Functional and immunological evaluation of two novel proteins of Leptospira spp.

This work shows the production and characterization of two novel putative lipoproteins encoded by the genes LIC10645 and LIC10731 identified in the genome sequences of Leptospira interrogans. In silico conservation analysis indicated that the proteins are well conserved among pathogenic leptospiral serovars and species. Recombinant proteins were obtained in Escherichia coli BL21(DE3) Star pLysS strain, purified by metal-affinity chromatography, and used for characterization and immunological evaluations. Recombinant proteins were capable of eliciting a combination of humoral and cellular immune responses in animal models, and could be recognized by antibodies present in human serum samples. The recombinant proteins Lsa44 and Lsa45 were able to bind laminin, and were named Lsa44 and Lsa45 for leptospiral surface adhesins of 44 and 45 kDa, respectively. The attachment to laminin was dose-responsive with KD values of 108.21 and 250.38 nM for Lsa44 and Lsa45, respectively. Moreover, these proteins interact with plasminogen (PLG) with KD values of 53.56 and 36.80 nM, respectively. PLG bound to the recombinant proteins could be converted to plasmin (PLA) in the presence of an activator. Cellular localization assays suggested that the Lsa44 and Lsa45 were surface-exposed. These are versatile proteins capable of interacting with laminin and PLG/PLA, and hence could mediate bacterial adhesion and contribute to tissue penetration.

Interaction of Leptospira interrogans with human proteolytic systems enhances dissemination through endothelial cells and protease levels.

We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date.

OmpL1 is an extracellular matrix- and plasminogen-interacting protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical ß-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with K(D) (equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a K(D) of 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system.

Intermediate and C-terminal regions of leptospiral adhesin Lsa66 are responsible for binding with plasminogen and extracellular matrix components.

Leptospirosis, a worldwide zoonotic infection, is an important human and veterinary health problem. We have previously identified a leptospiral multipurpose adhesin, Lsa66, capable of binding extracellular matrix (ECM) components and plasminogen (PLG). In this work, we report the cloning, expression, purification and characterization of three fragments derived from the full-length Lsa66: N-terminal, intermediate and C-terminal regions. We employed Escherichia coli BL21-SI as expression cells. The recombinant fragments tagged with N-terminal His6 were purified by metal-charged chromatography to major protein bands that were recognized by anti-His-tag mAbs. The recombinant fragments were evaluated for their capacity to attach to ECM components and to PLG. The intermediate region bound to laminin, plasma fibronectin and PLG. Laminin also bound to the C-terminal region. Antibodies in leptospirosis-positive serum samples recognized Lsa66, being the immune epitopes located at the N-terminal and intermediate fragments. The data confirm that Lsa66 is expressed during infection and that this protein might have a role in bacterial infection.

Characterization of three novel adhesins of Leptospira interrogans.

We report cloning, expression, purification, and characterization of three predicted leptospiral membrane proteins (LIC11360, LIC11009, and LIC11975). In silico analysis and proteinase K accessibility data suggest that these proteins might be surface exposed. We show that proteins encoded by LIC11360, LIC11009 and LIC11975 genes interact with laminin in a dose-dependent and saturable manner. The proteins are referred to as leptospiral surface adhesions 23, 26, and 36 (Lsa23, Lsa26, and Lsa36), respectively. These proteins also bind plasminogen and generate active plasmin. Attachment of Lsa23 and Lsa36 to fibronectin occurs through the involvement of the 30-kDa and 70-kDa heparin-binding domains of the ligand. Dose-dependent, specific-binding of Lsa23 to the complement regulator C4BP and to a lesser extent, to factor H, suggests that this protein may interfere with the complement cascade pathways. Leptospira spp. may use these interactions as possible mechanisms during the establishment of infection.