Pathogenic Leptospira are potentially able to control all pathways of the complement system by acquiring soluble negative host regulators. Control of the AP is achieved by interaction of Leptospira with FH, a 155 kDa plasma glycoprotein [443 ± 106 μg/mL in human serum (12)] composed of 20 globular domains (termed complement control protein domains, CCPs). FH inhibits AP activation by preventing binding of Factor B (FB) to C3b, by accelerating the decay of the C3-convertase C3bBb and by acting as a cofactor for the cleavage of C3b by FI (13–15). Serum-resistant Leptospira strains bind three members of the FH family: FH itself, Factor H-like protein 1 (FHL-1), and Factor H-related protein 1 (FHR-1) (9, 16). Once bound to the Leptospira surface, FH remains functional and promotes FI-mediated cleavage of C3b, thus generating the iC3b fragment (9, 16). Moreover, Leptospira survival in FH-depleted serum was shown to be impaired by 60%, and reconstitution of this serum with purified FH up to physiological concentrations restored bacterial survival in a dose-dependent manner, further supporting a role for FH in Leptospira serum resistance (16). Control of the CP by pathogenic Leptospira is mediated by surface-bound C4BP, a 570 kDa plasma glycoprotein [335 ± 83 μg/ml (12)] that is found in three isoforms with different subunit composition. The major isoform, α7/β1, is a complex of seven α-chains and one β-chain. The other C4BP isoforms in plasma are α7/β0 and α6/β1. Each α-chain is comprised of eight CCPs, and the β-chain is comprised of three CCPs (17). C4BP inhibits CP and LP activation by interfering with the assembly and decay of the C3 convertase (C4bC2a) and by acting as a cofactor for FI in the proteolytic inactivation of C4b (18, 19). Both virulent and culture-attenuated Leptospira strains express ligands for C4BP, in contrast to non-pathogenic strains, which have been shown to bind insignificant amounts of this complement regulator (10). As expected, C4b is more efficiently cleaved by FI in the presence of C4BP bound to Leptospira interrogans virulent strains, which may probably explain their higher survival rate in normal human serum as compared to culture-attenuated strains (10). Leptospires also acquire vitronectin (Vn) on their surfaces (20). Vn is a glycoprotein that circulates in the bloodstream as a monomer [65–75 kDa, 104 ± 25 μg/mL (12)] or is deposited in the extracellular matrix (ECM) as a multimer that interacts with several macromolecular components, including glycosaminoglycans and collagens (21, 22). Vn plays multiple roles in many biological processes including the regulation of the terminal pathway of complement by inhibiting C5b7 complex formation and C9 polymerization. Once bound to the bacterial surface, it may protect the microorganism against lysis by impairing MAC formation. A number of strains belonging to different Leptospira species have been shown to interact with human Vn (20). Acquisition of this terminal pathway regulatory protein may assist Leptospira to evade complement attack.

Proteolytic activity is a fundamental tool employed by diverse pathogens to both overcome tissue barriers and evade the immune system (37). Degradation of ECM components favors pathogen spreading and dissemination, while cleavage and inactivation of immune effector molecules dampen the host defense system, allowing an effective establishment of the infection (38, 39).

Pathogenic leptospires circumvent complement attack by the cleavage and inactivation of key complement molecules from the three activation pathways. The degradation of complement proteins may occur indirectly, using host-acquired proteases such as PLG, or directly, by the activity of endogenous proteases produced by pathogenic Leptospira strains, as discussed in the next section.

It is well-known that leptospires are able to bind human PLG (40, 41). PLG is a single-chain glycoprotein (92 kDa) that is a key component of the host fibrinolytic system. This proenzyme is found in plasma and extracellular fluids at concentrations of 180–200 µg/mL (42).

Although both saprophytic and pathogenic leptospires bind purified PLG, only pathogenic strains are able to acquire PLG from human plasma (40, 41). The interaction of PLG with Leptospira is mediated by bacterial membrane proteins (Table 1), and involves lysine residues, which are probably positioned at the PLG kringle domains. Another interesting finding is that Leptospira cell integrity is preserved, since cellular growth is not impaired by PLG binding (40). Once bound to the Leptospira surface and in the presence of urokinase-type plasminogen activator (uPA), PLG is converted to enzymatically active plasmin (40, 41). Plasmin is a serine protease that cleaves diverse important biological substrates, including ECM proteins, like fibrinogen, and complement molecules, such as C3b and C5 (43). In this way, pathogenic leptospires coated with plasmin showed reduced deposition of C3b and IgG on their surface, which was probably related to proteolytic degradation of these molecules, potentially reducing opsonization (44). Furthermore, L. interrogans serovar Pomona also displayed enhanced survival in human serum when bound to plasmin (44), which reinforces its role in complement resistance.

Several Leptospira membrane proteins have been described as PLG ligands (33, 41, 45–56). However, only a few of them were indeed shown to directly interfere with complement activation: the elongation factor Tu [EF-Tu (35)]; LigA and LigB (36) and Leptospira 23 kDa surface adhesion [Lsa23 (34)], whose interactions with PLG resulted in the cleavage of C3b, C4b, and/or C5 (Table 1).

The Leptospira evasion strategies described until know focused on host molecules hijacked by the pathogen to inactivate complement on its surface. Recently, we demonstrated that pathogenic Leptospira can produce molecules that are able to directly interfere with the complement system, in a manner independent of the host machinery. It was observed that the culture supernatants of pathogenic, but not of saprophytic Leptospira strains, were able to specifically inhibit all the three activation pathways of complement. The inhibitory effect observed could be directly correlated to the proteolytic activity present in these culture supernatants. Indeed, the leptospiral proteases were able to target a wide range of substrates including C3, a key factor in the amplification of the complement cascade, FB from AP, C2, and C4, from CP and LP. These cleavages were observed both with purified complement proteins or normal human serum, which indicates that the leptospiral proteases exert their function in a physiological context and may contribute to bacterial virulence (57).

The proteolytic activity found exclusively in pathogenic Leptospira supernatants was almost completely abolished by 1.10-phenanthroline, indicating a major role of metalloproteases in the degradation of complement proteins. A recombinant metalloprotease from the thermolysin family, present only in Leptospira pathogenic species, seems to contribute to these cleavages, since it was able to degrade the central complement protein C3 (57).

The degradation and functional inactivation of complement is a key strategy for attenuating diverse immune responses that are dependent on the proper activation of this system (58). The secretion of proteases that directly cleave complement proteins may contribute to Leptospira immune evasion, as demonstrated for a wide range of other pathogens (59).