Functional characterization of methionine sulfoxide reductases from Leptospira interrogans.

BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.

Leptospira interrogans Bat proteins impair host hemostasis by fibrinogen cleavage and platelet aggregation inhibition.

Leptospirosis is a worldwide spread zoonosis, caused by pathogenic Leptospira. Evidences suggest that compromised hemostasis might be involved in the leptospirosis pathophysiology. In the genome of L. interrogans serovar Copenhageni, we found two genes coding for proteins which comprise von Willebrand factor (VWF) A domains (BatA and BatB). As VWF A domains exhibit multiple binding sites which contributes to human VWF hemostatic functions, we hypothesized that the L. interrogans BatA and BatB proteins could be involved in the hemostatic impairment during leptospirosis. We have cloned, expressed in Escherichia coli, and purified recombinant BatA and BatB. The influence of recombinant BatA and BatB on different in vitro hemostatic assays evaluating the enzymatic activity, platelet aggregation and fibrinogen integrity was investigated. We describe BatB as a new serine protease which is able to cleave thrombin chromogenic substrate, fibrin, fibrinogen, gelatin and casein; while BatA is active only towards fibrinogen. BatA and BatB interfere with the platelet aggregation induced by VWF/ristocetin and thrombin. Our results suggest an important role of the L. interrogans serovar Copenhageni Bat proteins in the hemostasis dysfunction observed during leptospirosis and contribute to the understanding of the leptospirosis pathophysiological mechanisms.

A bacterial 2[4Fe4S] ferredoxin as redox partner of the plastidic-type ferredoxin-NADP+ reductase from Leptospira interrogans.

BACKGROUND: Ferredoxins are small iron-sulfur proteins that participate as electron donors in various metabolic pathways. They are recognized substrates of ferredoxin-NADP+ reductases (FNR) in redox metabolisms in mitochondria, plastids, and bacteria. We previously found a plastidic-type FNR in Leptospira interrogans (LepFNR), a parasitic bacterium of animals and humans. Nevertheless, we did not identify plant-type ferredoxins or flavodoxins, the common partners of this kind of FNR. METHODS: Sequence alignment, phylogenetical analyses and structural modeling were performed for the identification of a 2[4Fe4S] ferredoxin (LepFd2) as a putative redox partner of LepFNR in L. interrogans. The gene encoding LepFd2 was cloned and the protein overexpressed and purified. The functional properties of LepFd2 and LepFNR-LepFd2 complex were analyzed by kinetic and mutagenesis studies. RESULTS: We succeeded in expressing and purifying LepFd2 with its FeS cluster properly bound. We found that LepFd2 exchanges electrons with LepFNR. Moreover, a unique structural subdomain of LepFNR (loop P75-Y91), was shown to be involved in the recognition and binding of LepFd2. This structural subdomain is not found in other FNR homologs. CONCLUSIONS: We report for the first time a redox pair in L. interrogans in which a plastidic FNR exchanges electron with a bacterial 2[4Fe4S] ferredoxin. We characterized this reaction and proposed a model for the productive LepFNR-LepFd2 complex. GENERAL SIGNIFICANCE: Our findings suggest that the interaction of LepFNR with the iron-sulfur protein would be different from the one previously described for the homolog enzymes. This knowledge would be useful for the design of specific LepFNR inhibitors.

Leptospira interrogans Secreted Proteases Degrade Extracellular Matrix and Plasma Proteins From the Host.

Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.

Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism.

Heme oxygenase from Leptospira interrogans is an important virulence factor. During catalysis, redox equivalents are provided to this enzyme by the plastidic-type ferredoxin-NADP+ reductase also found in L. interrogans. This process may have evolved to aid this bacterial pathogen to obtain heme-iron from their host and enable successful colonization. Herein we report the crystal structure of the heme oxygenase-heme complex at 1.73 Å resolution. The structure reveals several distinctive features related to its function. A hydrogen bonded network of structural water molecules that extends from the catalytic site to the protein surface was cleared observed. A depression on the surface appears to be the H+ network entrance from the aqueous environment to the catalytic site for O2 activation, a key step in the heme oxygenase reaction. We have performed a mutational analysis of the F157, located at the above-mentioned depression. The mutant enzymes were unable to carry out the complete degradation of heme to biliverdin since the reaction was arrested at the verdoheme stage. We also observed that the stability of the oxyferrous complex, the efficiency of heme hydroxylation and the subsequent conversion to verdoheme was adversely affected. These findings underscore a long-range communication between the outer fringes of the hydrogen-bonded network of structural waters and the heme active site during catalysis. Finally, by analyzing the crystal structures of ferredoxin-NADP+ reductase and heme oxygenase, we propose a model for the productive association of these proteins.

Structural and Enzymatic Characterization of a cAMP-Dependent Diguanylate Cyclase from Pathogenic Leptospira Species.

Leptospira interrogans serovar Copenhageni is a human pathogen that causes leptospirosis, a worldwide zoonosis. The L. interrogans genome codes for a wide array of potential diguanylate cyclase (DGC) enzymes with characteristic GGDEF domains capable of synthesizing the cyclic dinucleotide c-di-GMP, known to regulate transitions between different cellular behavioral states in bacteria. Among such enzymes, LIC13137 (Lcd1), which has an N-terminal cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA (GAF) domain and a C-terminal GGDEF domain, is notable for having close orthologs present only in pathogenic Leptospira species. Although the function and structure of GGDEF and GAF domains have been studied extensively separately, little is known about enzymes with the GAF-GGDEF architecture. In this report, we address the question of how the GAF domain regulates the DGC activity of Lcd1. The full-length Lcd1 and its GAF domain form dimers in solution. The GAF domain binds specifically cAMP (KD of 0.24µM) and has an important role in the regulation of the DGC activity of the GGDEF domain. Lcd1 DGC activity is negligible in the absence of cAMP and is significantly enhanced in its presence (specific activity of 0.13s-1). The crystal structure of the Lcd1 GAF domain in complex with cAMP provides valuable insights toward explaining its specificity for cAMP and pointing to possible mechanisms by which this cyclic nucleotide regulates the assembly of an active DGC enzyme.

Kinetic and structural characterization of a typical two-cysteine peroxiredoxin from Leptospira interrogans exhibiting redox sensitivity.

Little is known about the mechanisms by which Leptospira interrogans, the causative agent of leptospirosis, copes with oxidative stress at the time it establishes persistent infection within its human host. We report the molecular cloning of a gene encoding a 2-Cys peroxiredoxin (LinAhpC) from this bacterium. After bioinformatic analysis we found that LinAhpC contains the characteristic GGIG and YF motifs present in peroxiredoxins that are sensitive to overoxidation (mainly eukaryotic proteins). These motifs are absent in insensitive prokaryotic enzymes. Recombinant LinAhpC showed activity as a thioredoxin peroxidase with sensitivity to overoxidation by H2O2 (Chyp 1% ~30 µM at pH 7.0 and 30°C). So far, Anabaena 2-Cys peroxiredoxin, Helicobacter pylori AhpC, and LinAhpC are the only prokaryotic enzymes studied with these characteristics. The properties determined for LinAhpC suggest that the protein could be critical for the antioxidant defense capacity in L. interrogans.

A highly stable plastidic-type ferredoxin-NADP(H) reductase in the pathogenic bacterium Leptospira interrogans.

Leptospira interrogans is a bacterium that is capable of infecting animals and humans, and its infection causes leptospirosis with a range of symptoms from flu-like to severe illness and death. Despite being a bacteria, Leptospira interrogans contains a plastidic class ferredoxin-NADP(H) reductase (FNR) with high catalytic efficiency, at difference from the bacterial class FNRs. These flavoenzymes catalyze the electron transfer between NADP(H) and ferredoxins or flavodoxins. The inclusion of a plastidic FNR in Leptospira metabolism and in its parasitic life cycle is not currently understood. Bioinformatic analyses of the available genomic and proteins sequences showed that the presence of this enzyme in nonphotosynthetic bacteria is restricted to the Leptospira genus and that a [4Fe-4S] ferredoxin (LB107) encoded by the Leptospira genome may be the natural substrate of the enzyme. Leptospira FNR (LepFNR) displayed high diaphorase activity using artificial acceptors and functioned as a ferric reductase. LepFNR displayed cytochrome c reductase activity with the Leptospira LB107 ferredoxin with an optimum at pH 6.5. Structural stability analysis demonstrates that LepFNR is one of the most stable FNRs analyzed to date. The persistence of a native folded LepFNR structure was detected in up to 6 M urea, a condition in which the enzyme retains 38% activity. In silico analysis indicates that the high LepFNR stability might be due to robust interactions between the FAD and the NADP(+) domains of the protein. The limited bacterial distribution of plastidic class FNRs and the biochemical and structural properties of LepFNR emphasize the uniqueness of this enzyme in the Leptospira metabolism. Our studies show that in L. interrogans a plastidic-type FNR exchanges electrons with a bacterial-type ferredoxin, process which has not been previously observed in nature.

Caracterização de proteínas de membrana de Leptospira interrogans expressas em Escherichia coli / Characterization of membrane proteins of Leptospira interrogans expressed in Escherichia coli

Leptospirose é uma zoonose altamente disseminada causada por espécies patogênicas do gênero Leptospira. Os roedores são os principais reservatórios da doença nos centros urbanos. Anualmente, são relatados no Brasil cerca de 5.000 casos os quais ocorrem como surtos epidêmicos nas épocas de chuva. O sequenciamento genômico da L. interrogans sorovar Copenhageni e os avanços das análises bioinformáticas permitiram a identificação de novos candidatos vacinais e novos fatores de virulência. Dessa forma, foram selecionadas através de bioinformática seis genes de L. interrogans sorovar Copenhageni LIC 10411, LIC12891, LIC 10827, LIC13305, LIC11469 e LIC11030, os quais foram submetidos a ensaios de conservação do DNA genômico, RNA mensageiro e proteína nativa correspondente em onze sorovares patogênicos prevalentes no Brasil em um saprovar saprofítico de Leptospira...

Leptospirosis is a widespread zoonosis caused by pathogenic species of the genus Leptospira. Rodents are the main reservoirs of the disease in the urban areas. Annually, arround 5,000 cases are reported in Brazil, wich occurs in endemic outbreaks during the rainy season. The genomic sequencing and the advances of bioinformatics analysis allowed the identification of new vaccine candidates and new virulence factors. Therefore, six genes from L. interrogans serovar Copenhageni: LIC 10411, LIC 12891, LIC 10827, LIC 13305, LIC 11469 and LIC11030, were selected through bionformatic tools and subjected to conservation assays employing genomic DNA, mRNA and native protein from eleven pathogenic serovars predominat in Brazil and one saprophytic serovar of Leptospira...

Evaluation of the expression and protective potential of Leptospiral sphingomyelinases.

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.

Functional characterization of GDP-mannose pyrophosphorylase from Leptospira interrogans serovar Copenhageni.

Leptospira interrogans synthesizes a range of mannose-containing glycoconjugates relevant for its virulence. A prerequisite in the synthesis is the availability of the GDP-mannose, produced from mannose-1-phosphate and GTP in a reaction catalyzed by GDP-mannose pyrophosphorylase. The gene coding for a putative enzyme in L. interrogans was expressed in Escherichia coli BL21(DE3). The identity of this enzyme was confirmed by electrospray-mass spectroscopy, Edman sequencing and immunological assays. Gel filtration chromatography showed that the dimeric form of the enzyme is catalytically active and stable. The recombinant protein was characterized as a mannose-1-phosphate guanylyltransferase. S (0.5) for the substrates were determined both in GDP-mannose pyrophosphorolysis: 0.20 mM (GDP-mannose), 0.089 mM (PPi), and 0.47 mM; and in GDP-mannose synthesis: 0.24 mM (GTP), 0.063 mM (mannose-1-phosphate), and 0.45 mM (Mg(2+)). The enzyme was able to produce GDP-mannose, IDP-mannose, UDP-mannose and ADP-glucose. We obtained a structural model of the enzyme using as a template the crystal structure of mannose-1-phosphate guanylyltransferase from Thermus thermophilus HB8. Binding of substrates and cofactor in the model agree with the pyrophosphorylases reaction mechanism. Our studies provide insights into the structure of a novel molecular target, which could be useful for detection of leptospirosis and for the development of anti-leptospiral drugs.

Lsa63, a newly identified surface protein ofLeptospira interrogans binds laminin and collagen IV

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic diseasethat affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona.This protein was predicted to be surface exposed by PSORT program and contains a p83/ 100 domain identified by BLAST analysis that is conserved in protein antigens of several strainsof Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 andexpressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. Therecombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this proteinis most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded byLIC10314, named Lsa63 (Leptospiral surface adhesin of 63 kDa), binds strongly to laminin andcollagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed.

Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+.

BACKGROUND: Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. RESULTS: The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. CONCLUSION: LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans.

Ferredoxin-NADP+ reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 angstroms resolution at a synchrotron source.