Presence Of Mg-EGTA Research Articles

Role of outer membrane proteins in serum resistance of A. actinomycetemcomitans and A. aphrophilus

ABSTRACTThe aim of this project was to study how outer membrane proteins contribute to serum resistance in A. actinomycetemcomitans and A. aphrophilus.Genes encoding Omp100 (A. actinomycetemcomitans) and OmpA (A. actinomycetemcomitans and A. aphrophilus) were knocked out through homologous recombination.Serum resistance was tested by incubating the bacterial strains in normal human serum (NHS; 50%) for 2h at 37°C. As control heat-inactivated (56°C) NHS was used. LC-MS/MS analysis was used to identify proteins in selected bands from SDS-PAGE.Whereas Omp100 appeared not to contribute significantly to serum resistance, we observed that the ompA mutants of both species had a markedly reduced capability to survive in 50% NHS. In presence of Mg-EGTA the ompA mutants survived clearly better in NHS suggesting that OmpA may act through the classical pathway of the complement system. Interestingly, when incubating A. actinomycetemcomitans ompA mutants in NHS, derivatives were obtained that had regained serum resistance. LC-MS/MS analysis revealed that these strains produced high levels of an OmpA-like protein (76% amino acid identity).Further experiments to investigate how OmpA and the OmpA-like protein may interact with complement system are being conducted.

The level of mannan-binding protein regulates the binding of complement-derived opsonins to mannan and zymosan at low serum concentrations

When sera diluted to 5% in a buffer containing calcium and magnesium were incubated with mannan-coated ELISA plates, C4 fragments, properdin and factor B were bound to the plates as well as the expected opsonic C3 fragments, C3b and C3bi. The calcium-dependent lectin mannan-binding protein, which is structurally similar to C1q, was also shown to bind in this assay and analysis of sera from 179 healthy blood donors revealed that the binding levels of all these proteins were highly significantly correlated. Results obtained with a previously described C3b opsonic assay using zymosan also correlated with the mannan-binding levels. When the sera were diluted to 5% in the presence of Mg-EGTA there was no detectable binding of complement proteins to the mannan surface, confirming that no alternative pathway activation occurred at this serum concentration. When sera were diluted to 5% in a buffer containing EDTA in order to study immunoglobulin binding in the absence of complement activation, the levels of bound IgG1, IgG2, IgG3, IgA and IgM antibodies were found to be completely unrelated to the C3bi binding levels previously observed. The results suggest that in this experimental system using low concentrations of serum, mannan-binding protein initiates an antibody-independent mechanism of cleavage of the classical pathway component C4, which subsequently regulates the degree of cleavage of C3 and recruitment of alternative pathway proteins.

Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland.

Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To study in vitro the bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonized Staphylococcus aureus (S. aureus) and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonized S. aureus and MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 on S. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg-EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf-dependent, antibody-independent and complement-mediated phagocytic killing of S. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.

Complement 1 inhibitor is a regulator of the alternative complement pathway.

We studied complement 1 inhibitor (C1-INH) as an inhibitor of the alternative complement pathway. C1-INH prevented lysis, induced by the alternative complement pathway, of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes in human serum. It inhibited the binding of both factors B and C3 to PNH and rabbit erythrocytes and blocked the ability of factor B to restore alternative-pathway function in factor B-depleted serum. C1-INH did not bind to factors B or D but did bind to immobilized C3b and cobra venom factor (CVF), a C3b analogue. C1-INH prevented factor B from binding to CVF-coated beads and dissociated bound factor B from such beads. Factor B and C1-INH showed cross competition in binding to CVF-coated beads. Factor D cleaved factor B into Bb and Ba in the presence of C3b. Cleavage was markedly inhibited when C3b was preincubated with C1-INH. C1-INH inhibited the formation of CVFBb and decreased the C3 cleavage. Removal of C1-INH from serum, in the presence of Mg-EGTA with an anti-C1-INH immunoabsorbant, markedly increased alternative-pathway lysis. C1-INH interacts with C3b to inhibit binding of factor B to C3b. At physiologic concentrations, it is a downregulator of the alternative pathway convertase.

Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis.

Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.

Characterization of group A streptococcal strains Sv and Su: determination of emm gene typing and presence of small vir regulon

The emm gene typing of GAS (group A streptococcus) strains Sv and Su and the molecular structure of the vir regulon were decided. An emm(-like) gene from the chromosomal DNA of GAS strain Sv was amplified with forward and reverse primers, which were selected from the best conserved portion in leader sequences of different strains and the C-terminal conserved portion, respectively, for determination of the M protein gene type. Strain Sv was defined as serotype M23, because deduced N-terminal amino acid positions of the products are identical to those of the M type 23 ( emml) gene derived from GAS strain M23-MEMPHIS (M serotype 23, GenBank accession number U11953). When the vir regulon of strain Sv was examined by polymerase chain reaction mapping and compared with that of GAS strain Su, they had a similar size in length. In addition, when sequencing analysis of the DNA fragment of 4 791 base pairs (bp) encoding three open reading frames ( orf, mga, and emm) and the upstream region of scpA from genomic DNAs of both strains was performed, the sequence of the DNA from strain Sv was, except for 1 bp (T for C at position 4 124), identical to that of the DNA from strain Su. These data show that both strains possess the genes in the order of mga ( virR or mry) - emm - scpA designated as the small vir regulon. The effect of the formation of alternative pathway C3 convertase of complement on the GAS strains Sv and Su was also examined. When GAS strains Sv and Su were incubated in NHS containing radiolabeled C3 in the presence of Mg-EGTA, binding of C3 to Su bacteria was dose-dependent, whereas less binding of C3 to Sv bacteria was seen. Taken together, the data suggest that M protein could be expressed on the surface of the Sv bacteria, but not on the Su bacteria.

Diversity of the third form of complement, C3, in fish: functional characterization of five forms of C3 in the diploid fish Sparus aurata.

We have recently shown that Sparus aurata, the gilthead sea bream (a diploid species), similarly to rainbow trout (a quasi-tetraploid species), possesses multiple forms of the third form of complement (C3). In the present study we have evaluated the ability of the gilthead sea bream proteins to function as active C3 molecules. All five C3 isoforms could be fixed covalently to sheep erythrocyte ghosts and were able to bind to various complement-activating surfaces in the presence of MgEGTA. In the absence of MgEGTA their binding capacities generally increased, presumably as a result of classical-pathway activation by the natural antibodies present in the serum. The presence of EDTA abrogated the binding of all C3 isoforms to the various surfaces tested. The C3 isoforms differed in the efficiency of their binding to complement-activating surfaces: the two most abundant C3 isoforms (C3-1 and C3-2) bound to zymosan as well as to sheep and rabbit erythrocyte ghosts, whereas C3-3, C3-4 and C3-5 were unable to bind to zymosan. Upon complement activation, all five C3 isoforms were cleaved to 'iC3b' by factor H and I-like proteins, generating fragments similar to those generated from C3 molecules of other species. Furthermore the degradation of methylamine-hydrolysed C3 isoforms to iC3b was significantly inhibited by EDTA. The structural and functional diversity that we have observed in the C3 isoforms of S. aurata would increase the capacity of this fish to recognize a broader spectrum of potential pathogens and reinforce a specific immune response, which in fish is delayed compared with that of higher vertebrates, and is based on a single Ig type (IgM).

Complement-mediated serum sensitivity among spirochetes that cause Lyme disease.

Borrelia burgdorferi-related isolates were tested for their sensitivity to normal human serum (NHS) and their ability to activate complement. By dark-field microscopy, electron microscopy, and subsurface plating, it was shown that exposure of a Borrelia garinii isolate to 10% or more NHS resulted in immobilization, blebbing, and killing of the spirochetes. These effects were mediated by complement, since they were not seen after heat treatment of NHS, in the presence of EDTA, or in an agammaglobulinemic serum. All seven B. garinii type 5 or 6 and all four VS116/M19 strains were serum sensitive, whereas all eight Borrelia afzelii, five of eight B. garinii type 4, and three of seven B. burgdorferi sensu stricto isolates were serum resistant. The other isolates were partially serum sensitive. Four serum-sensitive B. garinii isolates had been isolated from human cerebrospinal fluid. Most likely, activation of both the alternative pathway and the classical pathway of complement was involved, since bactericidal activity was diminished in properdin-deficient sera as well as in a C1q-depleted serum and in a C4-deficient serum. Bactericidal activity could be restored when a serum lacking C1q or C4 was mixed with a properdin-deficient serum. Isolates with various genetic backgrounds were equally able to activate C3 as measured by enzyme-linked immunosorbent assay. In the presence of Mg-EGTA, C3 was activated by all isolates after exposure to > or = 10% NHS. This study shows that B. burgdorferi-related spirochetes can be either serum sensitive or serum resistant in vitro and that this characteristic is associated with their genetic background.

Complement-mediated opsonisation and phagocytosis of Renibacterium salmoninarum

Renibacterium salmoninarum depletes complement-mediated haemolytic activity of human and chinook salmon ( Oncorhynchus tshawytscha ) serum, and the extent of depletion depends on bacterial density. The bacterium also consumes human, chinook salmon, channel catfish ( Ictalurus punctatus ), and rainbow trout ( O, mykiss ) complement in the presence of Mg-EGTA indicating the involvement of the alternative complement pathway. Bacteria incubated in human serum with or without Mg-EGTA cause human erythrocytes to agglutinate. This immune adherence is blocked by incubation of the opsonised bacteria in the presence of a goat anti-human C3 antibody or incubation of the human erythrocytes with a monoclonal antibody against the human complement receptor CR1. Both results indicate that the bacteria have been opsonised with C3b. Furthermore, incubation of R. salmoninarum in haemolytically-active rainbow trout serum is required for the adherence of the bacterium to rainbow trout kidney macrophages prior to their phagocytosis.

Activation of rat complement by soluble and insoluble rat IgA immune complexes.

The ability of rat monoclonal IgA, specific for 2,4-dinitrophenyl (DNA), to activate the complement (C) system of the rat was investigated using aggregated IgA or IgA immune complexes (IC). IgA was coated onto a solid phase, and tested for its capacity to bind C3 upon incubation at 37 degrees C in normal rat serum (NRS) in the presence of Mg-EGTA. Binding of C3 was observed dependent on the dose of dimeric (d-), polymeric (p-) and secretory IgA tested. In contrast, little C3 fixation was observed in this system with monomeric (m-) rat IgA or with mouse m- and d-IgA (MOPC315). Soluble and insoluble rat IgA IC were prepared using dinitrophenylated rat serum albumin (DNP8RSA) as antigen (Ag), and assessed for C activation. It was shown that insoluble IC (immune precipitates; IP) containing m-, d- or pIgA of rat origin activate the alternative pathway of rat C, as demonstrated by their capacity to induce C consumption in NRS in the presence of Mg-EGTA. When p- and m-IgA IP were compared for their capacity to activate C, it was found that p-IgA activated C four times as efficiently as m-IgA IP (at 2 mg/ml). Soluble rat IgA IC were prepared in an excess of DNP8RSA, fractionated by gel filtration on Sepharose 6B, and analyzed for C activation and antibody (Ab)/Ag ratio. In contrast to m-IgA IP, soluble m-IgA did not activate C. On the other hand soluble d-IgA IC activated C dependent on their concentration and size: at a concentration of 0.1 mg/ml high-molecular weight d-IgA IC with a high Ab/Ag ratio were four times as efficient as low-molecular weight IC with a low Ab/Ag ratio, and twice as efficient as IP prepared at equivalence. To demonstrate the induction by IgA of the assembly of the terminal membrane attack complex, trinitrophenyl (TNP)-conjugated rat red blood cells (TNP-RRBC) coated with d- or p-IgA were shown to be lysed in NRS in the presence of Mg-EGTA. No lysis of m-IgA-coated TNP-RRBC was observed. The results in this study demonstrate that both soluble and insoluble rat IgA IC activate the alternative pathway of homologous rat C. Alternative pathway activation by soluble rate IgA IC is dependent on the size of the IC. The degree of polymerization of the IgA Ab itself also influences C activation.

Activation of complement by human serum IgA, secretory IgA and IgA1 fragments

Activation of the complement (C) system by human IgA was studied. Both subclasses of IgA, IgA1 and IgA2, and secretory IgA were shown to activate C, as determined by deposition of C3 on glutaraldehyde-activated microwells coated with IgA. The activation of the C system occurred in the presence of MgEGTA and not in D̄-deficient serum. In addition to C3, deposition of properdin (P) but not of C4 was detected. These results indicate that C activation, as determined by measuring deposition of C3 and P, occurred by the alternative pathway (AP). The data further show that the major part of the hinge region, which is deleted in IgA2 as compared with IgA1 and which forms the major structural difference between the two subclasses, is not involved in C activation. Reduction and alkylation destroyed the ability of IgA to activate C, as has also been demonstrated for IgG. In order to define the C activating region of the IgA molecule, several fragments of IgA1 were tested. The four-chain molecules F(ab') 2 and F(abc) 2 were shown to activate the AP. No activation was observed with the two-chain fragments Fab and Fc. The Fc fragment of IgA also did not activate the CP, as does the Fc fragment of IgG. This indicates that activation of the AP of C by IgA is dependent on the presence of the F(ab') 2 fragment. In conclusion: human IgA does activate C by the AP. This activation requires an intact F(ab') 2 fragment.

Interaction of complement-solubilized immune complexes with CR1 receptors on human erythrocytes. The binding reaction.

The binding of complement (C)-solubilized 125I bovine serum albumin (BSA) anti-BSA immune complexes (IC) to CR1 receptors on human red blood cells (RBC-CR1) was studied. The binding of IC to CR1 was strongly dependent on the molar antigen to antibody ratio, and IC formed in moderate antigen excess showed no binding. IC solubilized in 50% human serum in the presence of autologous RBC bound rapidly to RBC-CR1, with maximal binding within less than 1 min at 37 degrees C. Release of CR1-bound IC under these conditions occurred slowly, requiring more than 30 min. Only binding of 'partially' solubilized, e.g., anti C3c (C4c), and conglutinin-reactive IC occurred, whereas fully solubilized complexes (IC-C3dg, C4d) showed virtually no binding. Solubilization of IC in the presence of Mg-EGTA or in C2-deficient serum resulted in a markedly delayed binding of IC to RBC, indicating the importance of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solubilized IC could be absorbed to solid-phase conglutinin or antibody to C3c and C4c, and these ligands were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect on the binding of presolubilized IC to RBC-CR1, whereas the binding was unaffected by the addition of monosaccharides or by the concentration of Ca2+ or Mg2+ ions.

Activation of the alternative and classical complement pathways by Entamoeba histolytica.

Entamoeba histolytica HM1 supported the activation of human alternative and classical complement pathways in the absence of ameba-reactive antibodies. Nonimmune serum depleted of C1q and factor D (NHS s C1q + D) and reconstituted with C1q was able to specifically deposit C3b onto trophozoites and produce lysis. This activity was not modified by the absorption of serum on E. histolytica. Serum depleted of factor B allowed C3b binding to amebae. Serum devoid of C4 effected only small amounts of C3 uptake. The kinetics of lysis of E. histolytica by serum in the presence of Mg-EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] (lacking classical pathway function) or by NHS s C1q + D and reconstituted with factor D was slow and only produced one-half the amount of lysis produced by NHS s C1q + D supplemented with C1q. These results indicate that the surface of the ameba can promote complement activation by the classical pathway, without the participation of specific antibodies, and that the magnitude of this activation is greater than that induced by the alternative pathway.

MOLECULAR PROPERTIES OF C3 CONVERTING FACTOR (C3 COF)

We have recently identified a yet undescribed factor in serum of an 8 year old girl with meningococcal septicemia. This factor caused hypercatabolism of the third component of complement, C3, in vivo and in vitro even in the absence of divalent cations and was termed C3 converting factor C3 CoF. Studies were undertaken to characterize this factor. On gel filtration using HPLC equipment and a TSK SW 4000 blue column two protein-containing fraction of serum were found to be able to induce C3 conversion in vitro: In the IgG-region significant activation of normal C3 occured in the presence of Mg-EGTA but not in EDTA. C3 CoF activity, in contrast, was cofiltrated with IgM. The IgG fraction was isolated from the patient's serum by ammonium sulphate precipitation, anion exchange chromatography and affinity chromatogra phy to apparent homogeneity on immunoelectrophoresis. It had all the properties required to define patient's IgG as C3 nephritic factor C3 NeF and, thus, no longer raised our interest. For the approximately 900 kD protein further biochemical data could be obtained: It is a β2-globulin, precipitable by low ionic strength buffer (=euglobulin), partly heat-resistant at 56°C, requires neither Mg nor Ca and cannot be inhibited by soy bean trypsin inhibitor at 5 mg/ml. This is, to our knowledge, the first case where two different activators of C3 simultaneously occur, one with known properties (C3 NeF) and one yet undescribed (C3 CoF).

The interaction of immune serum globulin and immune globulin intravenous with complement

The in vitro anticomplementary activity of untreated and heat-aggregated (63°C, 10 min) immune serum globulin (ISG) and immune globulin intravenous (IGIV) prepared by partial reduction and alkylation have been evaluated by three assays, C3 activation, binding to Clq and enhancement of alternative pathway lysis of rabbit erythrocytes. Crossed immunoelectrophoresis was used to quantitatively measure the ability of ISG and IGIV to activate endogenous C3 in normal serum. Binding to Clq was determined according to the ability to inhibit binding of 125I-Clq to solid phase IgG. ISG and IGIV enhancement of lysis of rabbit erythrocytes by normal human serum adsorbed with rabbit erythrocytes in the presence of MgEGTA was used to determine activity in the alternative complement pathway. Unheated IGIV at 10mg/ml only marginally activated endogenous C3 in normal serum, had about a 5-fold lower affinity for 125I-Clq ( K i = 138 to 356 μM vs K i = 62.5 μM for ISG), but was very similar in ability to ISG on a weight basis in enhancing complement alternative pathway activity ( RCH50 = 0.23 to 0.40 mg for IGIV vs 0.17mg for ISG). Heat-aggregated IGIV at 5 mg/ml in normal human serum was about 2-fold less effective than heat-aggregated ISG in the activation of C3 in normal serum and had approximately 2- to 3-fold lower affinity in the Clq binding assay ( K i = 45 to 83 n M for heat-aggregated IGIV vs K i = 14.6 n M for heat-aggregated ISG). These data suggest that IGIV prepared by chemical modification retains sufficient specific receptor activity to allow in vivo efficacy in complement-mediated amplification of host defense reactions, but is safe for intravenous use due to a lower capacity to initiate nonspecific complement activation.

The Modulation of the Alternative Pathway of Complement in C2-Deficient Human Serum by Changes in Concentration of the Component and Control Proteins

Abstract The opposing actions of component and regulatory proteins of the alternative pathway of C on activation of the sequence were demonstrated in C2-deficient serum utilizing two initiating particles and incremental additions of proteins purified to homogeneity. Activation of the alternative pathway in C2-deficient serum by zymosan was assessed by measurement of the inactivation of C3 and B, while activation by rabbit erythrocytes (ER) was measured by their lysis. Participation of the classical C-component sequence was excluded by the presence of Mg-EGTA and by the failure of reconstitution of C2 to have a measurable effect. Incremental additions of either purified C3bINA or β1H, which increased the serum concentrations of the proteins by 43% to 175% or 15% to 30%, respectively, produced dose-related suppression of zymosan-induced B and C3 inactivation throughout the 45 min period of observation. Additions of either C3 or B to C2-deficient serum so as to increase their concentrations by 35% reversed the combined 50%-inhibitory effect of added C3bINA and β1H on the zymosan-induced inactivation of C3 and B. Dose-dependent inhibition of the rate and extent of lysis of ER in C2-deficient serum was obtained with increases of C3bINA concentrations of 25 to 100% and of β1H concentrations of 12.5 to 50%. As little as a 25% increase in the concentration of B increased the rate of lysis of ER in C2-deficient serum and partially reversed the inhibitory effect of the combined addition of C3bINA and β1H. Thus, the disequilibrium between component and control proteins of the alternative pathway created by the surface of an activating particle, that permits transition to the amplification phase of C3 cleavage, can be modulated by alterations in the concentrations of these proteins. Modest increases in the concentrations of C3bINA and β1H served to maintain balance, whereas comparable augmentations in the concentrations of C3 and B facilitated activation by the initiating particle.