Gabriela H. Siqueira

Leptospiral extracellular matrix adhesins as mediators of pathogen–host interactions

Leptospirosis is been considered an important infectious disease that affects humans and animals worldwide. This review summarizes our current knowledge of bacterial attachment to extracellular matrix (ECM) components and discusses the possible role of these interactions for leptospiral pathogenesis. Leptospiral proteins show different binding specificity for ECM molecules: some are exclusive laminin-binding proteins (Lsa24/LfhA/LenA, Lsa27), while others have broader spectrum binding profiles (LigB, Lsa21, LipL53). These proteins may play a primary role in the colonization of host tissues. Moreover, there are multifunctional proteins that exhibit binding activities toward a number of target proteins including plasminogen/plasmin and regulators of the complement system, and as such, might also act in bacterial dissemination and immune evasion processes. Many ECM-interacting proteins are recognized by human leptospirosis serum samples indicating their expression during infection. This compilation of data should enhance our understanding of the molecular mechanisms of leptospiral pathogenesis.

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.

Adhesins of Leptospira interrogans Mediate the Interaction to Fibrinogen and Inhibit Fibrin Clot Formation In Vitro

We report in this work that Leptospira strains, virulent L. interrogans serovar Copenhageni, attenuated L. interrogans serovar Copenhageni and saprophytic L. biflexa serovar Patoc are capable of binding fibrinogen (Fg). The interaction of leptospires with Fg inhibits thrombin- induced fibrin clot formation that may affect the haemostatic equilibrium. Additionally, we show that plasminogen (PLG)/plasmin (PLA) generation on the surface of Leptospira causes degradation of human Fg. The data suggest that PLA-coated leptospires were capable to employ their proteolytic activity to decrease one substrate of the coagulation cascade. We also present six leptospiral adhesins and PLG- interacting proteins, rLIC12238, Lsa33, Lsa30, OmpL1, rLIC11360 and rLIC11975, as novel Fg-binding proteins. The recombinant proteins interact with Fg in a dose-dependent and saturable fashion when increasing protein concentration was set to react to a fix human Fg concentration. The calculated dissociation equilibrium constants (KD) of these reactions ranged from 733.3±276.8 to 128±89.9 nM for rLIC12238 and Lsa33, respectively. The interaction of recombinant proteins with human Fg resulted in inhibition of fibrin clot by thrombin-catalyzed reaction, suggesting that these versatile proteins could mediate Fg interaction in Leptospira. Our data reveal for the first time the inhibition of fibrin clot by Leptospira spp. and presents adhesins that could mediate these interactions. Decreasing fibrin clot would cause an imbalance of the coagulation cascade that may facilitate bleeding and help bacteria dissemination

Leptospira interrogans Lsa23 protein recruits plasminogen, factor H and C4BP from normal human serum and mediates C3b and C4b degradation.

It has been reported that pathogenic Leptospira are resistant to normal human serum (NHS) due to their ability to evade the complement immune system by interacting with factor H (FH) and C4b-binding protein (C4BP) regulators. Moreover, plasmin generation on the leptospiral surface diminishes C3b and IgG deposition, decreasing opsonophagocytosis by immune competent cells. We have previously reported that Lsa23 (LIC11360) is a multipurpose protein capable of binding purified extracellular matrix molecules, FH, C4BP and plasminogen (PLG)/plasmin in the presence of PLG activators. In this work, we provide further evidence that Lsa23 is located at the bacterial surface by using immunofluorescence microscopy. We show that Lsa23 has the ability to acquire FH, C4BP and PLG from NHS, and use these interactions to evade innate immunity. The binding with the complement regulators FH and C4BP preserves factor I (FI) activity, leading to C3b and C4b degradation products, respectively. C3b and C4b alpha-chain cleavage was also observed when Lsa23 bound to PLG generating plasmin, an effect blocked by the protease inhibitor aprotinin. Lsa23 also inhibited lytic activity by NHS mediated by both classical and alternative complement pathways. Thus, Lsa23 has the ability to block both pathways of the complement system, and may help pathogenic Leptospira to escape complement-mediated clearance in human hosts. Indeed, NHS treated with Lsa23 confers a partial serum resistance phenotype to Leptospira biflexa, whereas blocking this protein with anti-Lsa23 renders pathogenic L. interrogans more susceptible to complement-mediated killing. Thus, Lsa23 is a multifunctional protein involved in many pathways, featuring C4b cleavage by plasmin, knowledge that may help in the development of preventive approaches to intervene with human complement escape by this versatile pathogen.

Leptospira spp.: Novel insights into host–pathogen interactions

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is an important infectious disease that affects humans and animals. The disease causes economic losses as it affects livestock, with decreased milk production and death. Our group is investigating the genome sequences of L. interrogans targeting surface-exposed proteins because, due to their location, these proteins are capable to interact with several host components that could allow establishment of the infection. These interactions may involve adhesion of the bacteria to extracellular matrix (ECM) components and, hence, help bacterial colonization. The bacteria could also react with the host fibrinolytic system and/or with the coagulation cascade components, such as, plasminogen (PLG) and fibrinogen (Fg), respectively. The binding with the first system generates plasmin (PLA), increasing the proteolytic power of the bacteria, while the second interferes with clotting in a thrombin-catalyzed reaction, which may promote hemorrhage foci and increase bacterial dissemination. Interaction with the complement system negative regulators may help bacteria to evade the host immune system, facilitating the invasion. This work compiles the main described leptospiral proteins that could act as adhesins, as PLG and fibrinogen receptors and as complement regulator binding proteins. We present models in which we suggest possible mechanisms of how leptospires might colonize and invade host tissues, causing the disease. Understanding leptospiral pathogenesis will help to identify antigen candidates that would contribute to the development of more effective vaccines and diagnostic tests.

Induction of boosted immune response in mice by leptospiral surface proteins expressed in fusion with DnaK.

Leptospirosis is an important global disease of human and veterinary concern. Caused by pathogenic Leptospira, the illness was recently classified as an emerging infectious disease. Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity. Several studies have suggested the use of DnaK as an antigen in vaccine formulation, due to an exceptional degree of immunogenicity. We focused on four surface proteins: rLIC10368 (Lsa21), rLIC10494, rLIC12690 (Lp95), and rLIC12730, previously shown to be involved in host-pathogen interactions. Our goal was to evaluate the immunogenicity of the proteins genetically fused with DnaK in animal model. The chosen genes were amplified by PCR methodology and cloned into pAE, an E. coli vector. The recombinant proteins were expressed alone or in fusion with DnaK at the N-terminus. Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins. The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures. We believe that this approach could be employed in vaccines to enhance presentation of antigens of Leptospira to professional immune cells.

Mammalian cell entry (Mce) protein of Leptospira interrogans binds extracellular matrix components, plasminogen and β2 integrin.

A severe re‐emergingzoonosis, leptospirosis, is caused by pathogenic spirochetes of the genus Leptospira. Several studies have identified leptospiral surface proteins with the ability to bind ECM and plasma components, which could mediate adhesion and invasion through the hosts. It has been shown that Mce of pathogenic Leptospira spp. is an RGD (Arg‐Gly‐Asp)‐motif‐dependent virulence factor, responsible for infection of cells and animals. In the present article, we decided to further study the repertoire of the Mce activities in leptospiral biological properties. We report that the recombinant Mce is a broad‐spectrum ECM‐binding protein, capable of interacting with laminin, cellular and plasma fibronectin and collagen IV. Dose–response interaction was observed for all the components, fulfilling ligand–receptor requirements. Mce is a PLG binding protein capable to recruit this component from NHS, generating PLA in the presence of PLG activator. Binding of Mce was also observed with the leukocyte cell receptors αLβ2 [(CD11a/CD18)‐LFA‐1] and αMβ2 [(CD11b/CD18)‐Mac‐1], suggesting the involvement of this protein in the host immune response. Indeed, virulent Leptospira L1‐130 was capable of binding both integrins, whereas culture‐attenuated M‐20 strain only bind to αMβ2 [(CD11b/CD18)‐Mac‐1]. To the best of our knowledge, this is the first work to describe that Mce surface protein could mediate the attachment of Leptospira interrogans to human cell receptors αLβ2(CD11a/CD18) and αMβ2(CD11b/CD18).

Characterization of two new putative adhesins of Leptospira interrogans

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and hypothetical. . The coding sequences (CDSs) LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasm membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix (ECM) components revealed that they are laminin-binding, and were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen (PLG) and generate plasmin in the presence of PLG-activator, urokinase (uPA). Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the surface playing a secondary role, probably mediating host-pathogen interaction, suggests that LIC11122 is a moonlight protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest Lsa19 is a potential candidate for future evaluation of its role for adhesion/colonization activities during L. interrogans infection.

14 Years of History on Sustainability: Waste Management FMUSP/IMT/SVOC

High regulation in environmental protection and sustainability was established in Brazil around 2003. From that time, the board of directors of the Faculty of Medicine, Tropical Medicine Institute, Death Verification Service, and Medical Investigation Laboratories, of the University of Sao Paulo created the Waste Management Committee to organize and implement actions to comply with Brazilian legislation. This paper outlines the main actions taken by the Committee and Board of directors of the institutions, presenting it in ages. During the first five years were developed works to know the passive/regular discharge of infectious agents, chemicals, radioactive compounds, recyclable and common garbage. From this data, were constructed stations for provisory disposal, transportation flow and general rules were established, and training was given to staff and students. During the following five years, or in the second phase of actions plans, efforts were focused on improving the management of the process and increasing the gathering of recyclable materials. Now, in the third phase, actions have been developed aiming to eliminate devices that use mercury and mercury vapor lamps from FMUSP building; furthermore, composting of food and garden material is in the evaluation process. These actions gave to FMUSP two Prize Friend of the Environment (2010 and 2016), offered by Health Secretary of Sao Paulo State, and these experience will be shared in this paper. However, it is necessary to have in mind that this work was done under Brazilian regulations, but certainly, it can be adjusted to all countries.

Binding of human plasminogen by the lipoprotein LipL46 of Leptospira interrogans

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira. Bacteria disseminate via the bloodstream and colonize the renal tubules of reservoir hosts. Leptospiral surface-exposed proteins are important targets, because due to their location they can elicit immune response and mediate adhesion and invasion processes. LipL46 has been previously reported to be located at the leptospiral outer membrane and recognized by antibodies present in serum of infected hamsters. In this study, we have confirmed the cellular location of this protein by immunofluorescence and FACS. We have cloned and expressed the recombinant protein LipL46 in its soluble form. LipL46 was recognized by confirmed leptospirosis human serum, suggesting its expression during infection. Binding screening of LipL46 with extracellular matrix (ECM) and plasma components showed that this protein interacts with plasminogen. The binding is dose-dependent on protein concentration, but saturation was not reached with the range of protein concentration used. Kringle domains of plasminogen and lysine residues of the recombinant protein are involved in the binding because the lysine analog, amino caproic acid (ACA) almost totally inhibited the reaction. The interaction of LipL46 with plasminogen generates plasmin in the presence of plasminogen activator uPA. Because plasmin generated at the leptospiral surface can degrade ECM molecules and decrease opsonophagocytosis, we tentatively infer that Lip46 has a role in helping the invasion process of pathogenic Leptospira.