Review

Complement evasion by Borrelia burgdorferi: it takes three to tango

Steven W. de Taeye1,2, Lieselotte Kreuk1, Alje P. van Dam3, Joppe W. Hovius1,4, and Tim J. Schuijt1,4

1 Center for Experimental and Molecular Medicine, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands 2 Experimental Virology, Academic Medical Center, University of Amsterdam, Amsterdam, 1105 AZ, The Netherlands 3 Department of Medical Microbiology, Onze Lieve Vrouwe Gasthuis, Amsterdam, 1091 AC, The Netherlands 4 Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06420, USA

The complement system is one of the major innate B. afzelii is primarily associated with dermatological man- defense mechanisms Borrelia burgdorferi sensu lato ifestations [1,6,7]. In a subset of Lyme patients, post-treat- has to overcome to establish an infection of mammalian ment symptom recurrence occurs, although it remains hosts and to cause Lyme borreliosis in humans. Borrelia unclear whether this is caused by a relapse or if it is due prevents complement-mediated killing during host col- to reinfection. Interestingly, a recent study including 17 onization through (i) recruitment of host complement patients with a recurrent erythema migrans showed that regulators by Borrelia, (ii) evasion mechanisms by Bor- by examining ospC genotypes these recurrent episodes were relia itself, and (iii) exploitation of tick proteins by Bor- due to a reinfection [8]. relia. These interactions with complement can be host The mammalian–tick life cycle of Borrelia is crucial for species-specific. This review provides an overview of its survival. Ixodes ticks employ a three-host life cycle, in interactions between Borrelia, tick, and host leading which nymphal and adult ticks are able to transmit Bor- to evasion of complement-mediated killing. relia to mammalian hosts including humans [9]. To survive and replicate during each phase in the mammalian–tick Borrelia and Lyme borreliosis life cycle, Borrelia spirochetes have acquired the ability to Lyme disease or Lyme borreliosis has become the most adapt to the hostile and changing environment via both common tick-borne disease in the USA and Europe [1].In differential expression of outer surface proteins and the endemic areas, the prevalence of Lyme borreliosis has been use of tick proteins during transmission from the tick to the estimated to range from 20 to 100/100 000 people in the USA mammalian host [10]. The capacity to resist clearance by and from 100 to 130/100 000 in Europe [2]. Lyme borreliosis the mammalian complement system is an important step is a zoonotic disease caused by spirochetes of the Borrelia for persistence of Borrelia in the host. The purpose of this burgdorferi sensu lato (B. burgdorferi s.l.) complex [3].Inthe review is to provide an overview of the interactions be- USA, B. burgdorferi sensu stricto (s.s.) causes Lyme borre- tween Borrelia, the tick, and the mammalian host in the liosis, whereas in Europe, in addition to B. burgdorferi s.s., perspective of complement evasion by Borrelia spirochetes. other genospecies, for example, Borrelia afzelii, Borrelia garinii, Borrelia bavariensis,andBorrelia spielmanii,are Complement pathways involved in eradication of the major Lyme borreliosis-causing species. Borrelia Borrelia valaisiana, Borrelia lusitaniae,andBorrelia bisettii are also Spirochetes need to circumvent recognition by host com- part of the B. burgdorferi s.s. complex but have only sporad- plement not only during but also after transmission from ically been isolated from patients with Lyme borreliosis. the tick to the mammalian host. The complement system is Borrelia is transmitted by Ixodes ticks and more specifically part of the innate immune system and consists of a complex by Ixodes scapularis (USA), Ixodes pacificus (USA), Ixodes network of plasma and membrane-associated proteins, ricinus (Europe), and Ixodes persulcatus (Asia and Europe) which can be activated in a cascade-like manner on the [3]. A typical early clinical manifestation of Lyme borreliosis surface of invading pathogens (Box 1). Borrelia genospecies is a local skin rash, characterized by an expanding skin differ in their ability to survive in the presence of human lesion with central clearing at the tick bite site (e.g., erythe- complement [11–16] and are classified as either serum- ma migrans) [4]. When the infection is left untreated, the resistant or serum-sensitive isolates. B. garinii serotypes 5 spirochete can disseminate throughout the body and cause and 6 are classified as serum sensitive, B. burgdorferi s.s. late clinical manifestations including neuroborreliosis (71% as moderately resistant, and B. afzelii, B. spielmanii, and of Lyme manifestations), Lyme arthritis (22%), and acro- B. garinii serotype 4 (also known as B. bavariensis)as dermatitis chronica atrophicans (5%) [5]. B. burgdorferi s.s. serum resistant [11–16]. Serum sensitivity of Borrelia is infection is often associated with Lyme arthritis, whereas B. determined by measuring the borreliacidal effect of serum garinii infection is mostly linked to neuroborreliosis; as a result of formation of the complement membrane

Corresponding author: Schuijt, T.J. ([email protected]). attack complex (MAC) on the outer membrane of Borrelia Keywords: complement; Borrelia; Lyme; borreliosis; Ixodes; tick; regulators. spirochetes (Figure 1).

1471-4922/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2012.12.001 Trends in Parasitology, March 2013, Vol. 29, No. 3 119 Review Trends in Parasitology March 2013, Vol. 29, No. 3

Box 1. Complement activation pathways

The classical pathway is initiated via the C1 complex, consisting of complex with MBL or ficolins and become autoactivated after ligand C1q, two molecules of C1r, and two molecules of C1s (C1qr2s2) [17]. binding. Similar to the serine protease C1s of the classical pathway, Binding of C1q to pathogen-bound C-reactive protein (CRP) or activated MASP-2 cleaves C2 and C4, resulting in the formation of C3 immunoglobulins or even directly to the pathogenic surface leads to convertase, C4bC2a [71]. Another pathway of complement activation autocatalytic activation of C1r. Then, C1r activates C1s and C1s via the lectin pathway occurs through direct cleavage of C3, which activation is followed by cleavage of C2 and C4 in the activation was proposed to be a ‘back up’ pathway of complement activation products C2a, C2b, C4a, and C4b. Surface-bound C4b and C2a form via the lectin route in humans with complement deficiencies, such as C3 convertase of the classical pathway, C4b2a, which cleaves C3 into C2 deficiency [64]. MASP-1 was found to support complement the anaphylatoxin C3a and opsonin C3b. C3 is the central serum activation via the alternative pathway by activating factor D [63]. protein in the complement system where all three complement Recent data suggest that MASP-3 is also involved in complement cascades converge. In addition to its function as an opsonin to activation through the alternative pathway [62]. MASP-3 can activate support phagocytosis, C3b can also form the C5 convertase of the factor D as well as factor B [62], which are both essential proteins in classical pathway together with C4b2a. The C5 convertase, C4b2a3b, the alternative pathway. Complement activation via the alternative induces the activation of the late complement components C6 to C9 pathway occurs functionally differently in comparison to activation on the cell membrane. Formation of the MAC, which consists of C5b- via the classical and lectin pathways. On all cell surfaces ‘tick over’, 8 and a polymer of C9 molecules, will initiate lysis of the cell. low level initiation occurs continuously through hydrolysis of C3 to

Cleavage products C4a, C3a, and especially C5a are inflammatory C3H2O [17].FactorBbindsC3H2O, and subsequently factor B is mediators that attract neutrophils and enhance production of cleaved by factor D, forming an initial solvent-based C3 convertase

proinflammatory cytokines. Initiation of the lectin pathway occurs C3H2OBb. Cleaving of C3 into C3b is followed by the formation of the when MBL or ficolins, for example, ficolin-1 (M-ficolin), ficolin-2 (L- predominant alternative pathway C3 convertase C3bBb on cell ficolin), and ficolin-3 (H-ficolin), recognize and bind carbohydrate surfaces. The C3 convertase is further stabilized by properdin and patterns on the cell surface of pathogens through their carbohydrate can eventually form the C5 convertase C3bBb3b. See also Figure 2 in recognition domains (CRDs) [71,80]. MASPs 1, 2, and 3 form a main text.

Activation of complement can be initiated via three classical pathway also bactericidal complement-indepen- different pathways: the classical, lectin, and alternative dent antibodies against Borrelia are described [19]. pathways, which, to date, have all been shown to be The alternative pathway initiates complement activa- involved in complement activation on Borrelia tion through spontaneous hydrolysis of C3 (Figure 2) [17]. (Figure 2). In 1997, van Dam et al. described that both Interestingly, in the presence of C1-inhibitor (C1-INH), a the classical and alternative pathways were involved in serine protease inhibitor of both the classical and lectin complement-mediated killing of Borrelia by means of C1q-, pathways but not the alternative pathway, complement- properdin- and C4-deficient human sera [14,17]. Besides mediated killing of Borrelia was completely abrogated, that activation of the classical pathway is triggered upon suggesting that the alternative pathway may mainly be recognition of antibodies to the pathogen surface, C1 acti- important for amplification of complement activation [18]. vation also occurs by a diverse range of other structures Recently, the discovery of the tick salivary protein including the bacterial surface itself. Indeed, complement- TSLPI (Tick Salivary Lectin Pathway Inhibitor), a protein mediated killing of serum sensitive Borrelia strains via the that inhibits the binding of mannose-binding lectin (MBL) classical pathway was described in human serum that to its ligand, revealed the role of the lectin pathway in lacked Borrelia-specific antibodies [14,18]. Moreover, be- complement activation on Borrelia [18,20]. Complement- [(Figure_1)TD$IG]sides antibody-initiated lysis through the complement mediated killing of serum-sensitive B. garinii spirochetes

10 µm 10 µm

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Figure 1. Complement-mediated killing of Borrelia spirochetes. Scanning electron microscope micrographs of the serum-sensitive Borrelia garinii A87S (left) and the serum-resistant Borrelia burgdorferi B31 (right) after 1.5 h of incubation with 12.5% human serum. Bleb formation on the outer surface of B. garinii, as a result of complement activation and subsequent formation of the lytic membrane attack complex (C5b–9), is indicated with white arrows [18]. Formation of membranous blebs is a result of membrane disruption leading to death of the target cell.

120 Review [(Figure_2)TD$IG] Trends in Parasitology March 2013, Vol. 29, No. 3

Classical pathway (CP)

C3 C4 C4a C3a C2 C1q C4b2a C3b C3b C3b C3b C3b

Common terminal pathway

Phagocyte chemotaxis Membrane aack Lecn pathway (LP) C4a C3a C5a complex (MAC) C3 C4 C4a C3a C2 FCN C6 C7 C8 MBL C9 C4b2a C3b C3b C3bBbC3b C3b C3b C3b (AP) C5 Opsonisaon and for phagocytosis C4b2a3b C5a C5b-9 C3b (LP+CP) C3b C3b C3b C3b C5b

Alternave pathway (AP)

Factor B C3-H2O C3 Ba C4a C3a Bypass acvaon Bypass via MASP -1 and -3 Spontaneous C4 C3-H2O hydrolysis + factor B C2 C3 Properdin Factor D C3bBb C4b2a C3b C3Bb C3b

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Figure 2. Complement activation pathways leading to the common terminal pathway. Complement can be activated on the outer membrane of Borrelia through three pathways: the classical (CP), lectin (LP), and alternative (AP) pathways. The classical pathway is activated when complement protein C1q binds to the pathogen surface, which activates complement C4 and C2. In the lectin pathway, mannose-binding lectin (MBL) or ficolin (FCN), in complex with serine protease MASP-1/-2/-3 (mannose- binding lectin-associated serine protease-1/-2/-3) and small mannose-binding lectin-associated protein (sMAP), binds to polysaccharide structures on a pathogen surface, leading to the autoactivation of MASP-2, permitting the cleavage of C2 and C4. Both pathways converge to produce C3 convertase consisting of C4b and C2a. C3 convertase can either cleave additional C3 into C3b, or bind to C3b, producing the C3/C5 convertase (C4bC2aC3b). MASP-1 and MASP-3 of the lectin pathway can activate the alternative pathway directly. The contribution of this ‘bypass’ activation route in complement activation on Borrelia is yet to be determined. The alternative pathway involves the continuous spontaneous hydrolysis of C3 into C3-H2O, which binds to factor B, producing Bb and Ba through the action of factor D. Properdin binds and stabilizes the alternative C3 convertase C3bBb. The latter can either cleave more C3 components or bind to C3b, producing the C5 convertase (C3bBbC3b). C5 convertase of either the alternative pathway, the classical or lectin pathways can go on to produce the membrane attack complex (MAC) through the terminal assembly of complement components C5b through C9, leading to cell lysis and death. In addition to MAC formation, activation of the complement cascade results in leukocyte chemotaxis and opsonization of the invading pathogen, leading to enhanced phagocytosis. was reduced in the presence of TSLPI or neutralizing MBL through its binding to the tick receptor of OspA (TROSPA) antibody [18], illustrating the contribution of this pathway. [23]. To successfully infect a mammalian host, Borrelia has In conclusion, all three pathways of complement activation to migrate from the midgut to the salivary glands of the play a role in host defense against B. burgdorferi s.l. tick. Hereto, the expression pattern of Borrelia changes infection, at which the classical and lectin pathways initi- upon tick feeding, leading to a decrease in ospA and ospB ate complement activation, whereas the alternative path- expression and an increase in ospC expression [24]. Upre- way amplificates this process. Importantly, in addition to gulation of OspC facilitates migration of Borrelia from the differences in serum sensitivity between B. burgdorferi s.l. tick midgut to the salivary glands [25]. Borrelia spirochetes isolates, it is relatively possible that there are interspecies expressing OspC [26] as well as ErpP/A/C [27] and a differences in initiation of complement activation as well. surface enolase [28] on their surface were observed to immobilize host plasminogen to their surface. Indeed, it Survival of Borrelia in tick–mammal–tick cycle was shown that vertebrate plasminogen is important in The complex mammalian–tick life cycle drives Borrelia to efficient dissemination of Borrelia in ticks and spirochete- be extremely flexible, resulting in adaptation to existing mia in mice [29]. In the presence of plasminogen activators, environmental challenges. Borrelia upregulates outer sur- enzymatically active plasmin is generated on the surface of face proteins (Osp) A and B as soon as the bacteria colonize Borrelia, which resulted in enhanced penetration of endo- the tick vector, allowing spirochetes to persist in the tick thelial monolayers [30]. between blood meals [21,22]. In 2004, Pal et al. showed that Another surface-exposed lipoprotein of Borrelia, VlsE, OspA mediates in the adherence of Borrelia to the tick’s gut is upregulated and contributes to immune evasion and

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Table 1. Resistance of Borrelia genospecies to sera from different animal speciesa,b B. burgdorferi s.s. B. afzelii B. garinii B. bavariensis B. spielmanii Human +/– + – + + Rodents +/– + – + ND Birds +/– – + – ND Dog + + +/– + ND Wolf + +/– +/– + ND Cat ++++ND Horse +/– – – – ND Deer/cattle ––––ND aTable 1 compiles data of several studies [32–34]. bAbbreviations: +, serum resistant; +/–, moderately serum resistant; –, serum sensitive; ND, not determined. persistence of Borrelia in infected mammalian hosts killing in different hosts. In the following paragraphs, we through an elaborate antigenic variation mechanism to will discuss the interactions between Borrelia and the tick escape the adaptive immune system of the host [31]. and/or host proteins leading to (partial) complement eva- sion, with a focus on the human and murine host proteins Adaptation to a diversity of mammalian hosts because little is known about the interactions with proteins Tick feeding on different hosts further enhances the com- from other host species. plexity of the interplay between Borrelia and tick and/or host proteins leading to immune evasion by Borrelia. Complement evasion of Borrelia burgdorferi sensu lato Adaptation of isolates to specific vertebrate hosts underlies Recruitment of mammalian host complement regulators the diverging transmission patterns of Borrelia. In Europe, Many pathogens have evolved mechanisms allowing them adaptations in B. garinii have made bird species its main to evade complement activation upon host infection. A host, whereas rodent species are competent hosts for B. frequently used mechanism by pathogens is the acquisition afzelii and B. bavariensis [32]. B. burgdorferi is carried and of host complement regulators on their surfaces [35]. Box 2 transmitted by a larger variety of hosts, consisting of both summarizes the various complement regulators identified birds and rodents [32]. The pattern of in vitro serum in humans. The capacity of in vitro cultured Borrelia sensitivity of different Borrelia genospecies corresponds isolates to survive in human serum was shown to be with the observed spirochete transmission patterns and correlated with the expression of complement regulator with the reservoir status of many vertebrate species for acquiring surface proteins (CRASPs) [15,36–39]. The ex- Borrelia (Table 1) [32–34]. Large mammals, such as cows pression and function of CRASPs in Borrelia genospecies and deer, are incompetent dead-end hosts for all Borrelia has been studied extensively and the mechanism of com- species, most probably caused by complement-mediated plement resistance via factor H binding has been reviewed killing of Borrelia [9,34]. The species-specific adaptation of elsewhere [40,41]. Therefore, we will provide a brief over- Borrelia is interesting and may reveal which specific adap- view of current knowledge on CRASP expression, function, tations are necessary to overcome complement-mediated and the role of CRASPs in complement resistance in

Box 2. Complement regulators of the host

To prevent excessive complement activation and to discriminate C3b is inhibited; (ii) fH acts as a cofactor for C3b cleavage by factor I; between the surface of pathogenic cells and healthy human cells, and (iii) fH supports dissociation of the C3 convertase C3bBb [42]. complement activation is tightly balanced by both soluble and FHL-1 and CFHR proteins are part of the factor H family and share membrane-bound complement regulators [81]. Complement activa- some regulatory mechanisms of factor H [42]. Together with C4bp tion via all three pathways is regulated at multiple steps in the and proteins of the factor H family, membrane cofactor protein (MCP/ activation cascade by individual complement regulators. C1-inhibitor, CD46) and decay accelerating factor (DAF/CD55) belong to the a member of the serpin family of protease inhibitors, regulates regulators of complement activation (RCA) family [35]. RCA proteins complement activation at the level of initiation via binding of serine typically consist of complement control protein (CCP)/short consen- proteases of the classical and lectin pathways (C1r, C1s, MASP-2) sus repeat (SCR) domains, which are essential for their regulatory [82]. sMAP and MAP-1, both non-proteolytic alternative splice activity [35]. MCP binds C3b/C4b and acts as a cofactor for factor I products of the MASP 2 and MASP 1/3 genes, respectively, compete mediated degradation of C3b and C4b, whereas DAF further regulates with MASP-2 for binding to MBL or ficolins and regulate complement complement activation by accelerating the decay of C3 convertases activation via the lectin pathway [80]. Complement activation is C4b2a and C3bBb, as well as C5 convertases [81]. Complement furthermore regulated via the inactivation of anaphylatoxins by activation is furthermore regulated at the step of MAC formation by carboxypeptidase N (CPN). The biological activity of C3a and C5a is both fluid and membrane-bound proteins. MAC formation was found 10- to 100-fold reduced when cleaved by CPN to C3adesArg and to be inhibited by vitronectin via two mechanisms, either via binding C5adesArg [83]. C4-binding protein (C4bp) and factor H (fH), both of the C5b–7 complex and preventing membrane insertion, or via soluble serum proteins, recognize host-specific cell surface patterns, direct binding of C9, so that lytic pore formation is affected. In such as glycosaminoglycans, and regulate complement activation to addition to vitronectin, clusterin also regulates complement activa- prevent self-attack. C4bp inhibits complement activation via the tion at the step of MAC formation. Via binding the late complement classical and lectin pathways in three ways: (i) C4bp binds C4b and components C7, C8b, and C9b, clusterin inhibits the formation of the thereby prevents C2 binding and C4b2a formation; (ii) C4bp accel- lytic pore on the cell membrane. The only membrane-bound erates the decay of C4b2a; and (iii) acts as a cofactor for factor I complement regulator that interferes with MAC formation is CD59 cleavage of C4b [81]. Factor H inhibits the alternative pathway using [81]. CD59 binds the complement components C8 and C9 and thereby functionally similar mechanisms as C4bp: (i) binding of factor B to prevents assembly of the MAC.

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(a) CFH Factor H Family proteins CFHR Inhibion alternave pathway acvaon: - Prevents binding of factor B to C3b CFHL - Cofactor for cleavage of C3b by factor I C4bp - Supports dissociaon of C3bBb complex amla otTick Mammalian host C4 binding protein Inhibion classical & lecn pathway acvaon: Accelerates decay of C3 convertase C4b2a - Prevents C2 binding and thus C4b2a formaon - C4bp as cofactor for factor I in cleavage of C4b - CRASP-1 CRASP-5 (CspA) CRASP-2 (ErpA) C4bp Binding (CspZ) CRASP-4 receptor (ErpC) (43 kDa) CRASP-3 (ErpP)

(b) Borrelial membrane composion Steric constraints Modified Lyc MAC by cell surface cell surface Membrane aack Non-lyc C5b-9 (MAC) complex (MAC) IgG/Fab Borrelia

CD59-like C9 C5b-8

(c) Tick salivary MAC proteins TSLPI Salp20 / IxAC family Salp15 Properdin

FCN

MBL Complement acvaon on Borrelia

Unstable C3bBb OspC

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Figure 3. Molecular mechanisms that contribute to complement evasion by Borrelia. Borrelia spirochetes have evolved several strategies to evade complement activation by co-opting mammalian host (a) and tick (c) complement regulators/inhibitors and by protecting itself (b) directly against complement-mediated killing. (a) Serum-resistant Borrelia isolates recruit host complement regulators that are part of the factor H family such as complement factor H (CFH), factor H-like 1 (CFHL), and factor H-related (CFHR) proteins by expressing Erps and Csps on their outer membrane. These Erps and Csps are collectively called complement regulator-acquiring surface proteins (CRASPs) and, to date, five CRASPs have been identified. CFH binds to all five CRASPs, whereas CHFL only binds to CRASP-1 and -2, and CFHR proteins solely bind to CRASP-3, -4, and -5. This figure illustrates binding interactions between CRASPs from Borrelia burgdorferi sensu stricto to factor H family proteins. These interactions are slightly different for certain other Borrelia genospecies as discussed in the text. In addition to recruiting factory H family proteins, Borrelia has also shown to bind C4- binding protein of the host. (b) Borrelia expresses CD59-like protein that inhibits the formation of the lytic membrane attack complex (MAC, left panel). Studies by Kochi et al. [54,55] showed that altering the borrelial cell surface by means of anti-Borrelia IgG or IgG Fab fragments allowed effective MAC formation, suggesting that the borrelial membrane composition sterically inhibits formation of lytic MAC (right panel). (c) Borrelia co-opts the tick salivary protein TSLPI (Tick Salivary Lectin Pathway Inhibitor) to evade clearance mediated by the lectin pathway of complement activation (left panel). Tick salivary proteins from the Ixodes Anti-Complement (IxAC) family, including salivary protein (Salp)20 bind and neutralize host-derived properdin, a positive regulator of the alternative pathway of complement. Neutralization of properdin has been shown to inhibit complement-mediated killing of Borrelia by early degradation of unstable C3bBb (middle panel). Direct binding of tick salivary protein Salp15 to the outer surface protein C (OspC) protects Borrelia against MAC formation and lysis of the spirochetes (right panel).

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Borrelia genospecies. CRASPs are lipoproteins expressed serum-resistant isolates was further questioned when B. on the outer surface of Borrelia that bind one or more host burgdorferi was found to infect factor H-deficient mice as proteins of the factor H family (Figure 3a). Factor H well as wild type mice [56], suggesting that factor H binding enables spirochetes to reduce alternative pathway binding is not crucially important for Borrelia infectivity complement activation and prevent formation of the MAC in mice. Of note, Pickering et al. revealed that factor H- on the cell surface [40,41]. Other members of the comple- deficient mice have markedly reduced amounts of C3 in ment factor H family are factor H-like protein (FHL-1), a plasma as a result of uncontrolled alternative pathway splice variant of factor H, and five complement factor H- complement activation [57]. Low plasma levels of C3 might related (CFHR 1–5) proteins. FHL-1 shares ligand binding explain why spirochetes establish infection in factor H- and complement regulatory activity with the N terminus of deficient mice. Additionally, CRASPs might still contribute factor H, whereas CFHR proteins lack short consensus to serum resistance by binding to other proteins in addition repeats (SCRs) homologous to the complement regulatory to factor H or preventing complement activation in a domains (SCRs 1–4) of factor H [42]. Although the exact different way. In factor H-deficient mice, CFHR proteins role of CFHR proteins in complement regulation is un- may have taken over the role of factor H, possibly explain- known, these might function as a cofactor for factor H [42]. ing similar Borrelia loads in wild type and factor H-defi- To date, five CRASPs are known: CRASP-1 (CspA), cient mice [56]. Although murine and human factor H are CRASP-2 (CspZ), CRASP-3 (ErpP), CRASP-4 (ErpC), functionally similar [58], the possibility remains that, and CRASP-5 (ErpA). All five CRASPs have been identified owing to small differences in biochemical properties, hu- in the serum-resistant B. afzelii (BaCRASPs) and B. burg- man factor H plays a more pronounced role in complement dorferi s.s. (BbCRASPs) [43]. Besides the ability of all evasion by Borrelia. Also, the binding of FHL-1, which mice BbCRASPs to bind factor H, BbCRASP-1 and -2 addition- and other mammals lack, might still play a role in comple- ally bind FHL-1 [44,45], whereas BbCRASP -3, -4, and -5 ment evasion in humans. were found to bind complement factor H-related proteins 1, In addition to hijacking members of the factor H protein 2, and 5 (CFHR-1, -2, and -5) [46,47]. Binding capacities of family, Borrelia also recruits C4b-binding protein (C4bp) BaCRASPs to factor H family proteins are slightly differ- via a 43-kDa putative receptor (Figure 3a) [59]. B. afzelii, ent compared with BbCRASPs. Whereas BbCRASP-3 B. burgdorferi, and B. garinii were all found to bind C4bp, binds factor H, BaCRASP-3 was found to solely bind although binding was strongest to B. garinii [59]. C4bp FHL-1 [43]. Furthermore, CFHR2 and CFHR5 binding inhibits complement activation via the classical and lectin was only shown for BbCRASP-3, -4, and -5 [46]. Serum- pathways by increasing breakdown of the C3 convertase resistant B. spielmanii isolates were identified to express C4bC2a (Box 2). Interestingly, C4bp prevents formation BsCRASP-1, BsCRASP-2, and BsCRASP-3, in which and accelerates decay of the lectin pathway C3/C5 con- BsCRASP-1 and BsCRASP-2 bind factor H, and FHL-1 vertase to a greater extent compared with the classical and BsCRASP-3 bind factor H and CFHR-1 [15,48]. Serum- pathway C3/C5 convertase, because C4bp has a tenfold sensitive B. garinii isolates were found to express ortho- greater affinity for C4b deposited via the lectin pathway logous proteins of CRASP-1 with only weak binding to compared with the classical pathway [60]. A more strict FHL-1 and no binding to factor H [49]. By contrast, ortho- regulation of the lectin pathway might be needed, because logous CRASP-1 proteins isolated from serum-resistant B. each deposited C4b via the lectin pathway is able to form bavariensis isolates were able to bind both factor H and the C4bC2a convertase, whereas only one in four deposited FHL-1 [50]. BbCRASP-1 was found to be expressed exclu- C4b molecules has the capacity to form the C4bC2a con- sively during Borrelia transmission from tick-to-host and vertase via the classical pathway [61]. Further studies are host-to-tick, whereas BbCRASP-2 was only expressed dur- needed to characterize receptor function and expression of ing mammalian infection [51]. Resistance to complement- the putative receptor in other Borrelia genotypes, as well mediated killing of Borrelia correlates with the expression as the role of C4bp binding in serum resistance in different of BbCRASP-1 [38,52]. Although BbCRASP-2 binds factor Borrelia species. Moreover, activation of the alternative H and FHL-1 and protects Borrelia against complement- pathway via MASP-1 (mannose-binding lectin-associated mediated killing [37], BbCRASP-2 mutants remain insen- serine protease-1) and MASP-3 of the lectin pathway sitive to complement-mediated killing in vitro and retain should be considered, while this pathway could circumvent full murine infectivity [53]. These data indicate that the complement regulation of C4bp [62–64]. To date, the con- function of BbCRASP-2 is replaceable and is not required tribution of this ‘bypass’ complement activation pathway for spirochete persistence in the murine host. In line with via the lectin pathway on Borrelia has not been studied. this, a recent study demonstrated that the expression of It is not unlikely that B. burgdorferi s.l. species avoid Erp or OspE-related proteins protected Borrelia against complement-mediated killing by recruiting additional complement-mediated killing by binding factor H [39]. complement regulators (Box 2) of the host, which already Reduced C3b deposition and C3/C5 convertase activity have shown to play a role in complement evasion by would be expected on the surface of Borrelia as a conse- various other pathogens. As an example, Borrelia recur- quence of factor H and FHL-1 binding on the cell surface, rentis, the causative agent of louse-borne relapsing fever, resulting in protection against killing by MAC. However, acquires C1-INH to their cell surface via its surface protein several studies showed similar C3b deposition on serum- CihC, which might contribute to serum resistance [65]. sensitive and -resistant Borrelia isolates, which questions Interestingly, expression of CihC in a serum-sensitive B. the importance of CRASPs in serum resistance burgdorferi strain resulted in resistance to complement- [12,13,54,55]. The importance of factor H binding by mediated killing [65]. Further studies are needed to reveal

124 Review Trends in Parasitology March 2013, Vol. 29, No. 3 a possible role of these additional host complement reg- B. burgdorferi [68]. Borrelia binds Salp15 via OspC to ulators in complement evasion by B. burgdorferi s.l. protect Borrelia from antibody-mediated [68,69] as well as complement-mediated [70] killing. Both serum-sensi- Borrelial factors tive B. garinii and moderately resistant B. burgdorferi s.l. In addition to recruitment of host complement regulators, isolates were protected against complement-mediated kill- several pathogens produce structural mimics of mem- ing after binding to either I. scapularis or I. ricinus Salp15 brane-bound complement regulators of the host to prevent (Figure 3c) [70]. Despite great heterogeneity of OspC be- complement-mediated lysis [35]. CD59, a host protein that tween different Borrelia genospecies, Salp15 binds with prevents MAC formation, was found to be mimicked by similar affinity to recombinant and native OspC from B. some Borrelia isolates (Figure 3b) [13]. This CD59-like burgdorferi, B. garinii, and B. afzelii [69]. However, in protein was expressed by a serum-resistant B. afzelii strain Borrelia-immune mice Salp15 only provided B. burgdorferi and not by a serum-sensitive B. garinii strain [13], sug- a survival advantage. The reason for this preferential gesting that the expression of CD59-like protein is an protection is unclear. It could be that the different homo- additional strategy to avoid complement-mediated killing logs that exist in I. ricinus preferentially bind to or protect by Borrelia. It would be interesting to further assess the other Borrelia genospecies from antibody-mediated com- function and expression of CD59-like protein in other plement-dependent killing, although preliminary data Borrelia genospecies and its role in serum resistance. does not point in that direction (T.J. Schuijt et al., unpub- There is evidence that the membrane composition of lished). Borrelia spirochetes plays a role in serum resistance as In addition to directly binding tick proteins, Borrelia well. In addition to comparable levels of C3 deposition, benefits indirectly by utilizing tick salivary proteins that similar levels of C8 and C9 were deposited on the outer inhibit complement activation at the tick bite site. Salivary membrane of serum-sensitive and serum-resistant isolates protein TSLPI inhibits complement-mediated killing of post-serum treatment [54]. Both anti-Borrelia IgG anti- Borrelia, by preventing MBL binding to its presently un- bodies [54] and anti-Borrelia IgG Fab fragments [55] in- known ligand on Borrelia (Figure 3c) [18]. In MBL-defi- duced complement-mediated killing of serum-resistant cient serum, TSLPI still diminished Borrelia killing, Borrelia strains. Because complement-mediated killing suggesting that another part of the lectin pathway is of these serum-resistant Borrelia isolates occurred when inhibited by TSLPI as well. In line with this, TSLPI was Borrelia-specific IgG or Fab fragments were added be- found to inhibit complement activation via human L-ficolin tween C5 convertase formation and C5 activation, Kochi [18]. L-ficolin is, in addition to MBL, known to activate the et al. suggested that this may be due to increased insertion lectin pathway of complement activation (Box 1). There- of the C5b–9 complex into the outer membrane of Borrelia fore, it is plausible that ficolins also play a role in recogni- [54] (Figure 3b). In line with this, differences in ‘bacteri- tion and complement activation on Borrelia. However, cidal efficiency’ of C5b–9 were shown to be caused by further research is needed to narrow down the role of differences in the molecular configuration of C5b–9 on ficolins in complement activation and subsequent eradica- the bacterial outer surface [66]. The exact mechanism of tion of Borrelia. In addition to initiating the lectin pathway how anti-Borrelia IgG and IgG Fab fragments alter the of complement activation, MBL and ficolins act as opsonins bacterial outer surface and subsequently the molecular to support phagocytosis through collectin receptors on configuration of C5b–9 remains unknown and needs fur- phagocytic cells of the immune system [71]. The exact role ther research. Contrary to the observations of Kochi et al., a of MBL, ficolins, and MBL-associated serine proteases different study showed reduction in C6 and C9 on serum- (MASPs) in complement activation, phagocytosis, and po- resistant isolates [12]. These contradictory findings could tentially other host defense mechanisms during Borrelia possibly be explained by the use of different Borrelia infection requires further characterization. isolates in these studies. Nevertheless, more extensive In comparison to uninfected ticks, TSLPI expression is research is needed to clarify contradictory results and to significantly elevated in Borrelia-infected ticks 24 h after further unravel the mechanisms underlying evasion of tick attachment [18]. At this time, Borrelia starts entering lytic pore formation. the feeding pit, suggesting that inhibition of the lectin pathway of host complement by Borrelia at the tick bite Tick proteins exploited by Borrelia site is especially important for its infectivity. TSLPI si- Tick feeding can take several days to complete, which has lencing in Borrelia-infected ticks as well as TSLPI vacci- pressured ticks to evolve novel mechanisms to evade im- nation of mice resulted in impaired murine Borrelia mune detection and coagulation. Tick salivary proteins are infection indicating a crucial role for TSLPI in the success- introduced into host skin and show specific anticoagulant ful tick–mouse transmission cycle of Borrelia [18]. and anti-inflammatory, including anticomplement, activi- Tick salivary protein Salp20 mediates complement in- ty [67]. Borrelia takes advantage of tick salivary proteins to hibition by destabilizing the C3 convertase through dis- evade complement-mediated killing. Indeed, Borrelia placement of properdin from the convertase complex influences the expression of these salivary proteins, show- (Figure 3c) [72,73]. Salp20 is part of the IxAC family of ing that there is interesting ‘teamwork’ in order to evade anticomplement proteins. Family members IRAC I and II complement activation and inflammation in both tick feed- from I. ricinus and its homolog Isac from I. scapularis were ing and in transmission of Borrelia. The differentially found to inhibit the alternative pathway of complement in expressed salivary protein Salp15 is significantly a similar manner [74–76]. Five additional proteins of the elevated in salivary glands of engorged ticks infected with IxAC family were subsequently discovered and, despite

125 Review Trends in Parasitology March 2013, Vol. 29, No. 3 their antigenic diversity and amino acid sequence diver- via recruitment and mimicry of host complement regulators, gence, functioned very similarly as the previously de- the protective effect of the membrane composition, and the scribed Isac and IRAC I/II [77]. Interestingly, IRAC I direct or indirect use of tick salivary proteins (Figure 3). and II showed different extents of inhibition of the alter- Nevertheless, one must consider that B. burgdorferi s.l. native pathway of complement among different host spe- isolates use different approaches in complement evasion. cies [75]. The adaptation of ticks to a variety of hosts, via New insights into the mechanisms used by Borrelia to expression of different tick proteins in the IxAC family, escape the attack of complement could help in identifying may also be beneficial for host colonization by Borrelia. novel vaccine candidates. Developing an effective vaccine to Because the biochemical properties of the individual mem- prevent Lyme borreliosis is challenging, especially as Bor- bers of IxAC family are comparable, their diversification is relia exploits tick proteins to successfully infect the mam- most probably not due to positive selection associated with malian host. As we previously postulated, one of the speciation. strategies to develop an effective vaccine is to target both tick proteins and Borrelia outer membrane proteins [78]. Concluding remarks Indeed, immunization with both Salp15 and OspA exerts a Over the past decade our understanding of the role of synergistic anti-Borrelia immune response [79]. Consider- complement during the Borrelia–mammalian–tick life cycle ing the complement evasion strategies of other pathogens, it has increased. However, several questions remain unan- is plausible that, in addition to the strategies mentioned swered (Box 3). During Borrelia transmission the comple- earlier, Borrelia species have developed additional mecha- ment system of the host is triggered in an attempt to nisms to cope with complement activation such as the eradicate Borrelia through complement-mediated killing, binding of other complement regulators (vitronectin, C1- phagocytosis, and recruitment of immune cells. Although INH) for its own benefit. Future research should focus on the both the classical and lectin pathways are important for molecular interactions of Borrelia with both host and tick initiation of complement activation, the alternative path- complement regulators and the contribution of individual way drives its amplification. B. burgdorferi s.l. isolates complement regulators to serum resistance of Borrelia. have been shown to avoid complement-mediated killing Moreover, the species-specific adaptation in Borrelia is an interesting platform to determine which complement eva- sion strategy is most important for the infectivity of different Box 3. Outstanding questions Borrelia genospecies. A better understanding of the role of  B. burgdorferi s.l. avoids complement-mediated killing through a the different complement pathways involved in eradication variety of strategies. Is/are there common mechanism(s) for of Borrelia and how these pathways are inhibited by ticks complement evasion necessary for survival of B. burgdorferi s.l. could help us in developing effective prevention strategies isolates in vivo? such as anti-tick vaccines and extend our knowledge of Lyme  Because complement evasion in vitro only appears to be partial, even in the case of serum-resistant isolates, how does Borrelia borreliosis pathogenesis and reservoir competence. manage to disseminate and survive systemically? And how do serum-sensitive isolates disseminate and survive in distant Acknowledgments compartments? We would like to thank Henk van Veen for excellent assistance in  What is the exact function of C4bp in serum sensitivity of Borrelia scanning electron microscopy (SEM) photography. genospecies, and is it predominantly recruited by B. garinii to resist complement-mediated killing? 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