observations (a) were consistent with the proposal that C5b-7 aggregation and membrane binding were competitive events and (b) defined conditions under which light-scattering intensity measurements could monitor C5b-9 assembly on vesicles without contribution from the fluid-phase assembly. The C8/C5b-7 ratio in the phospholipid-C5b-8 complex was 0.97 +/- 0.12, and the maximum ratio of C9/C5b-8 in the final complex was 16.2 +/- 2.0. One C9 molecule associated rapidly with each phospholipid-C5b-8, followed by slower incorporation of the remaining C9 molecules. The initial velocity of the slow phase of C9 addition was easily saturated with C9 and gave an activation energy of 37 kcal/mol. This was identical with the value measured for the analogous process in the fluid-phase assembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membranolysis by the ninth component of human complement

The lytic property of isolated C9 was shown by using heat (48°C) to induce polymerization of C9, which was then exposed to egg lecithin vesicles containing carboxyfluorescein. In this environment, C9 penetrated the vesicles and released the marker. C5b-9 and C5b-8 also released carboxyfluorescein at 48°C, as did C5b-7 to a lesser extent. However, neither isolated C5b-6, C7, C8 nor albumin caused such release. At 30°C, the C9 remained in the monomeric form and could not lyse the vesicles.C9 polymers formed at 48°C were ring-shaped with an internal diameter of approximately 100 Å and an outer diameter of approximately 180 Å. These rings of C9 polymers strikingly resembled the ultrastructures formed by the membrane attack complex of complement, viewed from the top.Our results indicate that polymeric C9 causes the membranolysis of phospholipid vesicles and, hence, that C9 alone is a cytotoxic molecule.     

Distinct localization of the complement C5b‐9 complex on Gram‐positive bacteria

The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo‐attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b‐9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram‐positive bacteria are protected from MAC‐dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram‐positive pathogens secrete small proteins that inhibit C5b‐9 formation. In this study, we found that complement activation on Gram‐positive bacteria in serum results in specific surface deposition of C5b‐9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS‐stable) forms, indicating the presence of ring‐structured C5b‐9. Surprisingly, confocal microscopy demonstrated that C5b‐9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b‐9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b‐9 complex and Gram‐positive bacteria, whichmight ultimately lead to a new model of MAC assembly and functioning.     

Inhibition of homologous complement by CD59 is mediated by a species-selective recognition conferred through binding to C8 within C5b-8 or C9 within C5b-9.

The capacity of the human complement regulatory protein CD59 to interact with terminal complement proteins in a species-selective manner was examined. When incorporated into chicken E, CD59 (purified from human E membranes) inhibited the cytolytic activity of the C5b-9 complex in a manner dependent on the species of origin of C8 and C9. Inhibition of C5b-9-mediated hemolysis was maximal when C8 and C9 were derived from human (hu) or baboon serum. By contrast, CD59 showed reduced activity when C8 and C9 were derived from dog or sheep serum, and no activity when C8 and C9 were derived from either rabbit or guinea pig (gp) serum. Similar specificity on the basis of the species of origin of C8 and C9 was also observed for CD59 endogenous to the human E membrane, using functionally blocking antibody against this cell surface protein to selectively abrogate its C5b-9-inhibitory activity. When E bearing human CD59 were exposed to C5b-8hu, CD59 was found to inhibit C5b-9-mediated lysis, regardless of the species of origin of C9, suggesting that the inhibitory function of CD59 can be mediated through recognition of species-specific domains expressed by human C8. Consistent with this interpretation, CD59 was found to bind to C5b-8hu but not to C5b67hu or C5b67huC8gp. Although CD59 failed to inhibit hemolysis mediated by C5b67huC8gpC9gp, its inhibitory function was observed for C5b67huC8gpC9hu, suggesting that, in addition to its interaction with C5b-8hu, CD59 also interacts in a species-selective manner with C9hu incorporated into C5b-9. Consistent with this interpretation, CD59 was found to bind both C5b67huC8gpC9hu and C5b-8huC9gp, but not C5b67huC8gpC9gp. Taken together, these data suggest that the capacity of CD59 to restrict the hemolytic activity of human serum complement involves a species-selective interaction of CD59, which involves binding to both the C8 and C9 components of the membrane attack complex. Although CD59 expresses selectivity for C8 and C9 of human origin, this "homologous restriction" is not absolute, and this human complement regulatory protein retains functional activity toward C8 and C9 of some nonprimate species.     

Mortalin/GRP75 Binds to Complement C9 and Plays a Role in Resistance to Complement-dependent Cytotoxicity

Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.     

Fluid-phase assembly of the membrane attack complex of complement

The dynamics and protein stoichiometry of the fluid-phase assembly of the membrane attack complex of complement were characterized by using light-scattering intensity measurements. The assembly proceeded in an ordered manner with generation of stable and highly reproducible intermediates. In the absence of phospholipid or C8, mixtures of C5b-6 and C7 self-associated to fluid phase-C5b-7 which had a weight-average molecular weight of (4.1 +/- 0.2) X 10(6). This corresponded to an average of nine C5b-7 complexes per particle. The particles appeared heterodisperse on sucrose gradients with S20,W values ranging from 21 to 39 S. Addition of C8 and C9 caused no further aggregation or disassembly of the particles. When excess C8 was added to the aggregated C5b-7, the ratio of C8 incorporated per C5b-7 moiety was 0.98 +/- 0.03. At saturating levels of C9, the C9/C5b-8 ratio in the particles was 7.2 +/- 0.6. Incorporation of C8 caused a small increase in the Z-averaged particle diffusion coefficient [(9.9-10.3) X 10(-8) cm2/s], indicating that it added in a manner that "filled in the gaps" in the C5b-7 particles. C9 caused only small decreases in the particle diffusion coefficient and substantially decreased the f/fmin ratio. The time course for C9 incorporation into fluid phase-C5b-8 indicated an initial rapid phase followed by a slow phase. The rapid phase corresponded to the incorporation of about one C9 for every two C5b-8 complexes. This suggested that one C9 binding site was accessible on about half of the C5b-8 complexes. This may imply that only about half of the C5b-8 complexes were capable of C9 polymerization so that the ratio of C9 incorporated per functional C5b-8 was (14 +/- 2)/1. The initial velocity of the slow phase of C9 addition gave an activation energy of 37 kcal/mol. The activation energy for C5b-8-independent polymerization of C9 had a similar value of 41 kcal/mol. Light-scattering intensity measurements seemed to be a highly reliable method for quantitative characterization of the fluid-phase assembly.     

Staphylococcal complement inhibitor: structure and active sites

The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 A resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31-Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.     

Kinetics of polymerization of a fluoresceinated derivative of complement protein C9 by the membrane-bound complex of complement proteins C5b-8

The fluorescence self-quenching by energy transfer of FITC-C9, a fluoresceinated derivative of human complement protein C9 [Sims, P.J. (1984) Biochemistry (preceding paper in this issue)], has been used to monitor the kinetics of C9 polymerization induced by the membrane-associated complex of complement proteins C5b-8. Time-based measurements of the fluorescence change observed during incubation of FITC-C9 with C5b-8-treated sheep red blood cell ghost membranes at various temperatures revealed that C9 polymerization induced by the C5b-8 proteins exhibits a temperature dependence similar to that previously reported for the complement-mediated hemolysis of these cells, with an Arrhenius activation energy for FITC-C9 polymerization of 13.3 +/- 3.2 kcal mol-1 (mean +/- 2 SD). Similar measurements obtained with C5b-8-treated unilamellar vesicles composed of either egg yolk phosphatidylcholine (egg PC), dipalmitoylphosphatidylcholine (DPPC), or dimyristoylphosphatidylcholine (DMPC) revealed activation energies of between 20 and 25 kcal mol-1 for FITC-C9 polymerization by C5b-8 bound to these membranes. Temperature-dependent rates of C9 polymerization were observed to be largely unaffected by the phase state of membrane lipid in the target C5b-8 vesicles. The significance of these observations of the mechanism of C9 activation of membrane insertion is considered.     

Elimination of terminal complement complexes in the plasma membrane of nucleated cells: influence of extracellular Ca2+ and association with cellular Ca2+

Nucleated cells, unlike erythrocytes, are able to survive limited complement attack by eliminating potentially cytolytic complement channels from the plasma membrane (PM) by processes that involve, plasma membrane (PM) by processes that involve, but may not be limited to, endocytosis. The observation that C5b-9 channels, as well as C5b-8 and C5b-7 intermediates, are rapidly eliminated from the cell surface of nucleated cells has prompted us to examine whether terminal complement complexes stimulate membrane events that lead to accelerated elimination of these complexes. We have suggested previously that ion flux through terminal complement complexes might influence the rate of elimination on the basis of our finding that terminal complement complexes with larger functional channel sizes are more rapidly eliminated. In this study, we examined the role of Ca2+ on the elimination rate of terminal complement complexes in the PM of Ehrlich cells, because changes in Ca2+ flux across the PM are known to influence many metabolic activities including endocytosis. To determine the elimination rate for terminal complement complexes by functional analysis, cells bearing C5b-7 or C5b-8 complexes with or without a sublytic dose of C9 were incubated at 37 degrees C for various time intervals before converting the remaining complexes to lytic C5b-9 channels. The initial elimination rates for the terminal complement complexes were compared in the presence of 0.015, 0.15, and 1.5 mM CaCl2 in the medium. Sufficient lowering of the extracellular Ca2+ concentration, (Ca2+)o, resulted in prolonging the elimination of each of the terminal complement complexes to a different extent. The effect of (Ca2+)o on the elimination rate was most pronounced for C5b-8 in the presence of a sublytic number of C5b-9, with less of an effect on C5b-8 alone, and the least effect with C5b-7. The elimination rates for terminal complement complexes were also determined by measuring the persistence of C5b antigen on the cell surface at 37 degrees C in the presence of various (Ca2+)o by using fluorescence-activated cell sorter analysis and were comparable with that obtained by functional analysis. Examination of the effect of terminal complement complexes on the cellular Ca2+ concentration, (Ca2+)i, revealed that these complexes increased the (Ca2+)i in proportion with the known functional pore size of the terminal complement complex in the PM. In addition, Quin 2, which can buffer internal Ca2+ transients, was found to increase the susceptibility of Ehrlich cells to lysis by C5b-9, further suggesting a relationship between the (Ca2+)i and the elimination process.(ABSTRACT TRUNCATED AT 400 WORDS)