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Infection and Immunity, March 2009, p. 1137-1143, Vol. 77, No. 3
0019-9567/09/$08.00+0     doi:10.1128/IAI.01310-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Immune Evasion of Leptospira Species by Acquisition of Human Complement Regulator C4BP

Angela S. Barbosa,^1* Patricia A. E. Abreu,¹ Sílvio A. Vasconcellos,² Zenaide M. Morais,² Amane P. Gonçales,² Aldacilene S. Silva,³ Mohamed R. Daha,⁴ and Lourdes Isaac³

Laboratório de Bacteriologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, São Paulo, Brazil,¹ Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brazil,² Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil,³ Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands⁴

Received 26 October 2008/ Returned for modification 30 November 2008/ Accepted 21 December 2008

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Leptospirosis is a spirochetal zoonotic disease of global distribution with a high incidence in tropical regions. In the last 15 years it has been recognized as an important emerging infectious disease due to the occurrence of large outbreaks in warm-climate countries and, occasionally, in temperate regions. Pathogenic leptospires efficiently colonize target organs after penetrating the host. Their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues. Therefore, they must be able to evade the innate host defense. The main purpose of the present study was to evaluate how several Leptospira strains evade the protective function of the complement system. The serum resistance of six Leptospira strains was analyzed. We demonstrate that the pathogenic strain isolated from infected hamsters avoids serum bactericidal activity more efficiently than the culture-attenuated or the nonpathogenic Leptospira strains. Moreover, both the alternative and the classical pathways of complement seem to be responsible for the killing of leptospires. Serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Surface-bound C4BP promotes factor I-mediated cleavage of C4b. Accordingly, we found that pathogenic strains displayed reduced deposition of the late complement components C5 to C9 upon exposure to serum. We conclude that binding of C4BP contributes to leptospiral serum resistance against host complement.

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Leptospirosis is a spirochetal disorder caused by pathogenic species belonging to the genus Leptospira and is regarded as the most widespread zoonotic disease due to the large spectrum of animal species that serve as reservoirs (18). The infection is transmitted by exposure to flood waters, moist soil, or vegetation contaminated with urine from animals infected with Leptospira species. Clinical manifestations range from a mild febrile illness to severe disease forms such as Weil's syndrome. Leptospirosis-associated severe pulmonary hemorrhagic syndrome has increasingly become recognized as an important manifestation of leptospiral infection (3, 8, 19, 27, 34, 41).

After penetrating the host, pathogenic leptospires efficiently colonize target organs, and their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues (22). Therefore, it is clear that these pathogens have evolved strategies to circumvent the immune defense systems of a variety of hosts. Complement is a major component of the innate immune system and is involved in protection against invading microorganisms due to its opsonic, inflammatory, and lytic activities (4). One strategy adopted by pathogens to avoid clearance and destruction by complement is to acquire host fluid-phase regulators, notably factor H and C4b-binding protein (C4BP), the soluble proteins of the alternative and classical pathways, respectively. Factor H, a 150-kDa plasma protein composed of 20 globular domains termed short consensus repeats (SCRs), inhibits the alternative pathway of complement by preventing binding of factor B to C3b, accelerating decay of the C3-convertase C3bBb and acting as a cofactor for the cleavage of C3b by factor I (28, 38, 39). C4BP, composed of seven -chains (each with eight SCRs) and one β-chain (with three SCRs) linked together by a central core, is a 570-kDa plasma glycoprotein that displays a spiderlike shape (6, 10, 31). It inhibits the classical pathway of complement by interfering with the assembly and decay of the C3-convertase C4bC2a and acts as a cofactor for factor I in the proteolytic inactivation of C4b (7, 31). Therefore, as a consequence of the acquisition of fluid-phase regulators on the surface of a given pathogen, complement activation is downregulated, preventing opsonization and the formation of the lytic membrane attack complex on its surface.

Several human pathogens use this strategy to survive, including spirochetes. In the case of Borrelia spp., the causative agent of Lyme disease and relapsing fever, the expression of outer surface lipoproteins known as complement regulatory-acquiring surface proteins (CRASP) which bind factor H and/or factor H-like protein 1 (FHL-1), is restricted to serum-resistant strains (9, 12-17, 37). It has been reported that Borrelia recurrentis and Borrelia duttonii also acquire C4BP on their surfaces, which may contribute to evasion of antibody-mediated clearance from blood circulation (21). Treponema denticola, a spirochete that contributes to the development of periodontal disease, specifically binds FHL-1 via a 14-kDa, surface-exposed protein termed FhbB (FHL-1 binding protein B). This interaction seems to facilitate adhesion, biofilm formation, and possibly tissue penetration (20). Binding of factor H and factor H-related protein 1 (FHR-1) has also been demonstrated for serum-resistant and serum-intermediate strains of Leptospira (22). Moreover, the outer membrane proteins LenA (leptospiral endostatinlike protein A), formerly called LfhA (for leptospiral factor H-binding protein A) (35) and Lsa24 (for leptospiral surface adhesin, 24 kDa) (2), and LenB (for leptospiral endostatinlike protein B) seem to contribute to serum resistance of pathogenic leptospires by interacting with factor H (33, 35).

The main purpose of the present study was to evaluate complement evasion by Leptospira spp. Our data indicate that both the alternative and the classical pathways seem to contribute to leptospire killing. Both serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Pathogen-bound C4BP retained its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance.

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Leptospira strains and culture. L. interrogans serovar Pomona strain Fronn, L. interrogans serovar Pomona strain Pomona, L. kirshneri serovar Cynopteri strain 3522 C, L. borgpetersenii serovar Javanica strain Veldrat Bataviae 46, L. noguchi serovar Panama strain CZ 214K, and L. biflexa serovar Patoc strain Patoc I were used in the assays. All strains are from the Laboratório de Zoonoses, Faculdade de Medicina Veterinária e Zootecnia, USP, São Paulo, Brazil. Except for the strain Fronn, whose virulence is maintained by iterative passages in hamsters, all other pathogenic strains were culture attenuated by successive passages in artificial medium. Leptospires were cultured at 29°C under aerobic conditions in liquid EMJH medium (Difco) with 10% rabbit serum, enriched with L-asparagine (0.015%), sodium pyruvate (0.001% [wt/vol]), calcium chloride (0.001% [wt/vol]), magnesium chloride (0.001% [wt/vol]), peptone (0.03% [wt/vol]), and meat extract (0.02% [wt/vol]) (2).

Sera and proteins. Normal human sera (NHS) were obtained from healthy human donors without a known history of leptospiral infection. The sera were pooled, divided into aliquots, and stored at –80°C until use. NHS were incubated at 56°C for 30 min to yield heat-inactivated NHS (HI-NHS). Mannose-binding lectin (MBL)-deficient serum was obtained from a healthy donor, and C1s-deficient serum was obtained as described by Amano et al. (1). All serum samples were screened for the presence of antibodies reacting with leptospires by the microagglutination test and found to be negative. C4BP was purified from normal human plasma by using affinity chromatography through goat anti-human C4BP linked to CNBr-Sepharose.

Serum susceptibility testing for Leptospira strains. Complement-mediated killing of leptospires was evaluated after incubation with nonimmune NHS, essentially as described by Meri et al. (22). Leptospires were grown to mid-log phase and washed once with phosphate-buffered saline (PBS). In each microtiter plate well, approximately 5 x 10⁶ bacteria were incubated at 37°C for 60 or 120 min with 40% NHS or 40% HI-NHS as a control in a final volume of 200 µl. To stop complement activation, 20-µl portions of all samples were incubated on ice for 1 min and then transferred to microtiter plates filled with 180 µl of EMJH medium/well. The plates were sealed with a sterile acetate film and incubated at 30°C for 4 days. Bacterial growth was determined by using dark-field microscopy (Nikon Eclipse 50i) and a Petroff-Hausser chamber. Serum sensitivity was evaluated by comparing the counts in NHS-incubated samples to those incubated in HI-NHS (100% growth). In order to assess the contribution of each complement pathway in leptospire killing, MBL- and C1s-deficient sera were used. NHS and HI-NHS were also included in the assays. Bacteria were incubated at 37°C for 120 min with each serum (40%), and complement-mediated killing was evaluated as described above. All assays were performed in triplicate. For statistical analyses, survival in each serum was compared to survival in NHS by using the Student two-tailed t test. A P value of <0.05 was considered statistically significant.

Serum adsorption assays using intact leptospires. Freshly harvested leptospires (10⁹) were washed three times with complement fixation diluent buffer (CFD; 4 mM sodium barbitone, 0.145 M NaCl, 0.83 mM MgCl₂, 0.25 mM CaCl₂ [pH 7.3]) and incubated in NHS containing 10 mM EDTA (NHS-EDTA) for 60 min at 37°C with gentle agitation. After five washes with CFD (the last wash fraction was collected), proteins bound to the surface of bacteria were eluted with 100 µl of 0.1 M glycine-HCl (pH 2.0), and supernatants were collected after centrifugation. One-fifth (20 µl) of the wash and eluate fractions was subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-10% PAGE) under nonreducing conditions, and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked by using 10% (wt/vol) dried milk in PBS-0.05% Tween (pH 7.4; PBST) overnight at 4°C. Subsequently, membranes were rinsed three times in PBST and incubated for 2 h at room temperature with a monoclonal mouse antibody recognizing C4BP (Quidel) at a 1:1,000 dilution. After three washes with PBST, membranes were incubated with a secondary peroxidase-conjugated anti-mouse immunoglobulin G (IgG) antibody for 60 min at room temperature. The positive signal was detected by enhanced chemiluminescence (West Pico; Pierce).

Assessment of surface deposition of complement components on leptospires by enzyme immunoassay. These assays were performed essentially as described by Meri et al. (22) with minor modifications. Leptospires grown to mid-log phase were harvested by centrifugation at 11,600 x g for 25 min and gently washed in 100 mM NaCl-50 mM Tris-HCl twice. Bacteria were incubated with 40% NHS or HI-NHS at 37°C for 15 min and put on ice for 1 min to stop complement activation. After centrifugation at 10,000 x g for 10 min at 4°C, the pellets were washed three times with a buffer containing 1.0 mM MgCl₂, 0.6 mM CaCl₂, and 1% glucose and then resuspended in 0.1 M NaHCO₃ (pH 9.6). A total of 10⁷ cells were used for coating enzyme-linked immunosorbent assay (ELISA) plate wells overnight at 4°C. The wells were washed three times with PBST and then blocked with 200 µl of 0.5% bovine serum albumin for 2 h at 37°C. Bound complement components were detected by adding 100 µl of a 1:10,000 dilution of sheep anti-human C3d (The Binding Site), a 1:1,000 dilution of mouse anti-human C4d (Quidel), or a 1:5,000 dilution of sheep anti-human C5, C6, C7, C8, or C9 (The Binding Site) in PBS. Incubation proceeded for 1 h and after three washes with PBST, and 100 µl of a 1:5,000 dilution of horseradish peroxidase-conjugated anti-sheep IgG (for determination of C3, C5, C6, C7, C8, and C9) or anti-mouse IgG (for determination of C4) in PBS was added per well for 1 h. All incubations took place at 37°C. The wells were washed three times, and o-phenylenediamine (0.04%) in citrate phosphate buffer (pH 5.0) plus 0.01% H₂O₂ was added. The reaction was allowed to proceed for 20 min and was then interrupted by the addition of 50 µl of 8 M H₂SO₄. The absorbance at 492 nm was determined in a microplate reader (Labsystems Uniscience Multiskan EX). For statistical analyses, the deposition of complement components on the strains Pomona and Fronn was compared to the deposition of these proteins on strain Patoc I by using the Student two-tailed t test.

Cofactor activity of Leptospira-bound C4BP. The cofactor activity of surface-attached C4BP was assayed by measuring factor I-mediated cleavage of C4b. Freshly harvested leptospires (10⁹) were washed with a binding buffer (100 mM NaCl, 50 mM Tris-HCl [pH 7.4]) and incubated with NHS-EDTA (dilution of 1:2), purified C4BP (0.1 to 2 µg), or binding buffer for 60 min at 37°C with gentle agitation. Bacteria were washed three times with washing buffer (100 mM NaCl, 50 mM Tris-HCl, 0.05% Tween 20 [pH 7.4]) and incubated with purified C4b (Calbiochem) at 250 ng/assay and factor I (Calbiochem) at 250 ng/reaction for up to 60 min at 37°C. The samples were centrifuged, and the supernatants were subjected to SDS-12% PAGE under reducing conditions and transferred to nitrocellulose membranes. Blocking treatment and incubations with specific antibodies were performed as described above. C4d, a factor I-dependent cleavage product of C4b, was detected using a monoclonal mouse anti-human C4d (Quidel) at a 1:1,000 dilution.

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Serum resistance of Leptospira spp. Serum sensitivity to complement-mediated killing of six strains belonging to five different species of Leptospira was assessed upon incubation in nonimmune NHS (40%) for 60 and 120 min. The survival percentage was calculated by comparing the number of surviving leptospires incubated in NHS to those incubated with HI-NHS. The results, as presented in Fig. 1, indicate that L. interrogans serovar Pomona strain Fronn and L. kirshneri serovar Cynopteri strain 3522 C can be classified as resistant, L. biflexa serovar Patoc strain Patoc I as sensitive, and L. interrogans serovar Pomona strain Pomona, L. borgpetersenii serovar Javanica strain Veldrat Bataviae 46, and L. noguchi serovar Panama strain CZ 214K as intermediate since, in these cases, viable leptospires could still be detected after 60 min of incubation.

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FIG. 1. Serum resistance of Leptospira strains. Serum sensitivity was evaluated by comparing the counts in NHS-incubated samples to those incubated in HI-NHS. The results, presented as relative survivals (ratios of the observed survival rates in NHS to the survival rates in HI-NHS), are shown as means ± the standard errors of the mean (SEM) for three to six independent experiments, each performed in triplicate.

Both the alternative and the classical pathways contribute to leptospire killing. To assess the contribution of each complement pathway in serum killing of leptospires, we used the sensitive L. biflexa serovar Patoc strain Patoc I and the intermediate L. interrogans serovar Pomona strain Pomona. Bacteria were incubated for 120 min with C1s-deficient serum, MBL-deficient serum, NHS, or HI-NHS. The survival percentage was calculated by comparing the number of surviving leptospires after incubation with each serum. For the free-living nonpathogenic Patoc I strain, survival in MBL- and C1s-deficient sera did not differ significantly from that observed after treatment with NHS (P = 0.2 and P = 0.09, respectively). This indicates that, for this particular strain, the classical and the lectin pathways do not play an important role in complement-mediated lysis (Fig. 2A). Consequently, serum bactericidal activity can be mainly attributed to the alternative pathway. On the other hand, it seems that the classical pathway contributes to killing of the intermediate strain Pomona, since the survival rate in C1s-deficient serum was higher than that observed upon incubation in NHS (P < 0.05) but was quite similar to that observed after treatment with HI-NHS (Fig. 2B).

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FIG. 2. Survival of two Leptospira strains in human sera. L. biflexa serovar Patoc strain Patoc I (A) and L. interrogans serovar Pomona strain Pomona (B) were subjected to serum bactericidal assays using HI-NHS, NHS, MBL-deficient serum (MBL-), and C1s-deficient serum (C1s-), incubated for 120 min. The results are presented as relative survival to the survival of bacteria in heat-inactivated serum (100% growth). Means ± the SEM for three independent experiments, each performed in triplicate, are shown.

Leptospira spp. acquire C4BP from human serum. To assay the binding of the classical complement pathway regulator C4BP from human serum, leptospires were incubated in NHS-EDTA or in CFD and, after extensive washing, surface-bound proteins were eluted and subjected to Western blotting with anti-C4BP monoclonal antibody (MAb). All four strains belonging to pathogenic species bound C4BP, but the nonpathogenic saprophytic strain Patoc I bound negligible amounts of this complement regulator to its surface (Fig. 3). Anti-C4BP MAb did not consistently bind to components of the elution fractions when leptospires had been incubated in CFD instead of NHS (Fig. 3).

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FIG. 3. Acquisition of C4BP from human serum by L. interrogans (strain Fronn), L. kirshneri (strain 3522 C), L. borgpetersenii (strain Veldrat Bataviae 46), L. noguchi (strain CZ 214K), and L. biflexa (strain Patoc I). Leptospires were incubated with NHS-EDTA or with buffer (CFD) and, after extensive washing, surface-bound proteins were eluted and subjected to nonreducing SDS-PAGE and analyzed with anti-C4BP MAb. W, aliquots of the last wash; E, aliquots of the eluted fractions; lane C, purified C4BP (100 ng) included as a positive control. *, C4BP isoforms 7β1, 7β0, and 6β1.

Deposition of the late complement components C5 to C9 occurs mainly in the serum-sensitive strain Patoc I. In order to investigate the mechanism of leptospiral serum resistance, deposition of C3, C4, C5, C6, C7, C8, and C9 on the surfaces of the sensitive strain Patoc I, the intermediate strain Pomona, and the resistant strain Fronn was evaluated by an enzyme immunoassay. Bacteria preincubated with 40% NHS or HI-NHS (as a control) were immobilized on microdilution wells, and antibodies against complement proteins were used to compare the amounts of specific components of the complement cascade deposited on the above-mentioned leptospiral strains. As shown in Fig. 4, all three strains bound C3 and C4 on their surfaces 15 min after incubation with 40% NHS. Deposition of C5 was significantly greater on strain Patoc I, and considerable amounts of C6, C7, C8, and C9 could be detected mainly in this serum-sensitive strain (Fig. 4).

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FIG. 4. Deposition of complement proteins on L. interrogans (strains Fronn and Pomona) and on L. biflexa (strain Patoc I). Bacteria were incubated with 40% NHS at 37°C for 15 min and, after extensive washing, they were used for coating ELISA plate wells. Antibodies against each component (C3, C4, C5, C6, C7, C8, and C9) were added, and bound antibodies were detected with secondary peroxidase-conjugated IgG antibodies. Absorbance was measured at 492 nm. Optical density values of controls incubated with heat-inactivated serum were subtracted from values shown. Means ± the SEM for three independent experiments, each performed in triplicate, are shown. *, Statistically significant (P < 0.05); **, highly statistically significant (P < 0.001).

C4BP bound to leptospires exhibits cofactor activity against C4b. Besides regulating the complement convertases C4b2a and C4b2a3b on cell surfaces by accelerating the decay of the preformed convertase and preventing association of its subunits (7), C4BP also acts as a cofactor for factor I, promoting cleavage and inactivation of C4b with the subsequent formation of C4c (145-kDa) and C4d (44.5-kDa) fragments. A monoclonal anti-C4d, which recognizes both the intact -chain of C4b and its cleaved product C4d was used to assess the cofactor activity of C4BP bound to leptospires. The serum-resistant L. interrogans serovar Pomona strain Fronn was incubated with NHS or purified C4BP and, after a washing step to remove unbound C4BP, C4b and factor I were added. Incubation proceeded for the indicated periods, and the bacteria were pelleted by centrifugation. The cleavage fragments of C4b in the supernatant were subjected to Western blotting with anti-C4d MAb. The presence of a band of 45 kDa indicates that acquired C4BP was able to promote factor I-mediated cleavage of C4b (Fig. 5A). A second assay was then performed with three strains presenting different susceptibilities to complement-mediated killing. As shown in Fig. 5B, the nonpathogenic serum-sensitive strain Patoc I was unable to efficiently cleave C4b, whereas the C4d fragment could be readily detected when both the intermediate and the resistant strains were used. For the virulent strain Fronn, complete cleavage of C4b was achieved with 0.1 µg of purified C4BP, thus indicating that this strain cleaves C4b more efficiently than the culture-attenuated strain Pomona (Fig. 5B).

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FIG. 5. Cofactor activity of C4BP bound to leptospires. (A) The serum-resistant L. interrogans serovar Pomona strain Fronn was incubated with NHS (1:2) or purified C4BP (2 µg) and, after washing to remove unbound C4BP, C4b and factor I were added. Incubation proceeded for the indicated periods, and the bacteria were pelleted by centrifugation. Samples from supernatants were subjected to reducing SDS-PAGE and analyzed with anti-C4d MAb. The presence of a band of 45 kDa indicates that acquired C4BP was able to promote factor I-mediated cleavage of C4b. (B) The serum-sensitive L. biflexa serovar Patoc strain Patoc I, the culture attenuated L. interrogans serovar Pomona strain Pomona and the virulent L. interrogans serovar Pomona strain Fronn were incubated with increasing amounts of purified C4BP (0.1-2.0 µg). The cleavage of C4b was analyzed as described above. *, Fragment resulting from partial cleavage of C4b.

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Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Efficient colonization of target organs is achieved by their capacity to escape complement (22) and their ability to interact with host cells or with the extracellular matrix (2, 5, 25, 33). Therefore, the acquisition of host fluid-phase complement regulators onto their surfaces as a strategy to evade complement confers an extraordinary survival advantage.

In the present study, we first assessed the serum sensitivities of six leptospiral strains belonging to five different species. Among the strains tested, L. interrogans serovar Pomona strain Fronn and L. kirshneri serovar Cynopteri strain 3522 C could be classified as resistant. Since the virulence of the Fronn strain is regularly maintained by iterative passages in hamsters, it was expected that it would fully resist human serum bactericidal activity. With regard to strain 3522 C, ca. 90% of bacteria survived in 40% NHS after 2 h of incubation. Despite being cultivated in vitro for several passages, a longer cultivation period did not significantly affect its capacity to survive in NHS. In a previous work, Meri et al. (22) reported two serum-resistant leptospiral strains that had been previously cultivated in artificial medium for more than 10 passages. These authors suggested that factors or properties of strains conferring serum resistance would be preserved after several passages in culture. Our data further support this hypothesis. Even the strains classified as intermediate (L. interrogans serovar Pomona strain Pomona, L. borgpettersenii serovar Javanica strain Veldrat Bataviae 46, and L. noguchi serovar Panama strain CZ 214K) presented different relative survival rates, thus indicating that intrinsic properties might affect survival in NHS.

A prominent role of the alternative pathway in lysing nonpathogenic leptospires has already been suggested (22), but the contribution of the classical and lectin pathways has not been assessed thus far. For that purpose, the sensitive strain Patoc I and the intermediate strain Pomona were selected. Patoc I was susceptible to lysis upon incubation in either MBL- or C1s-deficient serum (Fig. 2), suggesting that the alternative pathway is the major complement pathway responsible for killing nonpathogenic leptospires. Concerning the intermediate strain Pomona, which presents a survival rate varying from 25 to 50% after a 2-h incubation in NHS (Fig. 1 and 2B), the classical pathway seems to contribute to partial serum susceptibility, since incubation in C1s-deficient serum significantly reduced the serum bactericidal activity (Fig. 2B). It is worth mentioning that the pooled normal sera used in these assays did not contain antibodies reactive to leptospires, as assessed by a microagglutination test. Nevertheless, leptospiral strains bound IgG present in nonimmune sera, as examined by ELISA using anti-human IgG-peroxidase conjugate (data not shown). Therefore, binding of C1q to the Fc portion of those antibody-antigen complexes on the bacterial surface may occur, thus initiating the complement classical pathway. A similar mechanism has been described for Escherichia coli K1, the leading cause of neonatal meningitis (40). Alternatively, C1q could bind directly to leptospires or to natural IgM bound on their surfaces (for a review, see reference 32). The mechanism by which the classical pathway is initiated in Leptospira remains to be elucidated.

Our analysis of the binding of C4BP to five Leptospira strains showed that all pathogenic strains efficiently acquired C4BP from human serum, whereas the nonpathogenic strain Patoc I bound negligible amounts of this complement regulator to its surface. It therefore seems that pathogenic strains whose virulence has been attenuated by successive passages in vitro continue to express ligands for C4BP. High-passage leptospiral strains, which have been shown to be intermediately resistant to complement-mediated killing, are also capable of binding factor H and FHR-1 from human serum (22).

The acquisition of fluid-phase regulators on the surface of a given pathogen normally results in downregulation of complement activation. We therefore evaluated the deposition of specific proteins of the complement cascade on three leptospiral strains after incubation with 40% NHS. Interestingly, the serum-sensitive, -intermediate, and -resistant strains bound significant amounts of both C3 and C4, but deposition of the late complement components C5, C6, C7, C8, and C9 occurred mainly on the serum-sensitive strain Patoc I, probably triggering the formation of the lytic membrane attack complex on its surface. Surface-bound C4BP confers additional protection against complement-mediated killing by acting as a cofactor for factor I in the cleavage of C4b. Our data indicate that C4BP bound to the virulent strain Fronn and to the intermediate strain Pomona are functionally active, since the C4d cleavage fragment could be detected in both cases. The ability of the strain Fronn to cleave C4b more efficiently may contribute to its higher survival rate in NHS compared to the strain Pomona.

To date, only a few pathogens have been reported to recruit both factor H and C4BP. Protection from the alternative and the classical pathways by binding those complement inhibitors has been demonstrated for Neisseria gonorrhoeae (29, 30), Streptococcus pyogenes (11, 26), Candida albicans (23, 24), Aspergillus fumigatus and Aspergillus terreus (36), and the relapsing fever spirochetes Borrelia recurrentis and Borrelia duttonii (21). As already mentioned, Meri et al. demonstrated acquisition of factor H and FHR-1 by pathogenic leptospires (22). In the present study, we show for the first time that these spirochetes also acquire C4BP from human plasma, and bound C4BP remains functionally active. This interaction may contribute to serum resistance of pathogenic leptospiral strains. Surviving in the host circulation for a longer period confers advantages to Leptospira in initiating and establishing an infection, contributing to their pathogenic potential. Future research focusing on leptospiral receptors for those complement regulators will be relevant for uncovering potential vaccine candidates.

 
We thank Shaker Chuck Farah (Instituto de Química, Universidade de São Paulo, São Paulo, Brazil) for reading the manuscript. We are also indebted to Rita de Cássia Paro Alli (Instituto de Pesquisas Tecnológicas, São Paulo, Brazil) for allowing use of the dark-field microscope.

This study has benefited from grants provided by FAPESP (06/54853-0), CNPq (478081/2007-3), and Fundação Butantan.

 
* Corresponding author. Mailing address: Laboratório de Bacteriologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, São Paulo, Brazil. Phone: 5511 37267222, x2271. Fax: 5511 37261505. E-mail: angelabarbosa{at}butantan.gov.br

Published ahead of print on 29 December 2008.

Editor: A. J. Bäumler

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Infection and Immunity, March 2009, p. 1137-1143, Vol. 77, No. 3
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