Leptospiral complement evasion
An antileptospiral activity in normal serum mediated by the complement system was first reported by Johnson and Muschel in the mid-1960s . The authors observed that serum from a variety of mammals exerted a bactericidal effect against different Leptospira serotypes. According to their studies, non-pathogenic forms could be distinguished from pathogenic ones by a greater susceptibility to normal serum. It became clear that virulence correlates with the ability to survive in mammalian hosts, which can be greatly attributed to the capacity of resisting complement-mediated killing . Further studies on the bactericidal effect of lower-vertebrate sera towards Leptospira revealed a different pattern of killing compared with that of mammalian sera . Curiously, normal serum from turtles (Chrysemys picta and Chelydra serpentina) and from the frog Rana pipiens displayed bactericidal activity against both pathogenic and saprophytic Leptospira strains. One plausible interpretation for these findings would be the presence of either specific or cross-reacting antibodies in the serum of these lower-vertebrates, enabling antibody-dependent complement-mediated lysis .
Although leptospiral serum resistance against host complement was described many decades ago, the mechanisms underlying this resistance started to be unraveled only recently. Pathogens have evolved sophisticated strategies to circumvent the immune defence systems of a variety of hosts, notably mechanisms to escape complement activation and/or lytic complement attack. Among these mechanisms are the acquisition of host fluid-phase complement regulators, notably Factor H and C4b-binding protein (C4BP), the secretion of proteases that inactivate key complement components, and the expression of proteins in the pathogens’ surface that may inhibit or modulate complement activation (reviewed in ). The first study reporting a complement evasion strategy in pathogenic Leptospira was carried out by Meri et al. . The authors demonstrated that serum-resistant and serum-intermediate strains of Leptospira were able to bind Factor H and Factor H-related protein 1 (FHR-1α and FHR-1β) from human serum. Bound Factor H remained functionally active, acting as a cofactor in the cleavage of C3b by Factor I  (Fig. 3). To date, only two leptospiral ligands for human Factor H have been described, LenA and LenB. LenA (leptospiral endostatin-like protein A), formerly called LfhA (for leptospiral Factor H-binding protein A)  and Lsa24 (for leptospiral surface adhesin, 24 kDa) , was first identified through the screening of a lambda phage expression library of L. interrogans serovar Pomona . Sequence analyses of genes from L. interrogans allowed identification of five additional lenA paralogs, designated lenB, lenC, lenD, lenE and lenF, which encode domains with putative structural and functional similarities with mammalian endostatins . All Len proteins have been shown to interact with the extracellular matrix components laminin and fibronectin (LenA is able to interact only with laminin), but binding specificities for human Factor H are displayed solely by LenA and LenB . Besides interacting with Factor H and laminin, LenA is a receptor for human plasminogen . A number of surface proteins of pathogenic microorganisms involved in complement escape may also bind other host molecules, such as plasminogen, fibrinogen, trombin, IgA, IgG and extracellular matrix components (reviewed by ), thus contributing to tissue degradation and adhesion to host cells as well.
Figure 3. Complement evasion strategies in Leptospira. Saprophytic Leptospira are susceptible to complement-mediated killing because they do not bind the host complement regulatory proteins Factor H (FH)  and C4b Binding Protein (C4BP) . By contrast, pathogenic Leptospira evade complement attack by acquiring these soluble proteins of the alternative and classical pathways on their surfaces. Factor H, a 150-kDa plasma protein, 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. LenA and LenB are leptospiral ligands for human FH [82, 83]. C4BP is a 570-kDa plasma glycoprotein that 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. LcpA is a leptospiral outer membrane protein which interacts with human C4BP . As a consequence of the acquisition of those fluid-phase regulators on the surface of a given pathogen, complement activation is down-regulated preventing opsonization and the formation of the lytic membrane attack complex on its surface. In the schematic representation of FH and C4BP, each circle represents one SCR domain. Open circles indicate the three SCR domains of the C4BP β-chain.
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Besides recruiting Factor H to its surface, Leptospira also have the ability to bind human C4BP, a key fluid-phase inhibitor of the classical and lectin pathways . Pathogenic leptospiral strains efficiently acquire C4BP from human serum, whereas the non-pathogenic strain Patoc I binds negligible amounts of this complement regulator to its surface. Surface-bound C4BP retains cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance  (Fig. 3). We recently identified a 20-kDa Leptospira outer membrane protein named LcpA which interacts with this complement regulator . LcpA is surface-exposed, binds both purified and soluble C4BP from human serum, and is expressed by leptospiral strains that are at least partially able to resist complement-mediated killing. Moreover, C4BP remains functionally active when bound to LcpA, acting as a cofactor for Factor I in the cleavage of C4b . Considering that pathogenic leptospires are able to widely spread in the host organism, it is reasonable to suppose that these pathogens may express many different surface receptors for complement and other host molecules. The identification and characterization of these proteins is of great relevance, as they may represent interesting targets for immune intervention.
One interesting observation is that, while pathogenic leptospires evade the complement system, purified peptidoglycan (PG) is able to activate complement . At this point, it is important to consider that PG is not exposed in leptospires, being overlaid by the outer membrane. As a result of mechanical shear stress or bacterial killing, the PG could be exposed and activate the complement system. However, in pathogenic leptospires this activation would be counter-balanced by the inhibitory effects of bound Factor H and C4BP.
Recently, mutations affecting L. interrogans LPS demonstrated that it plays a crucial role in leptospiral virulence. Although previous studies have demonstrated that an intact LPS is essential for complement resistance, the above-mentioned LPS mutants did not present a higher susceptibility to complement-mediated killing .
Immune evasion strategies involved in renal colonization
Leptospires colonize the proximal renal tubules of reservoir animals, where they are able to replicate and persist, being constantly eliminated in the urine. A wide range of mechanisms possibly involved in the ability of leptospires to survive in the kidneys has been suggested .
Despite the recent finding that the absence of LPS did not reduce the complement resistance of leptospires in sera , it is possible that the mechanisms underlying persistence in the renal tubules are different. Leptospiral LPS recovered from rat kidneys presents a higher content of the O antigen compared with the LPS of leptospires isolated from guinea pig liver with acute infection . This increased content of LPS O antigen in chronically infected kidneys could constitute an immune evasion strategy. Indeed, the O antigen expression was associated with complement resistance in Francisella tularensis, a facultative intracellular Gram-negative pathogen . Therefore, the role of the LPS O antigen in the leptospiral immune evasion should be evaluated.
Besides increased LPS O antigen content, proteomic analysis revealed a reduced expression of antigenic proteins in leptospires from rat kidneys in contrast to in vitro cultured bacteria . This antigenic reduction could also reflect a means of escaping from host immune responses.
Saprophytic and pathogenic leptospires are able to form biofilms, helping them to survive in environmental habitats and to colonize the hosts . Indeed, biofilms can constitute a barrier against the immune effector cells and molecules, including antibodies and complement, and represent one of the major mechanisms of Pseudomonas aeruginosa persistence in chronic infections . Investigations on biofilm formation by leptospires in renal tubule cells from resistant and susceptible hosts could certainly contribute to our understanding of immune evasion strategies and disease pathology.