Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.

Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7'-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective K(d)s of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface for L. interrogans to efficiently counteract the multiple homeostatic processes of the host.

A LigA three-domain region protects hamsters from lethal infection by Leptospira interrogans.

The leptospiral LigA protein consists of 13 bacterial immunoglobulin-like (Big) domains and is the only purified recombinant subunit vaccine that has been demonstrated to protect against lethal challenge by a clinical isolate of Leptospira interrogans in the hamster model of leptospirosis. We determined the minimum number and location of LigA domains required for immunoprotection. Immunization with domains 11 and 12 was found to be required but insufficient for protection. Inclusion of a third domain, either 10 or 13, was required for 100% survival after intraperitoneal challenge with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. As in previous studies, survivors had renal colonization; here, we quantitated the leptospiral burden by qPCR to be 1.2×10(3) to 8×10(5) copies of leptospiral DNA per microgram of kidney DNA. Although renal histopathology in survivors revealed tubulointerstitial changes indicating an inflammatory response to the infection, blood chemistry analysis indicated that renal function was normal. These studies define the Big domains of LigA that account for its vaccine efficacy and highlight the need for additional strategies to achieve sterilizing immunity to protect the mammalian host from leptospiral infection and its consequences.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin.

BACKGROUND: In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. RESULTS: The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. CONCLUSIONS: This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

The multifunctional LigB adhesin binds homeostatic proteins with potential roles in cutaneous infection by pathogenic Leptospira interrogans.

Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9-11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats.

The OmpL37 surface-exposed protein is expressed by pathogenic Leptospira during infection and binds skin and vascular elastin.

Pathogenic Leptospira spp. shed in the urine of reservoir hosts into freshwater can be transmitted to a susceptible host through skin abrasions or mucous membranes causing leptospirosis. The infection process involves the ability of leptospires to adhere to cell surface and extracellular matrix components, a crucial step for dissemination and colonization of host tissues. Therefore, the elucidation of novel mediators of host-pathogen interaction is important in the discovery of virulence factors involved in the pathogenesis of leptospirosis. In this study, we assess the functional roles of transmembrane outer membrane proteins OmpL36 (LIC13166), OmpL37 (LIC12263), and OmpL47 (LIC13050), which we recently identified on the leptospiral surface. We determine the capacity of these proteins to bind to host tissue components by enzyme-linked immunosorbent assay. OmpL37 binds elastin preferentially, exhibiting dose-dependent, saturating binding to human skin (K(d), 104±19 nM) and aortic elastin (K(d), 152±27 nM). It also binds fibrinogen (K(d), 244±15 nM), fibrinogen fragment D (K(d), 132±30 nM), plasma fibronectin (K(d), 359±68 nM), and murine laminin (K(d), 410±81 nM). The binding to human skin elastin by both recombinant OmpL37 and live Leptospira interrogans is specifically enhanced by rabbit antiserum for OmpL37, suggesting the involvement of OmpL37 in leptospiral binding to elastin and also the possibility that host-generated antibodies may promote rather than inhibit the adherence of leptospires to elastin-rich tissues. Further, we demonstrate that OmpL37 is recognized by acute and convalescent leptospirosis patient sera and also by Leptospira-infected hamster sera. Finally, OmpL37 protein is detected in pathogenic Leptospira serovars and not in saprophytic Leptospira. Thus, OmpL37 is a novel elastin-binding protein of pathogenic Leptospira that may be promoting attachment of Leptospira to host tissues.

Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement.

The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM) components. The LenA outer surface protein (formerly called LfhA and Lsa24) was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF. All six genes encode domains predicted to bear structural and functional similarities with mammalian endostatins. Sequence analyses of genes from seven infectious L. interrogans serovars indicated development of sequence diversity through recombination and intragenic duplication. LenB was found to bind human factor H, and all of the newly-described Len proteins bound laminin. In addition, LenB, LenC, LenD, LenE and LenF all exhibited affinities for fibronectin, a distinct host extracellular matrix protein. These characteristics suggest that Len proteins together facilitate invasion and colonization of host tissues, and protect against host immune responses during mammalian infection.

Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen.

Transmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenic Leptospira spp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of the Staphylococcus aureus FnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests.

Reduced atherosclerosis in hormone-sensitive lipase transgenic mice overexpressing cholesterol acceptors.

Macrophage-specific overexpression of cholesteryl ester hydrolysis in hormone-sensitive lipase transgenic (HSL Tg) female mice paradoxically increases cholesterol esterification and cholesteryl ester accumulation in macrophages, and thus susceptibility to diet-induced atherosclerosis compared to nontransgenic C57BL/6 mice. The current studies suggest that whereas increased cholesterol uptake could contribute to transgenic foam cell formation, there are no differences in cholesterol synthesis and the expression of cholesterol efflux mediators (ABCA1, ABCG1, apoE, PPARgamma, and LXRalpha) compared to wild-type macrophages. HSL Tg macrophages exhibit twofold greater efflux of cholesterol to apoA-I in vitro, suggesting the potential rate-limiting role of cholesteryl ester hydrolysis in efflux. However, macrophage cholesteryl ester levels appear to depend on the relative efficacy of alternate pathways for free cholesterol in either efflux or re-esterification. Thus, increased atherosclerosis in HSL Tg mice appears to be due to the coupling of the efficient re-esterification of excess free cholesterol to its limited removal mediated by the cholesterol acceptors in these mice. The overexpression of cholesterol acceptors in HSL-apoA-IV double-transgenic mice increases plasma HDL levels and decreases diet-induced atherosclerosis compared to HSL Tg mice, with aortic lesions reduced to sizes in nontransgenic littermates. The results in vivo are consistent with the effective efflux from HSL Tg macrophages supplemented with HDL and apoA-I in vitro, and highlight the importance of cholesterol acceptors in inhibiting atherosclerosis caused by imbalances in the cholesteryl ester cycle.