Quantitation of the third component of human complement attached to the surface of opsonized bacteria: opsonin-deficient sera and phagocytosis-resistant strains.

The role of the third component of human complement (C3) in the opsonization of bacteria in nonimmune human sera was evaluated. The amount of C3 that becomes attached to the surface of bacteria upon incubation in serum was measured in a quantitative fluorescent immunoassay using fluorescein-conjugated monospecific antiserum to human C3. The intensity of the fluorescence from opsonized bacteria was found to be directly proportional to the absolute amount of C3 fixed, and this enabled the detection of as few as 300 molecules of bound C3 per bacterium. In normal serum the rate of C3 fixation was closely correlated with an increase in opsonization of the bacteria for human PMNs. Both C3 fixation and opsonization were maximal after 15 min of incubation. C3 fixation was also observed, albeit at a significantly slower rate, in human serum with a nonfunctional classical pathway but an intact alternative complement pathway and in serum deficient in immunoglobulins. Again, the kinetics of C3 fixation correlated with bacterial opsonization. Using a total of 21 strains of several bacterial species, including Staphylococcus aureus and Escherichia coli, encapsulation of bacteria was found to interfere with the process of C3 fixation in normal human serum, rendering these organisms resistant to subsequent phagocytosis by human polymorphonuclear leukocytes.     

Opsonization of four Bacteroides species: role of the classical complement pathway and immunoglobulin

Previous investigators have suggested that opsonization of two Bacteroides species is mediated exclusively by the alternative complement pathway and requires immunoglobulins. In this study, the nature of the opsonic factors in nonimmune human serum for four species of Bacteroides was investigated by measuring uptake of [(3)H]thymidine-labeled bacteria by human polymorphonuclear leukocytes. Normal human serum, C2-deficient serum, immunoglobulin-deficient serum, and serum chelated with ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid (EGTA), MgEGTA, and ethylenediaminetetraacetic acid (EDTA) were used as opsonic sources. Heat inactivation of each of these sera significantly reduced its opsonic activity for all four Bacteroides species, suggesting that serum complement was essential for effective opsonization. All strains were opsonized in the absence of the classical complement pathway; however, kinetics studies revealed that opsonization proceeded at a significantly faster rate when the classical complement pathway was intact. Although two strains were opsonized in immunoglobulin-deficient sera, opsonization was less efficient and appeared to occur via the alternative complement pathway. Unexpectedly, all strains were well opsonized by the classical complement pathway in 10% serum which had been effectively chelated with EGTA or EDTA. The explanation for this finding is unknown; however, it is possible that cell wall cations of Bacteroides species may participate in the activation of complement in chelated serum, resulting in effective opsonization. It was also found that Bacteroides, when incubated with an Escherichia coli strain in normal serum, could compete for opsonins and thereby reduce phagocytosis of E. coli. It is possible that competition for opsonins among bacterial species contributes to the synergistic role these organisms share in mixed floral infections.     

Influence of growth temperature of Escherichia coli on K1 capsular antigen production and resistance to opsonization.

When Escherichia coli strains that produce K1 capsular polysaccharide antigen at 37 degrees C were grown at 22 degrees C, K1 antigen was not detected in the supernatant or washed-cell fraction of broth cultures. Significant amounts of K1 polysaccharide were detected only when the organism was grown at temperatures of 30 degrees C or higher. Rabbits immunized with an E. coli K1 strain (serotype O18ac:K1:H7) grown at 37 degrees C produced agglutinating antibody to somatic antigen and precipitating and agglutinating antibody to capsular K1 antigen; those immunized with this strain grown at 22 degrees C produced antibody to somatic antigen, but not to K1 antigen. Antibody to somatic antigen was markedly reduced by adsorption with the organism grown at 22 degrees C, while antibody to capsular antigen was not. E. coli K1 strains grown at 37 degrees C (K1 present) resisted phagocytosis and killing if they were opsonized solely by the alternative complement pathway (ACP) using magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid-chelated serum. When these strains were grown at 22 degrees C (K1 absent), they were opsonized efficiently by the ACP (28 versus 94% killing, respectively; P less than 0.001). In addition, a non-K1 mutant of an E. coli K1 strain was opsonized efficiently by the ACP although its encapsulated K1 parent was not. Sensitivity of E. coli strains to the bactericidal activity of serum was observed in strains with and without K1 capsular antigen. These studies demonstrated that production of K1 polysaccharide antigen was regulated by environmental temperature and that K1 capsule plays an essential role in rendering the organism resistant to opsonization by the ACP.     

Antibodies raised against rough mutants of Escherichia coli and Salmonella strains are opsonic only in the presence of complement

The opsonic capacity of antisera raised in rabbits against rough (R) mutants and smooth (S) parent strains of Escherichia coli and Salmonella typhimurium were studied. All specific antibodies in the antisera belonged to the immunoglobulin G (IgG) class. Radioactively labeled bacteria were preincubated in various dilutions of antisera, in which complement was inactivated. Fresh normal rabbit serum, as a standard complement source, was used in some experiments. After preincubation, washed bacteria were added to normal human neutrophils. Opsonization of R mutants for 5 min in 5% fresh normal rabbit serum resulted in effective phagocytosis; S strains needed at least a 30-min opsonization time or 20 to 50% serum. After incubation for 5 min in diluted, homologous antisera, phagocytosis of S strains was optimal, but preincubation of R mutants in diluted, homologous antisera did not lead to amelioration of phagocytosis compared with that of bacteria preincubated in buffer only. However, when fresh normal serum was added to homologous antisera, uptake of R mutants occurred at a faster rate than that of bacteria opsonized in fresh serum alone. Using six clinical isolates of members of the family Enterobacteriaceae, we found that, with or without complement, antisera raised against E. coli J5 or S. typhimurium Re had, with the exception of one strain, no opsonic activity for these strains. Thus, the protective effect of R antisera in gram-negative bacteremia, as shown by several investigators, is unlikely to be mediated through enhanced opsonization of invading bacteria by IgG antibodies directed against these R mutants.     

Evaluation of phagocytosis of alpha hemolytic strains of Escherichia coli using the fluorescence method

The authors investigated phagocytosis in 37 alpha haemolytic strains of E. coli by the fluorescent method, after opsonization of bacteria with 5% and concentrated human serum. The strains were isolated from infants with gastrointestinal and extraintestinal infections and belonged to eleven serogroups. During opsonization of the strain with concentrated serum, as compared with opsonization of strains with 5% serum, higher rates of killing were recorded. As compared with a control strain of E. coli K12 after opsonization with concentrated serum in the examined strains the value of killing was on average by 20% lower. Differences in the killing of examined strains were recorded also within a single serogroup which may be ascribed to the different provision of strains with plasmids.     

Evidence for quantitative variability of bacterial opsonic requirements.

We studied human serum opsonins by using combinations of heat inactivation and chelation to inhibit complement, adsorption to remove antibody, and trypan blue to inactivate the C3 receptor of human polymorphonuclear leukocytes. Streptococcus pneumoniae, serotype 25, required both complement and immunoglobulin for opsonization, even though that strain activated the alternative complement pathway. Both strains of Escherichia coli required antibody and complement, but varied in the degree of dependence on the C3 opsonin, since trypan blue moderately inhibited the killing of E. coli-1 and markedly inhibited the killing of E. coli-2. Serratia marcescens was opsonized in heat-inactivated serum (limited complement) or serum absorbed at 0 degrees C with S. marcescens (limited antibody), but depended on the C3 receptor in absorbed serum. S. marcescens activated the alternative pathway. Thus, opsonic requirements varied with the availability of opsonins. Requirements for bacterial opsonization vary with species and strains within species, perhaps reflecting quantitative relationships among alternative and classical pathway activation of C3, efficiency of adsorption of C3 or immunoglobulin G to bacterial surfaces, and efficiency of attachment of these ligands to polymorphonuclear leukocyte receptors. Furthermore, although not always sufficient for opsonization, the C3 opsonin (activated through either the classical or alternative pathway) appears necessary for effective phagocytosis and killing of all strains studied.     

Staphylococcus aureus opsonization mediated via the classical and alternative complement pathways. A kinetic study using MgEGTA chelated serum and human sera deficient in IgG and complement factors C1s and C2.

Staphylococcus aureus opsonization was studied kinetically by: (1) determination of the uptake of [3H]-thymidine labelled bacteria by human PMN's; (2) fluorescent anti-C3 and anti-IgG staining of opsonized bacteria; and (3) measuring bacterial complement consumption. Maximum opsonization in normal serum occurred within 5 min of incubation. About 80% of staphylococci were then taken up by PMN's, and IgG and C3b could be detected on the bacterial surface. In the absence of a functional classical complement pathway, as in sera deficient in C1s and C2 and in MgEGTA chelated serum, maximal opsonization was only achieved after 30--60 min incubation. Opsonization in IgG deficient serum occurred at a rate similar to that found in C2 deficient or MgEGTA chelated serum. Opsonization was greatly enhanced when sera were reconstituted. It was concluded that in IgG deficient serum Staphylococcus aureus opsonization is mediated via the alternative complement pathway. Dilution of normal serum primarily affected the classical complement pathway, resulting in a decreased rate of opsonization. In normal serum IgG did not appear to be a rate-limiting factor. S. Aureus opsonization was best studied by the phagocytosis assay and the fluorescent-antibody technique. Measuring haemolytic complement consumption was found to be an insensitive indicator of bacterial complement activation and opsonization.     

Availability of complement bound to Staphylococcus aureus to interact with membrane complement receptors influences efficiency of phagocytosis

Complement-mediated opsonization of encapsulated Staphylococcus aureus (CP+) of the predominant capsule types, 5 and 8, remains poorly understood. Our previous work showed that complement is important for mouse survival of CP+ type 5 bacteremia and that the type 5 capsule inhibits the binding of opsonic C3 fragments to the organism. The importance of complement-mediated opsonization of CP+ was tested by neutrophil phagocytosis assays. Complement-mediated opsonization of CP+ increased phagocytosis by 57% compared to opsonization in complement-inhibited serum. Agar-grown CP+, enhancing capsule expression, was phagocytosed only one-tenth as well as the capsule-negative organisms (CP-), supporting the belief that staphylococcal polysaccharide capsules impair phagocytosis. Despite relatively poor phagocytosis of CP+ compared to CP-, complement activation increased the phagocytosis of CP+ by 103%. Thus, complement in normal human serum may have an important role in opsonizing CP+, even when capsule expression is strong. The ability of bound C3 fragments to interact with complement receptor 1 (CD35) on the membrane of human erythrocytes was tested in an immune adherence assay. S. aureus capsule was able to mask C3 fragments on the organism from binding to complement receptor 1. The inhibition of C3 binding to CP+ and the masking of deposited C3 fragments caused by the presence of capsule was associated with markedly decreased phagocytosis. The addition of anti-capsule antibodies to normal human serum was found to markedly improve the recognition of deposited C3 fragments by complement receptor 1 even when the absolute number of C3 molecules bound to S. aureus was not increased.     

Immunological specificity of natural opsonins and their role in the cross-reactivity between Staphylococcus aureus Mardi and Escherichia coli 101.

The immunochemical specificity of the observed cross-reactivity between Escherichia coli strain 101 and Staphylococcus aureus strain Mardi was examined. The cross-reactivity was shown to be dependent upon mucopeptide antibodies which are present in normal and immune sera. Although both organisms contained surface antigens with immunodominant glucuronic acid residues, in vitro phagocytosis studies indicated that antibodies directed against these antigens were not significantly involved in the opsonization process. Rather, antibodies with mucopeptide specificity were shown to be involved in the in vitro phagocytosis of these organisms by polymorphonuclear leukocytes. The mucopeptide antibodies, which were found in both nonimmune and immune sera, were shown to be effective in opsonizing both the S. aureus strain and the E. coli strain. The ubiquitous distribution of E. coli strains containing mucopeptide antigens common to most bacteria suggests that these organisms may be responsible for the wide prevalence of natural staphylococcal opsonins with mucopeptide specificity in normal sera.     

The role of complement C3 opsonization, C5a receptor, and CD14 in E. coli-induced up-regulation of granulocyte and monocyte CD11b/CD18 (CR3), phagocytosis, and oxidative burst in human whole blood

The relative role of complement and CD14 in Escherichia coli-induced leukocyte CD11b up-regulation, phagocytosis, and oxidative burst in human whole blood was examined. The highly specific thrombin inhibitor lepirudin was used as anticoagulant, as it does not affect complement activation. Complement inhibition at the level of C3 (anti-C2 and anti-factor D) and C5 (C5a receptor antagonist and anti-C5/C5a) efficiently inhibited CD11b up-regulation, phagocytosis, and oxidative burst in granulocytes. Monocyte activation was generally less complement-dependent, but when C3 activation was blocked, a pronounced inhibition of phagocytosis and oxidative burst was obtained. Only the combination of anti-C2 and antifactor D blocked E. coli C3 opsonization completely. Whole E. coli, disrupted E. coli, and the C3-convertase activator cobra venom factor up-regulated CD11b rapidly on both cell types, proportional to their complement activation potential in the fluid phase. In comparison, purified LPS at concentrations comparable with that present in the E. coli preparations did not activate complement. Oxidative burst was induced only by whole bacteria. Finally, the combination of complement inhibition and anti-CD14 completely blocked E. coli-induced granulocyte and monocyte CD11b up-regulation and quantitatively, virtually abolished phagocytosis. The results indicate that complement and CD14, despite differential effects on granulocytes and monocytes, are the two crucial, quantitative factors responsible for E. coli-induced CD11b, phagocytosis, and oxidative burst in both cell types.     

Influence of encapsulation on staphylococcal opsonization and phagocytosis by human polymorphonuclear leukocytes

In previous studies, encapsulated Staphylococcus aureus strains have been shown to resist phagocytosis. In this investigation, the nature of the interference with phagocytosis by human polymorphonuclear leukocytes was examined by studying the opsonization of two pairs of unencapsulated (Smith compact and M variant) and encapsulated (Smith diffuse and M) S. aureus strains. The uptake of [3H]glycine-labeled bacteria by normal leukocytes was quantitatively measured after incubation of bacteria in pooled serum, C2-deficient serum, immunoglobulin-deficient serum, and serum from a rabbit immunized with S. aureus M. The presence of a capsule was found to interfere with opsonization by both the classical and alternative pathways of complement as well as by heat-stable opsonic factors in nonimmune human serum. This interference was significantly greater in the case of the S. aureus M strain than in the case of the Smith diffuse strain. The only effective opsonic source for S. aureus M was immune rabbit serum. It is proposed that encapsulation of S. aureus strains interferes with phagocytosis by preventing effective bacterial opsonization.     

Bactericidal, bacteriolytic and opsonic activity of human serum against Escherichia coli

The effect of human serum on Escherichia coli was studied with serum-sensitive and serum-resistant strains. The bactericidal effect of human serum on serum-sensitive strains of E. coli depended on the activation of the classical complement pathway. The role of activation of the alternative pathway was less important. After incubation in sub-bactericidal concentrations of serum these strains were also easily phagocytosed by polymorphonuclear leukocytes (PMNL). Strains of E. coli of certain O-types required not only an intact classical pathway but also the presence of specific antibodies for effective killing by serum and effective phagocytosis by PMNL, despite rapid activation of complement and rapid deposition of C3 on the bacterial surface in the absence of antibody. Capsulate strains O1K1 and O78K80 resisted the bactericidal effect of serum even in the presence of specific antibodies; phagocytosis by PMNL only occurred after opsonisation with specific antibodies.     

Capsular K1 polysaccharide of Escherichia coli: relationship to virulence in newborn rats and resistance to phagocytosis.

The virulence of Escherichia coli strains for newborn rats was related to opsonic requirements of the strains, sensitivity to the bactericidal activity of serum, and K1 capsular polysaccharide content. K1 E. coli strains were more virulent than non-K1 strains after intraperitoneal injection in newborn rats (P less than 0.05) and were more resistant to phagocytosis than non-K1 strains when the classical complement pathway was blocked with Mg-ethyleneglycoltetraacetic acid (P less than 0.0005). Sensitivity to the bactericidal activity of serum was similar among K1 and non-K1 E. coli strains. Two groups of K1 E. coli strains were defined on the basis of opsonic requirements. Group I strains were efficiently opsonized by the alternative complement pathway, while group II strains required the classical complement pathway for opsonization. Group I strains had less detectable K1 polysaccharide in the washed whole cell fraction than group II strains (10.3 versus 18.9 microgram of K1 polysaccharide per 10(10) colony-forming units) and were less virulent than group II strains (mortality, 44 versus 77%, P less than 0.05). The K1 capsular polysaccharide appears to play an important role in determining virulence in newborn rats and opsonic requirements of these strains, but does not contribute to the sensitivity of strains to the bactericidal activity of serum.     

Influence of Encapsulation on Phagocytosis of Pasteurella multocida by Bovine Neutrophils

The effect of encapsulation on phagocytosis of Pasteurella multocida by bovine neutrophils was examined by using two encapsulated strains, NA77 (capsular type A) and C42 (capsular type B), and comparing them with an unencapsulated counterpart strain, 1173. The uptake of [(3)H]thymidine-labeled bacteria by neutrophils was quantitatively measured after incubation of the bacteria in normal bovine serum, heat-inactivated serum, and hyperimmune sera (anti-NA77 and anti-C42). Results showed that all three strains of P. multocida were ineffectively opsonized by the heat-labile serum complement system. The unencapsulated strain was completely phagocytized in the presence of heat-stable opsonins found in normal serum. Although encapsulation of strain C42 was found to interfere with opsonization by normal serum, this strain was completely phagocytized when hyperimmune serum (anti-C42) was used as the opsonin source. These results suggest that specific anticapsular antibodies found in the hyperimmune serum readily opsonized the encapsulated strain C42 and enhanced phagocytosis. The presence of a thick capsule on strain NA77 interfered with phagocytosis in the presence of normal or hyperimmune serum (anti-NA77). This interference was due to the presence of hyaluronic acid which was a major component of the capsule. Treatment of this encapsulated strain with hyaluronidase decapsulated the bacteria. Bacteria treated in this way were almost completely phagocytized (90%) in the presence of heat-stable opsonins. The exact mechanism by which the capsule of P. multocida NA77 interfered with phagocytosis was not demonstrated; perhaps the slimy nature of the hyaluronic acid makes the phagocytic act difficult by changing the physiochemical surface properties, or it may prevent opsonization.     

Opsonization of various capsular (K) E. coli by the alternative complement pathway

A virulence factor of E. coli K-1 is its capacity to avoid opsonization via the alternative complement pathway (ACP). Since it is not known if E. coli with other capsular (K) antigens have similar properties we examined various capsular E. coli for opsonization by the ACP. To assess opsonization we used whole blood luminol-dependent chemiluminescence (CL) and the magnesium salt of ethyleneglycol tetraacetic acid to block the classical pathway. E. coli K-types 6, 7, 27, 30, 42, 53, 57 and 75 were effectively opsonized via the ACP (greater than 65% of CL obtained with unchelated normal serum). K types 2 and 13 were opsonized by the ACP in both the high range greater than 65% and intermediate range 36-65%. Only K-types 1, 3, 5, 12 and 92 were poorly opsonized (less than 35%) by the ACP. The data demonstrate that most E. coli K-types were opsonized via the ACP. The poor opsonization of E. coli K3, 5, 12 and 92 by the ACP may be virulence factor for these bacteria.     

Opsonic activity in human serum chelated with ethylene glycoltetra-aectic acid

The influence of the alternative pathway of complement activation on opsonization of S. aureus, E. coli and Candida albicans for phagocytosis was selectively studied by chelating serum with 10 mM EGTA and 10 mM MgC1₂. Opsonic activity of the chelated serum depended on the alternative pathway of complement activation. The classical pathway was inhibited. There was no phagocytosis of S. aureus in chelated normal serum, suggesting that activation of complement activity by the classical pathway is necessary for opsonization of this micro-organism. In control experiments when opsonization of S. aureus was accomplished by IgG separated from hyperimmune serum, without the requirement of complement activity, there was no inhibition of phagocytosis by EGTA and magnesium chloride. E. coli and C. albicans were opsonized in chelated normal serum, indicating that the alternative pathway of complement activation is involved in opsonization of these micro-organisms.     

The influence of the O and K antigens of Klebsiella aerogenes on surface hydrophobicity and susceptibility to phagocytosis and antimicrobial agents

The surface hydrophobicity of Klebsiella aerogenes is influenced by the presence of capsular (K) and lipopolysaccharide (O) antigens. Loss of both K and O antigens (K-O-), but not the K antigen alone (K-O+), increased surface hydrophobicity and susceptibility to phagocytosis. Unheated serum (i.e., containing complement) increased the surface hydrophobicity and phagocytosis of the K-O+ and K-O- strains, but not of the K+O+ encapsulated parent strain. Despite the altered susceptibility to phagocytosis caused by the presence or absence of the K and O antigens, their loss did not influence sensitivity to a range of hydrophilic, hydrophobic or cationic antimicrobial agents.     

Virulence-plasmid is associated with the inhibition of opsonization in Yersinia enterocolitica and Yersinia pseudotuberculosis.

Plasmid-cured variants of virulent strains of Yersinia enterocolitica and Y. pseudotuberculosis were obtained by selection after growth in calcium-deficient medium. To obtain antigen preparations consisting of whole bacteria the original plasmid-containing strains and the plasmid-cured variants were grown in conditions favouring expression of the temperature-inducible outer membrane proteins of Yersinia (YOP) (37 degrees C, calcium-deficient culture medium). The presence or absence of the YOP on the bacteria was verified by immunoblotting. Opsonophagocytosis of YOP-negative Yersinia preparations (YOP-) was compared to that of YOP-containing ones (YOP+) in human polymorphonuclear leukocyte (PMN) chemiluminescence (CL) assay. The attachment of complement C3b on the surface of the bacteria after opsonization with normal human serum was determined by using a fluorescent anti-C3c-antibody and flow cytometry. YOP+ bacteria resisted opsonization in the absence of specific antibodies, as indicated by diminished C3b-fixation on bacteria and weaker CL response. This implies that virulence-plasmid-coded structures provide Y. enterocolitica and Y. pseudotuberculosis with an ability to avoid complement-mediated opsonization and phagocytosis.     

Impaired opsonization with C3b and phagocytosis of Streptococcus pneumoniae in sera from subjects with defects in the classical complement pathway

Results from studies using mice deficient in specific complement factors and clinical data on patients with an inherited deficiency of the classical complement pathway component C2 suggest that the classical pathway is vital for immunity to Streptococcus pneumoniae. However, the consequences of defects in classical pathway activity for opsonization with C3b and the phagocytosis of different S. pneumoniae serotypes in human serum are not known, and there has not been a systematic analysis of the abilities of sera from subjects with a C2 deficiency to opsonize S. pneumoniae. Hence, to investigate the role of the classical pathway in immunity to S. pneumoniae in more detail, flow cytometry assays of opsonization with C3b and the phagocytosis of three capsular serotypes of S. pneumoniae were performed using human sera depleted of the complement factor C1q or B or sera obtained from C2-deficient subjects. The results demonstrate that, in human serum, the classical pathway is vital for C3b-iC3b deposition onto cells of all three serotypes of S. pneumoniae and seems to be more important than the alternative pathway for phagocytosis. Compared to the results for sera from normal subjects, C3b-iC3b deposition and total anti-S. pneumoniae antibody activity levels in sera obtained from C2(-/-) subjects were reduced and the efficiency of phagocytosis of all three S. pneumoniae strains was impaired. Anticapsular antibody levels did not correlate with phagocytosis or C3b-iC3b deposition. These data confirm that the classical pathway is vital for complement-mediated phagocytosis of S. pneumoniae and demonstrate why subjects with a C2 deficiency have a marked increase in susceptibility to S. pneumoniae infections.     

Logarithmic phase Escherichia coli K1 efficiently avoids serum killing by promoting C4bp-mediated C3b and C4b degradation

Meningitis caused by Escherichia coli K1 is a serious illness in neonates with neurological sequelae in up to 50% of survivors. A high degree of bacteremia is required for E. coli K1 to cross the blood-brain barrier, which suggests that the bacterium must evade the host defence mechanisms and survive in the bloodstream. We previously showed that outer membrane protein A (OmpA) of E. coli binds C4b-binding protein (C4bp), an inhibitor of complement activation via the classical pathway. Nevertheless, the exact mechanism by which E. coli K1 survives in serum remains elusive. Here, we demonstrate that log phase (LP) OmpA+ E. coli K1 avoids serum bactericidal activity more effectively than postexponential phase bacteria. OmpA- E. coli cannot survive in serum grown to either phase. The increased serum resistance of LP OmpA+ E. coli is the result of increased binding of C4bp, with a concomitant decrease in the deposition of C3b and the downstream complement proteins responsible for the formation of the membrane attack complex. C4bp bound to E. coli K1 acts as a cofactor to factor I in the cleavage of both C3b and C4b, which shuts down the ensuing complement cascade. Accordingly, a peptide corresponding to the complement control protein domain 3 of C4bp sequence, was able to compete with C4bp binding to OmpA and cause increased deposition of C3b. Thus, binding of C4bp appears to be responsible for survival of E. coli K1 in human serum.