Complement attack of altered outer membrane areas synthesized after inhibition of the 3-deoxy-D-manno-octulosonate pathway leads to cell death

Salmonella typhimurium containing specific genes coding for either temperature-sensitive (TS) 3-deoxy-D-manno-octulosonate (KDO) 8-phosphate synthetase or TS cytidine monophosphate-KDO synthetase grow normally when incubated at 30 degrees C and are resistant to C-mediated killing. However, bacteria become avirulent and sensitive to C-mediated killing upon thermal inhibition of TS KDO-8-phosphate synthetase (incubation at 38 degrees C) or TS cytidine monophosphate-KDO synthetase (incubation at 42 degrees C). Such thermal inhibition concurrently causes synthesis of an altered outer membrane which we now show is the site that renders cells susceptible to C-mediated killing. After incubation of cells in serum, the altered outer membrane area contains C9 in a trypsin-resistant state and membrane attack complex (MAC) lesions observable by electron microscopy. Trypsin-resistant C9 and MAC lesions were also observed in the inner membrane fraction from such serum-treated cells. In contrast, little C9 and few MAC lesions were associated with unaltered outer membrane areas present on these same serum treated cells. Control cells, grown at 30 degrees C and treated with serum (1) bound one-fifth as much C9 as was bound to cells incubated at 42 degrees C, (2) contained only a rare MAC lesion in the outer membrane, and (3) no observable MAC lesions in the inner membrane. We conclude that the altered outer membrane area is the site that renders cells susceptible to insertion of the MAC into both the outer and inner membrane resulting in cell death.     

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.     

The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing

We have examined the killing of E. coli and kinetics of lipopolysaccharide (LPS) release after the exposure of the bacteria to normal human serum (NHS) and sera deficient in complement components, or with inactivated complement components. LPS of the galactose epimerase-deficient strain E. coli J5 were specifically radiolabeled by growing the bacteria in a medium containing [3H]galactose. Exposure of the washed bacteria to NHS resulted in a significant reduction (greater than 99%) in viability within 15 min and the concomitant release of radiolabeled LPS. However, maximal release of LPS was consistently 30% of the total radiolabel incorporated into the LPS molecules. The amount of tritium-labeled LPS released was shown to be directly proportional to the concentration of bacteria exposed to NHS, suggesting that release of LPS was not limited by the availability of some critical serum component(s). The consumption of complement in NHS by incubation with E. coli was demonstrated by decreased alternative and classical pathway-specific hemolytic activity. The use of Factor D-depleted and VEM-treated human sera demonstrated that, with these bacteria, both the alternative and classical pathways of complement contribute to bacterial killing and release of LPS. It is noteworthy that, in VEM-treated and Factor D-depleted sera, the rate of killing and the kinetics of LPS release were somewhat slower as compared to control serum. Bacterial killing in C7-depleted and C9-deficient human sera was minimal. Neither killing nor LPS release occurred in heat-inactivated (56 degrees C, 30 min) human serum. The amount of [3H]LPS released by C9-deficient serum was qualitatively similar to the amount released by the action of NHS. Tritium-labeled LPS was not released in C7-depleted serum. These data indicate that bacterial killing can be dissociated from LPS release, and suggest that, whereas LPS release may be necessary for the bactericidal effects of serum complement, it is probably not sufficient to effect killing. Furthermore, a significant fraction of LPS can be removed from the outer membrane of the bacteria without an apparent affect on viability.     

Bactericidal activity of C9-deficient human serum.

Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.     

Structural investigation of Borrelia burgdorferi OspB, a bactericidal Fab target

Certain antibody Fab fragments directed against the C terminus of outer surface protein B (OspB), a major lipoprotein of the Lyme disease spirochete, Borrelia burgdorferi, have the unusual property of being bactericidal even in the absence of complement. We report here x-ray crystal structures of a C-terminal fragment of B. burgdorferi OspB, which spans residues 152-296, alone at 2.0-A resolution, and in a complex with the bactericidal Fab H6831 at 2.6-A resolution. The H6831 epitope is topologically analogous to the LA-2 epitope of OspA and is centered around OspB Lys-253, a residue essential for H6831 recognition. A beta-sheet present in the free OspB fragment is either disordered or removed by proteolysis in the H6831-bound complex. Other conformational changes between free and H6831-bound structures are minor and appear to be related to this loss. In both crystal structures, OspB C-terminal fragments form artificial dimers connected by intermolecular beta-sheets. OspB structure, stability, and possible mechanisms of killing by H6831 and other bactericidal Fabs are discussed in light of the structural data.     

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.     

Susceptibility of the gray wolf (Canis lupus) to infection with the Lyme disease agent, Borrelia burgdorferi

Four juvenile gray wolves (Canis lupus) were inoculated with live Borrelia burgdorferi. One received an intravenous inoculum, a second was inoculated subcutaneously, and two more were fed Peromyscus maniculatus sucklings which had earlier been inoculated with B. burgdorferi. The intravenously inoculated wolf developed a generalized lymphadenopathy and a persistent serum antibody titer to the spirochete which peaked at 1:512. Borrelia burgdorferi was visualized in liver sections of this wolf using direct immunofluorescent staining. The subcutaneously inoculated wolf showed a low and transient antibody response which peaked at 1:64, and manifested no clinical or postmortem abnormalities. The wolves which were fed inoculated mice showed no detectable antibody response. They were clinically normal throughout the project, and there were no detectable lesions at necropsy. Two control wolves were inoculated intravenously with formalin killed B. burgdorferi. Serum antibody titers of these controls peaked at 1:64 and 1:32, respectively, and fell to 1:16 by day 48 postinoculation. A survey of serum samples from 78 wild-trapped wolves from Wisconsin and Minnesota revealed that one was positive and another was suspect for B. burgdorferi infection based on presence of antibody to the spirochete. We conclude that the wolf is susceptible to infection by B. burgdorferi and that wolves are being infected in the wild.     

Outer surface protein polymorphisms linked to host‐spirochete association in Lyme borreliae

Lyme borreliosis is caused by multiple species of the spirochete bacteria Borrelia burgdorferi sensu lato. The spirochetes are transmitted by ticks to vertebrate hosts, including small‐ and medium‐sized mammals, birds, reptiles, and humans. Strain‐to‐strain variation in host‐specific infectivity has been documented, but the molecular basis that drives this differentiation is still unclear. Spirochetes possess the ability to evade host immune responses and colonize host tissues to establish infection in vertebrate hosts. In turn, hosts have developed distinct levels of immune responses when invaded by different species/strains of Lyme borreliae. Similarly, the ability of Lyme borreliae to colonize host tissues varies among different spirochete species/strains. One potential mechanism that drives this strain‐to‐strain variation of immune evasion and colonization is the polymorphic outer surface proteins produced by Lyme borreliae. In this review, we summarize research on strain‐to‐strain variation in host competence and discuss the evidence that supports the role of spirochete‐produced protein polymorphisms in driving this variation in host specialization. Such information will provide greater insights into the adaptive mechanisms driving host and Lyme borreliae association, which will lead to the development of interventions to block pathogen spread and eventually reduce Lyme borreliosis health burden.     

The role of C9 in complement-mediated killing of Neisseria

During the routine examination of a healthy 31-yr-old woman, we found an incomplete deficiency of the 9th component of complement (C9). By hemolytic assay her serum C9 activity was 10 to 15% of normal. Limited family studies suggested that she inherited the deficiency as an autosomal codominant trait.She had no history of unusual or severe infections. When tested for bactericidal activity against serum-sensitive Neisseria gonorrhoeae and N. meningitidis, her serum reacted comparably to normal serum. Normal serum depleted immunochemically of C9 and sera from congenitally C9-deficient patients were also bactericidal against serum-sensitive Neisseria but required 120 min to kill the same numbers of gonococci that intact serum killed within 30 min. In the electron microscope, N. gonorrhoeae incubated with C9-depleted serum were fragmented but lacked the typical C lesions. Therefore, serum lacking C9 can kill serum-sensitive Neisseria, unlike sera deficient in the other terminal C components.     

Complement consumption by Photobacterium damselae subsp. piscicida in seabream, red porgy and seabass normal and immune serum. Effect of the capsule on the bactericidal effect

A virulent strain of Photobacterium damselae subsp. piscicida (Pdp) was grown without (C form) or with (C+ form) glucose supplementation, the latter to enhance capsule formation. Both forms were resistant to killing by normal serum of seabream, red porgy and seabass. However, the C form was killed by immune serum of all three fish species while the C+ form was killed only by seabream and red porgy sera and to a lesser extent than the C form. Both C and C+ forms consumed complement in normal serum and this consumption was enhanced by precoating the bacteria in specific fish antibody. Complement consumption was greatest in seabass serum, especially with antibody-coated C+ form yet in this case the bacteria were not killed. The killing of the C form in immune serum of all three fish species was completely inhibited by EGTA/Mg(2+), indicating that the mechanism of complement activation leading to killing of the bacteria was by the classical pathway. The results suggest that immune serum killing by the classical complement pathway may provide some degree of protection against pasteurellosis, but enhanced expression of the capsule by Pdp in vivo may restrict complement-mediated killing, especially in immunised seabass.     

Mechanisms of stable serum resistance of Neisseria gonorrhoeae

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent sera from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by immunoglobulin G (IgG) or F(ab)₂ isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, contained most of the blocking activity in IgG. In addition, immune convalescent DGI serum, which did not exhibit bactericidal activity, was restored to killing by selective immunodepletion of protein III antibodies. Blocking IgG or F(ab)₂ prepared from IgG, partially inhibited binding of bactericidal antibody to N. gonorrhoeae. Also, binding of a monoclonal antibody recognizing N. gonorrhoeae outer membrane protein PIII was almost completely inhibited by blocking F(ab)₂.Presensitization of N. gonorrhoeae with increasing concentrations of blocking IgG or F(ab)₂ before incubation with bactericidal antibody and an antibody free source of complement, increased consumption and deposition of the third component of human complement (C3) and the ninth component of complement (C9) but inhibited killing in dose-related fashion.     

Susceptibility of Helicobacter pylori to the Bactericidal Activity of Human Serum

Background. Human serum represents an important barrier to the entry of most mucosal organisms into tissues and to the systemic circulation. If at all present, Helicobacter pylori within gastric tissue is rare, and bacteremia for this organism has been described only once.
Methods. To assess the susceptibility of H. pylori to the bactericidal activity present in normal human serum (NHS), we examined 13 H. pylori isolates. To assess the contributions of the classical and alternative complement pathways to killing, we added either C2-deficient or factor B-deficient serum, respectively, to heat-inactivated NHS. Also we assessed the ability of the strains to bind ¹²⁵I-C3.
Results. After incubation for 60 minutes at 37°C, all 13 H. pylori strains were killed by NHS; heating to 56°C for 30 minutes ablated killing, indicating complement dependence for this phenomenon. In the absence of an antibody source, there was no killing when either an alternative or classical complement pathway source was used. Adding B-deficient serum to heat-inactivated normal human serum did not restore killing, but adding C2-deficient serum permitted partial killing. All of the 13 strains bound ¹²⁵I-C3. Although the kinetics varied from strain to strain, C3 bound was significantly correlated ( r = 0.61, p = 0.03) with serum susceptibility.
Conclusions. H. pylori are susceptible to complement, alternative pathway activation appears critical, and C3 binding is a major locus of variability.     

Borrelia burgdorferi EbfC, a novel, chromosomally encoded protein, binds specific DNA sequences adjacent to erp loci on the spirochete's resident cp32 prophages

All examined isolates of the Lyme disease spirochete, Borrelia burgdorferi, naturally maintain numerous variants of a prophage family as circular cp32 episomes. Each cp32 carries a locus encoding one or two different Erp outer membrane, surface-exposed lipoproteins. Many of the Erp proteins bind a host complement regulator, factor H, which is hypothesized to protect the spirochete from complement-mediated killing. We now describe the isolation and characterization of a novel, chromosomally encoded protein, EbfC, that binds specific DNA sequences located immediately 5' of all erp loci. This is one of the first site-specific DNA-binding proteins to be identified in any spirochete. The location of the ebfC gene on the B. burgdorferi chromosome suggests that the cp32 prophages have evolved to use this bacterial host protein for their own benefit and that EbfC probably plays additional roles in the bacterium. A wide range of other bacteria encode homologs of EbfC, none of which have been well characterized, so demonstration that B. burgdorferi EbfC is a site-specific DNA-binding protein has broad implications across the eubacterial kingdom.     

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.     

Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F(ab')2 mediate killing of E. coli 0111B4 by the alternative complement pathway without increasing C5b-9 deposition

The mechanism of antibody-dependent complement-(C) mediated killing of Escherichia coli 0111B4, strain 12015 (12015), was examined. 12015 was resistant to serum killing when incubated in hypogammaglobulinemic serum (H gamma S) or pooled normal human serum (NHS) that had been previously adsorbed to remove specific antibody (Abs NHS). Presensitization with immune rabbit serum or purified immune rabbit IgG resulted in 1 to 3 log killing when 5 X 10(8) colony forming units (CFU)/ml were incubated in 10 to 40% Abs NHS. Binding of 125I-C3 and 131I-C9 to the bacterial surface of the presensitized and the nonpresensitized strain was quantitated when these organisms were incubated in 10, 20, and 40% Abs NHS. Stable binding of up to 3.0 X 10(5) molecules of C3 and 8.0 X 10(4) molecules of C9 to presensitized and nonpresensitized isolates occurred in the highest concentration of serum, but there was no killing without presensitization. Similar results were found when Abs NHS was chelated with ethylene bis glycoltetraacetic acid containing 2 mM MgCl2 (Mg EGTA) to block classical pathway activation, indicating that antibody mediated the bactericidal reaction through the alternative pathway. Deposition of C3 and C9 and killing of 120 15 in 10% Abs NHS or 10% H gamma S was measured after presensitization with increasing amounts of IgG, F(ab')2, or Fab'. There was a dose-dependent increase in C3 deposition and killing, but only minimal change in C9 binding when 1.0 X 10(3) to 3.2 X 10(4) IgG or F(ab')2/CFU were bound to the bacterial surface. In contrast, there was no increase in C3 or C9 binding and no bacterial killing when 1 X 10(3) to 3.4 X 10(4) molecules Fab'/CFU were bound to the bacterial surface. These experiments show that immune IgG and F(ab')2 can mediate killing of E. Coli 0111B4 by the alternative pathway without changing the extent of terminal C component attachment to the bacterial surface.     

Multimeric C9 within C5b-9 deposits in unique locations in the cell wall of Salmonella typhimurium

We have shown previously that multimeric C9 within C5b-9 (C9:C5b-8 greater than 3:1) is needed for killing of a rough strain of Escherichia coli. We now extend these studies using serum sensitive, rough (R) and serum resistant, wild type (WT) strains of Salmonella typhimurium as well as a mutant S. typhimurium strain (TS) with a temperature sensitive mutation in synthesis of keto-deoxy-octulosonate, a constituent within the deep core structure of Salmonella LPS. Both R and TS required multimeric C9 within C5b-9 to be killed. Addition at 37 degrees C of increasing inputs of C9 to TS or R bearing C5b-9 led to a dose-related increase in C9 binding and killing. In contrast, addition of high inputs of C9 to the same strains at 4 degrees C, a procedure that limits the C9:C5b-8 ratio to 1:1, resulted in low C9 binding and minimal killing. Bactericidal C5b-9 formed at 37 degrees C on R and TS with high inputs of C9 co-sedimented with the bacterial outer membrane on sucrose density gradient analysis. Non-bactericidal C5b-9 on R, WT, and TS co-sedimented near the inner membrane, despite the presumed lack of association between these constituents. Whereas 125I C9 within the non-bactericidal pools immunoprecipitate with anti-C5, 125I C9 within bactericidal pools did not immunoprecipitate with anti-C5, anti-C7, or anti-C9. These findings suggest that bactericidal C5b-9 may be deposited in a unique location or configuration within the bacterial cell wall.     

A murine IgG1 monoclonal antibody thatbinds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi

Abstract A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi , has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii . The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA aas well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.     

Live cell imaging of outward and inward vesiculation induced by the complement c5b-9 complex

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.     

Repetition, conservation, and variation: the multiple cp32 plasmids of Borrelia species

Members of the spirochete genus Borrelia contain large numbers of extrachromosomal DNAs. Sequence analysis of the B. burgdorferi strain B31 genome indicated that its many plasmids contain large quantities of repeated sequences, the most obvious of which are the cp32 plasmid family. Individual spirochetes may carry nine or more different, but homologous, cp32 plasmids. Every other species of Borrelia examined thus far also contains multiple plasmids related to the B. burgdorferi cp32s. These plasmids are arguably the best characterized of all the borrelial plasmids, and epitomize the apparent redundancy evident in the many plasmids carried by these bacteria. Despite their extensive similarities, cp32 plasmids contain some open reading frames whose sequences often vary between plasmids, and which encode proteins synthesized by the bacteria during vertebrate infection. In this review, we analyze the hypervariable and conserved regions of the cp32 plasmid family, and discuss possible reasons why borreliae harbor multiple gene paralogs.     

Human IgA1 initiates complement-mediated killing of Neisseria meningitidis

We studied the effect of human IgA1, the predominant IgA subclass in serum, on C-mediated killing of Neisseria meningitidis. We purified monomeric IgA1 from normal human serum and tetravalent meningococcal polysaccharide vaccinate serum by using the following successive chromatographic steps: jacalin lectin affinity, Superose 12 FPLC gel filtration, Mono Q FPLC anion exchange, and anti-IgG affinity. SDS-PAGE, ELISA, and Western immunoblot analyses of the IgA1 detected no trace of contaminating IgG or IgM. IgA1 initiated partial or complete lysis (62 to 100%) of nine group C strains by using either normal, hypogammaglobulinemic, factor B-depleted, or properdin-deficient human serum as a C source, but IgA1 was unable to effect killing in serum chelated with 10 mM MgCl2 and 10 mM EGTA. Lytic activity was dependent on the group C strain and the source of the IgA1; neither IgA1 preparation was bactericidal for all nine strains. Removal of the Fc portion of IgA1 with pepsin completely abolished bactericidal activity. We purified and radiolabeled C component C3, and found that IgA1 did not increase C3 deposition. With the use of a group C polysaccharide ELISA, we found that the vaccinate IgA1 had a high titer of group C polysaccharide antibody, whereas the IgA1 purified from normal human serum had no detectable group C polysaccharide specificity. Absorption of the vaccinate IgA1 with alum-bound group C polysaccharide did not affect the killing of a sensitive strain, but it did potentiate the killing of a previously resistant strain. Western immunoblots of whole cell lysates, outer membrane complex, and purified lipooligosaccharide showed that the bactericidal IgA1 was specific for several outer membrane proteins. Four of the proteins recognized by both IgA1 preparations had apparent Mr of 29, 42, 66, and 74 kDa. We conclude that IgA1, when bound to specific outer membrane proteins, can initiate lysis of group C meningococci via the classical C pathway, and that initiation of lysis is an Fc-dependent event which occurs without an increase in C3 deposition.