- Email: [email protected]
Activation of the complement cascade by Bordetella pertussis
FEMS Microbiology Letters 220 (2003) 271^275
www.fems-microbiology.org
Activation of the complement cascade by Bordetella pertussis Michael G. Barnes, Alison A. Weiss
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 231 Sabin Way, ML 0524, Cincinnati, OH 45267, USA Received 3 July 2002; received in revised form 5 February 2003 ; accepted 7 February 2003 First published online 7 March 2003
Abstract Bordetella pertussis must survive the defenses of the human respiratory tract including the complement system. The BrkA (Bordetella resistance to killing) protein prevents killing by the antibody-dependent classical pathway. In this study, the ability of B. pertussis to activate the human complement cascade by other pathways was examined. B. pertussis was not killed in serum depleted of C2, however serum depleted for factor B killed B. pertussis as efficiently as intact serum, suggesting complement activation occurred exclusively by the classical pathway. B. pertussis was not killed by serum depleted of antibody, suggesting the bacteria fail to activate the antibodyindependent branches of the classical pathway, including the mannose binding lectin pathway. Mutants lacking the terminal trisaccharide of lipopolysaccharide retained the complement-resistant phenotype, suggesting this structure does not influence activation of complement. . 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Complement; Classical pathway; Alternative pathway; Bordetella pertussis
1. Introduction The complement cascade is a potent antimicrobial immune defense that can be activated several ways (Fig. 1). The classical pathway of complement is usually initiated by binding of antibody to a bacterial surface. Complement protein C1 binds the Fc regions from two antibodies in close proximity, and undergoes a conformational change to become proteolytically active. Activated C1 cleaves C4 and C2 to form the C3-convertase, C2b4a, which cleaves C3 leading to production of C3b and activation of the rest of the complement cascade. C1 can activate the classical pathway in the absence of antibody by directly binding to bacterial targets [1^5], for example porin proteins. Lipopolysaccharide (LPS) can a¡ect C1 deposition and killing [4]. Complement activation can also occur if the mannosebinding-lectin (MBL) binds to mannose residues on the surface of a bacterium. The mannose-binding-protein-associated-serine-proteases activate C4 and C2 of the classical pathway in a manner similar to that used by the antibody/C1 complex (reviewed in [6]). Gram-negative pathogens, such as B. pertussis, have de-
* Corresponding author. Tel. : +1 (513) 558 2820; Fax : +1 (513) 558 7484. E-mail address : [email protected] (A.A. Weiss).
veloped various methods to avoid killing by complement. B. pertussis produces the BrkA protein which protects the bacteria from killing by complement [7]. BrkA prevents activation of the complement cascade at deposition of C4, but interestingly, puri¢ed C1 was shown to bind equivalently to wild-type and BrkA mutants in the absence of antibody [8]. However the ability of the C1 to activate complement in the absence of antibody was not assessed in this study. The alternative pathway is activated by the interaction of C3i with factor B (Fig. 1) leading to formation of C3iBb, the alternative-pathway-C3-convertase. The alternative pathway can also provide ampli¢cation of the classical pathway using C3b generated by the classical pathway to create more alternative-pathway-C3-converase complexes (C3bBb) [9]. The surface of B. pertussis appears to lack the ability to activate the alternative pathway of complement [7]. For some bacterial pathogens, resistance to the alternative pathway has been shown to be due to modi¢cation of their LPS, speci¢cally the addition of sialic acid [10]. B. pertussis does not appear to modify its LPS. The LPS of B. pertussis lacks repeating O-side chains, and the core has a unique branched structure [11]. In addition, the LPS of B. pertussis contains a linear trisaccharide with unusual sugars, including N-acetylglucosamine, a mannuronic acid residue (diNAcManA), and a fucosyl residue (FucNAcMe) [11,12]. The LPS of B. pertussis has been
0378-1097 / 03 / $22.00 . 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00132-0
FEMSLE 10896 19-3-03
272
M.G. Barnes, A.A. Weiss / FEMS Microbiology Letters 220 (2003) 271^275
Scholte (SS) broth overlaid on BG agar as previously described [8,18]. Antibiotics were used as follows : 30 Wg ml31 nalidixic acid for all strains, 50 Wg ml31 kanamycin for BP347 and the wlb mutants, and 30 Wg ml31 gentamicin for RFBP2152. 2.2. Human serum and complement Human sera were collected from healthy adult volunteers as previously described [8]. Heat inactivation, when appropriate, was carried out at 56‡C for 30 min. All serum samples were stored at 380‡C until use. Human serum with intact complement activity was depleted of immunoglobulin by sequential incubation with agarose-conjugated immunoglobulin binding proteins, including protein A (Sigma, St. Louis, MO, USA; Lot #60K7018; protein-G (Sigma ; Lot #40K7025) ; protein-L (Pierce, Rockford, IL, USA; Lot #BE43650) ; and protein LA (Sigma ; Lot #119H1653), as previously described [18]. 2.3. Serum killing assay Fig. 1. Activation of the complement cascade. The complement cascade consists of the classical and alternative pathways. The classical pathway can be activated by C1 in the presence of speci¢c antibody (Ab), C1 binding to bacterial structures such as porins, or the MBL without the need for C1. Depletion of antibody (Ab, circled) will prevent killing by the antibody-dependent classical pathway, and depletion of C2 (circled) will inactivate the entire classical pathway. The alternative pathway is always induced at a low level, and ampli¢cation is controlled by complex interactions between positive and negative regulatory factors. Depletion of factor B (circled) will inactivate the alternative pathway. The classical and alternative pathways converge at C3, leading to activation of C5 through C9 and formation of the membrane attack complex (MAC), shown in the box. The formation of the convertases is depicted by the open arrows.
shown to be a target for bactericidal antibodies [13], however it could also serve as a protective structure against the alternative pathway. In this study we examined how B. pertussis activates complement.
2. Materials and methods 2.1. Bacterial strains and growth conditions B. pertussis strains used in this study include virulentphase, wild-type stain, BP338 [14], and two derivatives of BP338: RFBP2152, a brkA mutant [15] and BP347, a bvgS mutant [14]. The role of LPS in complement resistance was assessed using wild-type strain BP536 and four derivatives : BP536vwlbD [16], BP536vwlbG [16], BP536vwlbH [17], and BP536vwlbL [17] expressing mutant LPS. B. pertussis was grown on Bordet^Gengou (BG) agar containing 15% de¢brinated sheep’s blood (Colorado Serum, Denver, CO, USA) and 1% glycerol with or without 7 ml Stainer^
Serum killing assays were performed essentially as described [8,18]. Brie£y, bacteria were in grown in SS broth overlaid on BG agar for 24 h at 37‡C. Approximately 105 bacteria with complement and antibody were brought to 20 Wl with SS broth in a 96-well U-bottom plate. Sources of complement were 2 Wl serum, factor B-depleted serum (Advanced Research Technologies, San Diego, CA, USA; Lot #6T), C2-depleted serum (Advanced Research Technologies, San Diego, CA, USA; Lot #5) or 4 Wl antibodydepleted serum. 2 Wl puri¢ed factor B (1.0 mg ml31 ) (Advanced Research Technologies, San Diego, CA, USA; Lot #7) or 4 Wl puri¢ed C2 (9.1 mg ml31 ) (Advanced Research Technologies, San Diego, CA, USA; Lot #12) was added as indicated. To ensure the presence of B. pertussis-speci¢c antibody, 2 Wl of heat-inactivated pooled human immune serum was added to the heat-inactivated serum, serum control, or complement-depleted sera. 5 Wl heat-inactivated pooled immune serum was added to the antibodydepleted serum to provide exogenous antibody. Samples were incubated at 37‡C for 1 h. PBS with 10 mM EDTA was added to stop the killing reaction and serial dilutions were plated onto BG agar and colony-forming units were determined to assess bacterial survival. Statistical analysis was performed using the Student’s t-test. P values 6 0.05 were assumed to be signi¢cant.
3. Results 3.1. B. pertussis is killed by complement in the absence of the alternative pathway B. pertussis was incubated with serum lacking complement activity or in the presence of serum with di¡erent
FEMSLE 10896 19-3-03
M.G. Barnes, A.A. Weiss / FEMS Microbiology Letters 220 (2003) 271^275
273
Serum-killing assays were performed with C2-depleted serum to assess bactericidal activity due to the alternative pathway. Viability was not signi¢cantly di¡erent from the heat inactivated, no killing control for any of the three strains (Fig. 2). Addition of puri¢ed C2 (1.8 mg ml31 ) restored killing to levels similar to the serum control. The ¢nding that C2-depleted serum did not kill B. pertussis demonstrates that killing by serum is entirely mediated by the classical pathway of complement and not other serum factors. 3.3. B. pertussis is not killed by direct C1q binding or the MBL pathway
Fig. 2. Role of alternative and classical pathways in killing of B. pertussis. WT (BP338), BrkA (RFBP2152) and Bvg-(BP347) were incubated for 1 h in the presence of 10% human serum with various types of complement activity. Immune antibody was added at 10% to all samples to standardize the classical pathway killing potential. Samples are as follows: 1, No CP, heat-inactivated serum lacking complement activity; 2, CP control, human serum with intact complement activity; 3, No factor B, serum depleted of factor B; 4, +factor B, serum depleted of factor B with addition of exogenous factor B; 5, No C2, serum depleted of complement protein C2; 6, +C2, serum depleted of complement protein C2 with addition of exogenous C2. Results are presented as log10 CFU and represent the average and standard error of four separate trials.
kinds of complement activity. No loss of bacterial viability was observed in heat-inactivated serum lacking complement activity (Fig. 2, No CP). Addition of serum with complement and antibody decreased viability of the wild-type strain BP338 by more than 2 logs (Fig. 2, CP control). Survival in serum of either the BrkA mutant, RFBP2152, or the Bvg mutant, BP347, lacking all virulence factors including BrkA, was signi¢cantly lower than wild-type (P 6 0.05 and 1036 respectively), con¢rming the role of BrkA in mediating resistance to complement. Factor B-depleted serum killed all three strains (Fig. 2, No factor B). Addition of puri¢ed factor B (0.1 mg ml31 ) to the factor B-depleted serum did not signi¢cantly increase killing of any of the three strains. These data demonstrate that B. pertussis is sensitive to killing by complement in the absence of the alternative pathway and ampli¢cation of the classical pathway by the alternative pathway is not required. 3.2. B. pertussis is not killed in the absence of the classical pathway Killing by the classical pathway requires C2 (Fig. 1).
C1q can bind porins (Fig. 1) and activate complement in the absence of antibody [1]. Alternatively, the MBL pathway can directly activate C4 and C2 (Fig. 1) in the absence of antibody or C1. The role of antibody-independent killing of B. pertussis was investigated. Survival of the wild-type strain or a BrkA mutant in antibody-depleted serum (Fig. 3, No antibody) was similar to survival in the heat inactivated, no complement control (Fig. 3, No CP). Addition of immune antibody restored killing (Fig. 3, +antibody) demonstrating that antibodies are required for killing, and ruling out a role for either direct C1q activation of the classical pathway or the MBL pathway in killing B. pertussis with intact LPS.
Fig. 3. Role of antibody in complement killing. WT (BP338) or BrkA (RFBP2152) B. pertussis were incubated for 1 h in serum as follows: No CP, heat-inactivated serum lacking complement activity; No antibody, antibody-depleted serum; +antibody, antibody-depleted serum with the addition of immune antibody. Results are presented as log10 CFUs and represent the average and standard error of at least six separate experiments. Asterisk indicates signi¢cance relative to heat-inactivated control (WT, P 6 0.02; BrkA, P 6 0.0002) ; # signi¢cantly di¡erent relative to wild-type (P 6 0.0007).
FEMSLE 10896 19-3-03
274
M.G. Barnes, A.A. Weiss / FEMS Microbiology Letters 220 (2003) 271^275
4. Discussion
Fig. 4. In£uence of LPS on susceptibility to complement. Wild-type BP536 (WT), and four LPS mutants: BP536vwlbH (WlbH), BP536vwlbG (WlbG), BP536vwlbD (WlbD), and BP536vwlbL (WlbL) were incubated for 1 h in serum as follows : No CP, heat-inactivated serum lacking complement activity; No antibody, antibody-depleted serum ; +antibody, antibody-depleted serum with the addition of immune antibody. Asterisk indicates signi¢cantly di¡erent relative to wild-type (P 6 0.03). Results are presented as log10 CFUs and represent the average and standard error of at least three separate experiments. The structure of LPS for the strains is diagrammed below. The sugars of the terminal trisaccharide (indicated in brackets) are N-acetylglucosamine (GlcNAc), a mannuronic acid residue (diNAcManA), and a fucosyl residue (FucNAcMe). In addition to the structure shown in the diagram, the WlbD mutant also produces a larger LPS band of unknown structure, as indicated by a question mark.
3.4. E¡ect of LPS mutations on serum resistance LPS can function as a target for complement mediated killing or it can provide protection from killing. Wild-type BP536 and LPS mutants, BP536vwlbH, BP536vwlbG, BP536vwlbD and BP536vwlbL (Fig. 4) each de¢cient in di¡erent genes required for synthesis of the O-antigen trisaccharide [16,17] were used to assess the contribution of the O-antigen to complement-susceptibility. Serum killing assays were performed using antibody-depleted serum as described for Fig. 3. Survival in antibodydepleted serum was not statistically di¡erent from the heat-inactivated no killing control for any of the strains (Fig. 4). Addition of pooled antibody from immune individuals restored killing. The wild-type strain BP536 and its LPS mutant derivatives displayed similar susceptibility to complement except BP536vwlbG, which displayed increased sensitivity to killing. The killing was mediated by the antibody-dependent pathway, suggesting the increased susceptibility of the wlbG mutant was due to antigenic di¡erences. The enzyme encoded by wlbG is proposed to be needed for the ¢rst step in polysaccharide synthesis, possibly the transfer of a sugar to a carrier lipid [16].
Complement is an important immune defense. Complement is present in serum, as well as on mucosal surfaces including the respiratory tract [19]. Like many immune defenses, the complement cascade has multiple avenues for activation, and this redundancy plays an important role in minimizing the ability of pathogens to escape immune clearance. The BrkA protein of B. pertussis mediates resistance to complement killing by the antibody-dependent classical pathway. BrkA inhibits activation of complement at an early stage of the classical complement pathway after C1 deposition [8]. In this study we investigated the ability of B. pertussis to activate the complement cascade by non-antibody mediated mechanisms. B. pertussis is only killed by the classical pathway, as evidenced by the failure of C2-depleted serum to kill the bacteria. Direct binding of C1 to surface structures such as porins [1^5] has been shown to activate complement in the absence of antibody. Puri¢ed C1 binds equally to wildtype and BrkA mutants of B. pertussis in the presence or absence of antibody [8] suggesting that activation of the classical pathway could proceed without a requirement for antibody, however the current study has shown that this is not the case. Antibody-independent killing of wild-type or BrkA mutants was not observed, and the antibody-independent C1 binding to B. pertussis must occur in a manner that fails to activate complement. Furthermore, the lack of killing in the absence of antibody also suggests B. pertussis fails to activate the MBL pathway. This result was not unexpected since mannose is not a component of the B. pertussis LPS, and has not been reported to be on the surface of the bacteria. Killing of B. pertussis by the alternative pathway was not detected, and furthermore, ampli¢cation of the classical pathway by the alternative pathway was not observed, since factor B-depleted serum killed B. pertussis as e⁄ciently as factor B-depleted serum supplemented with factor B. The failure to detect killing by the alternative pathway is consistent with a previous study that suggested that only the classical pathway of complement mediates killing of B. pertussis [7]. In that study no killing was observed in the presence of a calcium chelator, and calcium ions are necessary for the classical pathway, but not the alternative pathway. Interestingly, the wild-type strain used in this study was killed by rabbit serum lacking antibodies to Bordetella bronchiseptica [20], suggesting killing by the alternative pathway of complement had occurred. Activation of the alternative pathway may occur di¡erently for di¡erent mammalian species. B. pertussis presents a surface to the human host that is not recognized by the innate pathways of the human complement system. Pathogens, such as Neisseria gonorrhoeae resist killing by the alternative pathway by adding sialic acid to their LPS [10], but such modi¢cations have not been observed for B. pertussis. The LPS of B. pertussis
FEMSLE 10896 19-3-03
M.G. Barnes, A.A. Weiss / FEMS Microbiology Letters 220 (2003) 271^275
lacks an O-side chain, and has a branched core structure and a trisaccharide with unusual sugars [11]. We had thought that the unusual sugars might play a role in blocking activation of the alternative pathway, however four mutants with di¡erent de¢ciencies in production of the trisaccharide were all resistant to killing in the absence of antibody. The mechanism used by B. pertussis to resist killing by the alternative pathway is currently unknown. B. pertussis is a strongly host-adapted pathogen, and our studies suggest this human pathogen has adapted to evade the human defenses. Only the most speci¢c pathway of complement activation, the antibody dependent classical pathway can initiate bacterial killing, but even then, the BrkA protein can protect B. pertussis from complement killing.
Acknowledgements This study was supported by Grant RO1 AI45715 to A.A.W. M.G.B. was the recipient of a Graduate Student Summer Research Fellowship from the University of Cincinnati. References [1] Alberti, S., Rodriquez-Quinones, F., Schirmer, T., Rummel, G., Tomas, J.M., Rosenbusch, J.P. and Benedi, V.J. (1995) A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding. Infect. Immun. 63, 903^910. [2] Butko, P., Nicholson-Weller, A. and Wessels, M.R. (1999) Role of complement component C1q in the IgG-independent opsonophagocytosis of group B Streptococcus. J. Immunol. 163, 2761^2768. [3] Loos, M., Wellek, B., Thesen, R. and Opferkuch, W. (1978) Antibody-independent interaction of the ¢rst component of complement with Gram-negative bacteria. Infect. Immun. 22, 5^9. [4] Merino, S., Nogueras, M.M., Aguilar, A., Rubires, X., Alberti, S., Benedi, V.J. and Tomas, J.M. (1998) Activation of the complement classical pathway (C1q binding) by mesophilic Aeromonas hydrophila outer membrane protein. Infect. Immun. 66, 3825^3831. [5] Mintz, C.S., Arnold, P.I., Johnson, W. and Schultz, D.R. (1995) Antibody-independent binding of complement component C1q by Legionella pneumophila. Infect. Immun. 63, 4939^4943.
275
[6] Wong, N.K., Kojima, M., Dobo, J., Ambrus, G. and Sim, R.B. (1999) Activities of the MBL-associated serine proteases (MASPs) and their regulation by natural inhibitors. Mol. Immunol. 36, 853^ 861. [7] Fernandez, R.C. and Weiss, A.A. (1994) Cloning and sequencing of a Bordetella pertussis serum resistance locus. Infect. Immun. 62, 4727^ 4738. [8] Barnes, M.G. and Weiss, A.A. (2001) BrkA Protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition. Infect. Immun. 69, 3067^3072. [9] Walport, M.J. (2001) Complement. First of two parts. N. Engl. J. Med. 344, 1058^1066. [10] Parsons, N.J., Andrade, J.R., Patel, P.V., Cole, J.A. and Smith, H. (1989) Sialylation of lipopolysaccharide and loss of absorption of bactericidal antibody during conversion of gonococci to serum resistance by cytidine 5P-monophospho-N-acetyl neuraminic acid. Microb. Pathog. 7, 63^72. [11] Caro¡, M., Brisson, J., Martin, A. and Karibian, D. (2000) Structure of the Bordetella pertussis 1414 endotoxin. FEBS Lett. 477, 8^14. [12] Brodeur, B.R., Martin, D., Hamel, J., Shahin, R.D. and Laferriere, C. (1993) Antigenic analysis of the saccharide moiety of the lipooligosaccharide of Bordetella pertussis. Springer Semin. Immunopathol. 15, 205^215. [13] Weiss, A.A., Mobberley, P.S., Fernandez, R.C. and Mink, C.M. (1999) Characterization of human bactericidal antibodies to Bordetella pertussis. Infect. Immun. 67, 1424^1431. [14] Weiss, A.A., Hewlett, E.L., Myers, G.A. and Falkow, S. (1983) Tn5induced mutations a¡ecting virulence factors of Bordetella pertussis. Infect. Immun. 42, 33^41. [15] Fernandez, R.C. and Weiss, A.A. (1998) Serum resistance in bvgregulated mutants of Bordetella pertussis. FEMS Microbiol. Lett. 163, 57^63. [16] Allen, A. and Maskell, D. (1996) The identi¢cation, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis. Mol. Microbiol. 19, 37^52. [17] Allen, A.G., Thomas, R.M., Cadisch, J.T. and Maskell, D.J. (1998) Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Mol. Microbiol. 29, 27^38. [18] Barnes, M.G. and Weiss, A.A. (2002) Growth phase in£uences complement resistance of Bordetella pertussis. Infect. Immun. 70, 403^ 406. [19] Persson, C.G. et al. (1991) Plasma exudation in the airways: mechanisms and function. Clin. Exp. Allergy 21, 17^24. [20] Harvill, E.T., Preston, A., Cotter, P.A., Allen, A.G., Maskell, D.J. and Miller, J.F. (2000) Multiple roles for Bordetella lipopolysaccharide molecules during respiratory tract infection. Infect. Immun. 68, 6720^6728.
FEMSLE 10896 19-3-03
www.fems-microbiology.org
Activation of the complement cascade by Bordetella pertussis Michael G. Barnes, Alison A. Weiss
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 231 Sabin Way, ML 0524, Cincinnati, OH 45267, USA Received 3 July 2002; received in revised form 5 February 2003 ; accepted 7 February 2003 First published online 7 March 2003
Abstract Bordetella pertussis must survive the defenses of the human respiratory tract including the complement system. The BrkA (Bordetella resistance to killing) protein prevents killing by the antibody-dependent classical pathway. In this study, the ability of B. pertussis to activate the human complement cascade by other pathways was examined. B. pertussis was not killed in serum depleted of C2, however serum depleted for factor B killed B. pertussis as efficiently as intact serum, suggesting complement activation occurred exclusively by the classical pathway. B. pertussis was not killed by serum depleted of antibody, suggesting the bacteria fail to activate the antibodyindependent branches of the classical pathway, including the mannose binding lectin pathway. Mutants lacking the terminal trisaccharide of lipopolysaccharide retained the complement-resistant phenotype, suggesting this structure does not influence activation of complement. . 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Complement; Classical pathway; Alternative pathway; Bordetella pertussis
1. Introduction The complement cascade is a potent antimicrobial immune defense that can be activated several ways (Fig. 1). The classical pathway of complement is usually initiated by binding of antibody to a bacterial surface. Complement protein C1 binds the Fc regions from two antibodies in close proximity, and undergoes a conformational change to become proteolytically active. Activated C1 cleaves C4 and C2 to form the C3-convertase, C2b4a, which cleaves C3 leading to production of C3b and activation of the rest of the complement cascade. C1 can activate the classical pathway in the absence of antibody by directly binding to bacterial targets [1^5], for example porin proteins. Lipopolysaccharide (LPS) can a¡ect C1 deposition and killing [4]. Complement activation can also occur if the mannosebinding-lectin (MBL) binds to mannose residues on the surface of a bacterium. The mannose-binding-protein-associated-serine-proteases activate C4 and C2 of the classical pathway in a manner similar to that used by the antibody/C1 complex (reviewed in [6]). Gram-negative pathogens, such as B. pertussis, have de-
* Corresponding author. Tel. : +1 (513) 558 2820; Fax : +1 (513) 558 7484. E-mail address : [email protected] (A.A. Weiss).
veloped various methods to avoid killing by complement. B. pertussis produces the BrkA protein which protects the bacteria from killing by complement [7]. BrkA prevents activation of the complement cascade at deposition of C4, but interestingly, puri¢ed C1 was shown to bind equivalently to wild-type and BrkA mutants in the absence of antibody [8]. However the ability of the C1 to activate complement in the absence of antibody was not assessed in this study. The alternative pathway is activated by the interaction of C3i with factor B (Fig. 1) leading to formation of C3iBb, the alternative-pathway-C3-convertase. The alternative pathway can also provide ampli¢cation of the classical pathway using C3b generated by the classical pathway to create more alternative-pathway-C3-converase complexes (C3bBb) [9]. The surface of B. pertussis appears to lack the ability to activate the alternative pathway of complement [7]. For some bacterial pathogens, resistance to the alternative pathway has been shown to be due to modi¢cation of their LPS, speci¢cally the addition of sialic acid [10]. B. pertussis does not appear to modify its LPS. The LPS of B. pertussis lacks repeating O-side chains, and the core has a unique branched structure [11]. In addition, the LPS of B. pertussis contains a linear trisaccharide with unusual sugars, including N-acetylglucosamine, a mannuronic acid residue (diNAcManA), and a fucosyl residue (FucNAcMe) [11,12]. The LPS of B. pertussis has been
0378-1097 / 03 / $22.00 . 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00132-0
FEMSLE 10896 19-3-03
272
M.G. Barnes, A.A. Weiss / FEMS Microbiology Letters 220 (2003) 271^275
Scholte (SS) broth overlaid on BG agar as previously described [8,18]. Antibiotics were used as follows : 30 Wg ml31 nalidixic acid for all strains, 50 Wg ml31 kanamycin for BP347 and the wlb mutants, and 30 Wg ml31 gentamicin for RFBP2152. 2.2. Human serum and complement Human sera were collected from healthy adult volunteers as previously described [8]. Heat inactivation, when appropriate, was carried out at 56‡C for 30 min. All serum samples were stored at 380‡C until use. Human serum with intact complement activity was depleted of immunoglobulin by sequential incubation with agarose-conjugated immunoglobulin binding proteins, including protein A (Sigma, St. Louis, MO, USA; Lot #60K7018; protein-G (Sigma ; Lot #40K7025) ; protein-L (Pierce, Rockford, IL, USA; Lot #BE43650) ; and protein LA (Sigma ; Lot #119H1653), as previously described [18]. 2.3. Serum killing assay Fig. 1. Activation of the complement cascade. The complement cascade consists of the classical and alternative pathways. The classical pathway can be activated by C1 in the presence of speci¢c antibody (Ab), C1 binding to bacterial structures such as porins, or the MBL without the need for C1. Depletion of antibody (Ab, circled) will prevent killing by the antibody-dependent classical pathway, and depletion of C2 (circled) will inactivate the entire classical pathway. The alternative pathway is always induced at a low level, and ampli¢cation is controlled by complex interactions between positive and negative regulatory factors. Depletion of factor B (circled) will inactivate the alternative pathway. The classical and alternative pathways converge at C3, leading to activation of C5 through C9 and formation of the membrane attack complex (MAC), shown in the box. The formation of the convertases is depicted by the open arrows.
shown to be a target for bactericidal antibodies [13], however it could also serve as a protective structure against the alternative pathway. In this study we examined how B. pertussis activates complement.
2. Materials and methods 2.1. Bacterial strains and growth conditions B. pertussis strains used in this study include virulentphase, wild-type stain, BP338 [14], and two derivatives of BP338: RFBP2152, a brkA mutant [15] and BP347, a bvgS mutant [14]. The role of LPS in complement resistance was assessed using wild-type strain BP536 and four derivatives : BP536vwlbD [16], BP536vwlbG [16], BP536vwlbH [17], and BP536vwlbL [17] expressing mutant LPS. B. pertussis was grown on Bordet^Gengou (BG) agar containing 15% de¢brinated sheep’s blood (Colorado Serum, Denver, CO, USA) and 1% glycerol with or without 7 ml Stainer^
Serum killing assays were performed essentially as described [8,18]. Brie£y, bacteria were in grown in SS broth overlaid on BG agar for 24 h at 37‡C. Approximately 105 bacteria with complement and antibody were brought to 20 Wl with SS broth in a 96-well U-bottom plate. Sources of complement were 2 Wl serum, factor B-depleted serum (Advanced Research Technologies, San Diego, CA, USA; Lot #6T), C2-depleted serum (Advanced Research Technologies, San Diego, CA, USA; Lot #5) or 4 Wl antibodydepleted serum. 2 Wl puri¢ed factor B (1.0 mg ml31 ) (Advanced Research Technologies, San Diego, CA, USA; Lot #7) or 4 Wl puri¢ed C2 (9.1 mg ml31 ) (Advanced Research Technologies, San Diego, CA, USA; Lot #12) was added as indicated. To ensure the presence of B. pertussis-speci¢c antibody, 2 Wl of heat-inactivated pooled human immune serum was added to the heat-inactivated serum, serum control, or complement-depleted sera. 5 Wl heat-inactivated pooled immune serum was added to the antibodydepleted serum to provide exogenous antibody. Samples were incubated at 37‡C for 1 h. PBS with 10 mM EDTA was added to stop the killing reaction and serial dilutions were plated onto BG agar and colony-forming units were determined to assess bacterial survival. Statistical analysis was performed using the Student’s t-test. P values 6 0.05 were assumed to be signi¢cant.
3. Results 3.1. B. pertussis is killed by complement in the absence of the alternative pathway B. pertussis was incubated with serum lacking complement activity or in the presence of serum with di¡erent
FEMSLE 10896 19-3-03
M.G. Barnes, A.A. Weiss / FEMS Microbiology Letters 220 (2003) 271^275
273
Serum-killing assays were performed with C2-depleted serum to assess bactericidal activity due to the alternative pathway. Viability was not signi¢cantly di¡erent from the heat inactivated, no killing control for any of the three strains (Fig. 2). Addition of puri¢ed C2 (1.8 mg ml31 ) restored killing to levels similar to the serum control. The ¢nding that C2-depleted serum did not kill B. pertussis demonstrates that killing by serum is entirely mediated by the classical pathway of complement and not other serum factors. 3.3. B. pertussis is not killed by direct C1q binding or the MBL pathway
Fig. 2. Role of alternative and classical pathways in killing of B. pertussis. WT (BP338), BrkA (RFBP2152) and Bvg-(BP347) were incubated for 1 h in the presence of 10% human serum with various types of complement activity. Immune antibody was added at 10% to all samples to standardize the classical pathway killing potential. Samples are as follows: 1, No CP, heat-inactivated serum lacking complement activity; 2, CP control, human serum with intact complement activity; 3, No factor B, serum depleted of factor B; 4, +factor B, serum depleted of factor B with addition of exogenous factor B; 5, No C2, serum depleted of complement protein C2; 6, +C2, serum depleted of complement protein C2 with addition of exogenous C2. Results are presented as log10 CFU and represent the average and standard error of four separate trials.
kinds of complement activity. No loss of bacterial viability was observed in heat-inactivated serum lacking complement activity (Fig. 2, No CP). Addition of serum with complement and antibody decreased viability of the wild-type strain BP338 by more than 2 logs (Fig. 2, CP control). Survival in serum of either the BrkA mutant, RFBP2152, or the Bvg mutant, BP347, lacking all virulence factors including BrkA, was signi¢cantly lower than wild-type (P 6 0.05 and 1036 respectively), con¢rming the role of BrkA in mediating resistance to complement. Factor B-depleted serum killed all three strains (Fig. 2, No factor B). Addition of puri¢ed factor B (0.1 mg ml31 ) to the factor B-depleted serum did not signi¢cantly increase killing of any of the three strains. These data demonstrate that B. pertussis is sensitive to killing by complement in the absence of the alternative pathway and ampli¢cation of the classical pathway by the alternative pathway is not required. 3.2. B. pertussis is not killed in the absence of the classical pathway Killing by the classical pathway requires C2 (Fig. 1).
C1q can bind porins (Fig. 1) and activate complement in the absence of antibody [1]. Alternatively, the MBL pathway can directly activate C4 and C2 (Fig. 1) in the absence of antibody or C1. The role of antibody-independent killing of B. pertussis was investigated. Survival of the wild-type strain or a BrkA mutant in antibody-depleted serum (Fig. 3, No antibody) was similar to survival in the heat inactivated, no complement control (Fig. 3, No CP). Addition of immune antibody restored killing (Fig. 3, +antibody) demonstrating that antibodies are required for killing, and ruling out a role for either direct C1q activation of the classical pathway or the MBL pathway in killing B. pertussis with intact LPS.
Fig. 3. Role of antibody in complement killing. WT (BP338) or BrkA (RFBP2152) B. pertussis were incubated for 1 h in serum as follows: No CP, heat-inactivated serum lacking complement activity; No antibody, antibody-depleted serum; +antibody, antibody-depleted serum with the addition of immune antibody. Results are presented as log10 CFUs and represent the average and standard error of at least six separate experiments. Asterisk indicates signi¢cance relative to heat-inactivated control (WT, P 6 0.02; BrkA, P 6 0.0002) ; # signi¢cantly di¡erent relative to wild-type (P 6 0.0007).
FEMSLE 10896 19-3-03
274
M.G. Barnes, A.A. Weiss / FEMS Microbiology Letters 220 (2003) 271^275
4. Discussion
Fig. 4. In£uence of LPS on susceptibility to complement. Wild-type BP536 (WT), and four LPS mutants: BP536vwlbH (WlbH), BP536vwlbG (WlbG), BP536vwlbD (WlbD), and BP536vwlbL (WlbL) were incubated for 1 h in serum as follows : No CP, heat-inactivated serum lacking complement activity; No antibody, antibody-depleted serum ; +antibody, antibody-depleted serum with the addition of immune antibody. Asterisk indicates signi¢cantly di¡erent relative to wild-type (P 6 0.03). Results are presented as log10 CFUs and represent the average and standard error of at least three separate experiments. The structure of LPS for the strains is diagrammed below. The sugars of the terminal trisaccharide (indicated in brackets) are N-acetylglucosamine (GlcNAc), a mannuronic acid residue (diNAcManA), and a fucosyl residue (FucNAcMe). In addition to the structure shown in the diagram, the WlbD mutant also produces a larger LPS band of unknown structure, as indicated by a question mark.
3.4. E¡ect of LPS mutations on serum resistance LPS can function as a target for complement mediated killing or it can provide protection from killing. Wild-type BP536 and LPS mutants, BP536vwlbH, BP536vwlbG, BP536vwlbD and BP536vwlbL (Fig. 4) each de¢cient in di¡erent genes required for synthesis of the O-antigen trisaccharide [16,17] were used to assess the contribution of the O-antigen to complement-susceptibility. Serum killing assays were performed using antibody-depleted serum as described for Fig. 3. Survival in antibodydepleted serum was not statistically di¡erent from the heat-inactivated no killing control for any of the strains (Fig. 4). Addition of pooled antibody from immune individuals restored killing. The wild-type strain BP536 and its LPS mutant derivatives displayed similar susceptibility to complement except BP536vwlbG, which displayed increased sensitivity to killing. The killing was mediated by the antibody-dependent pathway, suggesting the increased susceptibility of the wlbG mutant was due to antigenic di¡erences. The enzyme encoded by wlbG is proposed to be needed for the ¢rst step in polysaccharide synthesis, possibly the transfer of a sugar to a carrier lipid [16].
Complement is an important immune defense. Complement is present in serum, as well as on mucosal surfaces including the respiratory tract [19]. Like many immune defenses, the complement cascade has multiple avenues for activation, and this redundancy plays an important role in minimizing the ability of pathogens to escape immune clearance. The BrkA protein of B. pertussis mediates resistance to complement killing by the antibody-dependent classical pathway. BrkA inhibits activation of complement at an early stage of the classical complement pathway after C1 deposition [8]. In this study we investigated the ability of B. pertussis to activate the complement cascade by non-antibody mediated mechanisms. B. pertussis is only killed by the classical pathway, as evidenced by the failure of C2-depleted serum to kill the bacteria. Direct binding of C1 to surface structures such as porins [1^5] has been shown to activate complement in the absence of antibody. Puri¢ed C1 binds equally to wildtype and BrkA mutants of B. pertussis in the presence or absence of antibody [8] suggesting that activation of the classical pathway could proceed without a requirement for antibody, however the current study has shown that this is not the case. Antibody-independent killing of wild-type or BrkA mutants was not observed, and the antibody-independent C1 binding to B. pertussis must occur in a manner that fails to activate complement. Furthermore, the lack of killing in the absence of antibody also suggests B. pertussis fails to activate the MBL pathway. This result was not unexpected since mannose is not a component of the B. pertussis LPS, and has not been reported to be on the surface of the bacteria. Killing of B. pertussis by the alternative pathway was not detected, and furthermore, ampli¢cation of the classical pathway by the alternative pathway was not observed, since factor B-depleted serum killed B. pertussis as e⁄ciently as factor B-depleted serum supplemented with factor B. The failure to detect killing by the alternative pathway is consistent with a previous study that suggested that only the classical pathway of complement mediates killing of B. pertussis [7]. In that study no killing was observed in the presence of a calcium chelator, and calcium ions are necessary for the classical pathway, but not the alternative pathway. Interestingly, the wild-type strain used in this study was killed by rabbit serum lacking antibodies to Bordetella bronchiseptica [20], suggesting killing by the alternative pathway of complement had occurred. Activation of the alternative pathway may occur di¡erently for di¡erent mammalian species. B. pertussis presents a surface to the human host that is not recognized by the innate pathways of the human complement system. Pathogens, such as Neisseria gonorrhoeae resist killing by the alternative pathway by adding sialic acid to their LPS [10], but such modi¢cations have not been observed for B. pertussis. The LPS of B. pertussis
FEMSLE 10896 19-3-03
M.G. Barnes, A.A. Weiss / FEMS Microbiology Letters 220 (2003) 271^275
lacks an O-side chain, and has a branched core structure and a trisaccharide with unusual sugars [11]. We had thought that the unusual sugars might play a role in blocking activation of the alternative pathway, however four mutants with di¡erent de¢ciencies in production of the trisaccharide were all resistant to killing in the absence of antibody. The mechanism used by B. pertussis to resist killing by the alternative pathway is currently unknown. B. pertussis is a strongly host-adapted pathogen, and our studies suggest this human pathogen has adapted to evade the human defenses. Only the most speci¢c pathway of complement activation, the antibody dependent classical pathway can initiate bacterial killing, but even then, the BrkA protein can protect B. pertussis from complement killing.
Acknowledgements This study was supported by Grant RO1 AI45715 to A.A.W. M.G.B. was the recipient of a Graduate Student Summer Research Fellowship from the University of Cincinnati. References [1] Alberti, S., Rodriquez-Quinones, F., Schirmer, T., Rummel, G., Tomas, J.M., Rosenbusch, J.P. and Benedi, V.J. (1995) A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding. Infect. Immun. 63, 903^910. [2] Butko, P., Nicholson-Weller, A. and Wessels, M.R. (1999) Role of complement component C1q in the IgG-independent opsonophagocytosis of group B Streptococcus. J. Immunol. 163, 2761^2768. [3] Loos, M., Wellek, B., Thesen, R. and Opferkuch, W. (1978) Antibody-independent interaction of the ¢rst component of complement with Gram-negative bacteria. Infect. Immun. 22, 5^9. [4] Merino, S., Nogueras, M.M., Aguilar, A., Rubires, X., Alberti, S., Benedi, V.J. and Tomas, J.M. (1998) Activation of the complement classical pathway (C1q binding) by mesophilic Aeromonas hydrophila outer membrane protein. Infect. Immun. 66, 3825^3831. [5] Mintz, C.S., Arnold, P.I., Johnson, W. and Schultz, D.R. (1995) Antibody-independent binding of complement component C1q by Legionella pneumophila. Infect. Immun. 63, 4939^4943.
275
[6] Wong, N.K., Kojima, M., Dobo, J., Ambrus, G. and Sim, R.B. (1999) Activities of the MBL-associated serine proteases (MASPs) and their regulation by natural inhibitors. Mol. Immunol. 36, 853^ 861. [7] Fernandez, R.C. and Weiss, A.A. (1994) Cloning and sequencing of a Bordetella pertussis serum resistance locus. Infect. Immun. 62, 4727^ 4738. [8] Barnes, M.G. and Weiss, A.A. (2001) BrkA Protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition. Infect. Immun. 69, 3067^3072. [9] Walport, M.J. (2001) Complement. First of two parts. N. Engl. J. Med. 344, 1058^1066. [10] Parsons, N.J., Andrade, J.R., Patel, P.V., Cole, J.A. and Smith, H. (1989) Sialylation of lipopolysaccharide and loss of absorption of bactericidal antibody during conversion of gonococci to serum resistance by cytidine 5P-monophospho-N-acetyl neuraminic acid. Microb. Pathog. 7, 63^72. [11] Caro¡, M., Brisson, J., Martin, A. and Karibian, D. (2000) Structure of the Bordetella pertussis 1414 endotoxin. FEBS Lett. 477, 8^14. [12] Brodeur, B.R., Martin, D., Hamel, J., Shahin, R.D. and Laferriere, C. (1993) Antigenic analysis of the saccharide moiety of the lipooligosaccharide of Bordetella pertussis. Springer Semin. Immunopathol. 15, 205^215. [13] Weiss, A.A., Mobberley, P.S., Fernandez, R.C. and Mink, C.M. (1999) Characterization of human bactericidal antibodies to Bordetella pertussis. Infect. Immun. 67, 1424^1431. [14] Weiss, A.A., Hewlett, E.L., Myers, G.A. and Falkow, S. (1983) Tn5induced mutations a¡ecting virulence factors of Bordetella pertussis. Infect. Immun. 42, 33^41. [15] Fernandez, R.C. and Weiss, A.A. (1998) Serum resistance in bvgregulated mutants of Bordetella pertussis. FEMS Microbiol. Lett. 163, 57^63. [16] Allen, A. and Maskell, D. (1996) The identi¢cation, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis. Mol. Microbiol. 19, 37^52. [17] Allen, A.G., Thomas, R.M., Cadisch, J.T. and Maskell, D.J. (1998) Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Mol. Microbiol. 29, 27^38. [18] Barnes, M.G. and Weiss, A.A. (2002) Growth phase in£uences complement resistance of Bordetella pertussis. Infect. Immun. 70, 403^ 406. [19] Persson, C.G. et al. (1991) Plasma exudation in the airways: mechanisms and function. Clin. Exp. Allergy 21, 17^24. [20] Harvill, E.T., Preston, A., Cotter, P.A., Allen, A.G., Maskell, D.J. and Miller, J.F. (2000) Multiple roles for Bordetella lipopolysaccharide molecules during respiratory tract infection. Infect. Immun. 68, 6720^6728.
FEMSLE 10896 19-3-03