Evaluation of immunoprotective activity of six leptospiral proteins in the hamster model of leptospirosis.

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of L. interrogans serovar Copenhageni together with bioinformatics tools represent a great opportunity to search for novel antigen candidates that could be used as subunit vaccine against leptospirosis. We focused on six genes encoding for conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from Leptospira interrogans genomic DNA and were cloned and expressed in Escherichia coli. The recombinant proteins tagged with N-terminal hexahistidine were purified by metal-charged chromatography. The immunization of hamsters followed by challenge with lethal dose of virulent strain of Leptospira showed that the recombinant proteins Lsa21, Lsa66 and rLIC11030 elicited partial protection to animals. These proteins could be used combined or in a mixture with novel adjuvants in order to improve their effectiveness.

Emprego de estirpes Leptospira spp. isoladas no Brasil na microtécnica de soroaglutinação microscópica aplicada ao diagnóstico da leptospirose em rebanhos bovinos de oito estados brasileiros / Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states

O objetivo do presente trabalho foi investigar a conveniência do emprego de estirpes de leptospiras autóctones isoladas no Brasil, na coleção de antígenos da microtécnica de soroaglutinação microscópica (SAM) aplicada a leptospirose. Foram amostradas por conveniência 109 propriedades e 9820 bovinos, fêmeas em idade reprodutiva, distribuídos em 85 municípios, dos Estados de Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Paraná, Rio Grande do Sul, Santa Catarina e São Paulo. Dos 9820 animais examinados, 5806 (59,12%) foram reagentes na SAM para pelo menos um sorovar com a coleção de 23 sorovares de referência. Com a coleção de antígenos de referência e dez estirpes autóctones houve 6400 (65,17%) reagentes, com diferença significativa entre as proporções (p=0,001). Os sorovares mais prováveis identificados com a coleção de antígenos de referência foram Hardjo (43,03%), Shermani (20 %), Wolffi (9,96%), Grippothyphosa (5,42%) e Pomona (4,28%). Com a coleção ampliada por dez estirpes isoladas no Brasil, os sorovares mais prováveis foram Hardjo (31,00%), Guaricura-M4/84 (22,50%), Shermani (15,43%), Wolffi (4,76%), Grippothyphosa (3,71%) e Autumnalis (3,24%). O sorovar Guaricura, estirpe M4/84, isolada de bovinos e búfalos no Estado de São Paulo, despontou como um dos três sorovares mais freqüentes nos Estados de Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais e São Paulo. A introdução de estirpes autóctones na coleção de antígenos da SAM propiciou a confirmação do diagnóstico de leptospirose em 594 animais (6,00%) classificados como não reagentes pela coleção de referência (p=0,001).

The aim of this study was to investigate the adequacy of the use of autochthonous strains of leptospires isolated in Brazil, added to antigen collection of the microscopic agglutination test (MAT) applied to the diagnosis of bovine leptospirosis. By means of non-probability sampling, 109 farms and 9,820 cattle, females at reproductive age were chosen from 85 municipalities in the states of Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Paraná, Rio Grande do Sul, Santa Catarina and São Paulo. Among the 9,820 examined animals, 5,806 (59.12%) were reactants at MAT for at least one serovar using the 23 reference serovars. Employing the collection of reference serovars and the ten autochthonous strains, 6,400 (65.24%) reactants and significant difference (p=0.001) was found. The most probable serovars identified by the collection of reference antigens were Hardjo (43.03%), Shermani (20%), Wolfi (9.96%), Grippothyphosa (5.42%) and Pomona (4.28%). With the collection amplified with the ten strains isolated in Brazil, the most probable serovars were Hardjo (31%), Guaricura-M4/84 (22.50%), Shermani (15.43%), Wolffi (4.76%), Grippothyphosa (3.71%) and Autumnalis (3.24%). The serovar Guaricura, strain M4/84, isolated from bovines and buffaloes in the State of São Paulo, was ranked as one of the three most probable serovars in the states of Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais and São Paulo. The addition of autochthonous strains to the MAT antigen collection provided the confirmation of the diagnosis of leptospirosis in 594 cattle (6%) which have been classified as non-reactants by the reference collection (p=0.001).

Failure of LIC13435 Protein of Leptospira interrogans SerovarCopenhageni to Confer Protection in Immunized Hamsters

Leptospirosis is a re-emergent zoonosis characterized by an acute febrile and systemic illness in humans caused by pathogenic spirochetes belonging to the genus Lepstospira. This disease has global distribution, and it is more frequent in tropical and subtropical areas. The complete genomic sequence of Leptospira species offered the possibility to identify potential vaccine candidates for leptospirosis, since environmental control measures are difficult to implement and there is not an ideal vaccine available for human use. Secreted and surface exposed molecules are potential targets for inducing protective immune response in the host. Although we selected six predicted sequences coding for putative outer membrane proteins with unknown function to be analyzed as vaccinal candidates against leptospirosis and for biological characterization, only the lic13435 gene was expressed and purified. The lic13435 gene is specific for pathogenic leptospires suggesting a possible virulence and/or pathogenicity associated function. The recombinant protein was purified and tested as vaccine candidate against leptospirosis. The immunization with the recombinant protein was able to produce a significant immune response in hamsters. Nevertheless, the animals were not protected against leptospirosis.

Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans.

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6xHis were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

Refolding of the recombinant protein OmpA70 from Leptospira interrogans from inclusion bodies using high hydrostatic pressure and partial characterization of its immunological properties.

Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects human populations worldwide. Available vaccines have demonstrated limited effectiveness, and therapeutic interventions are complicated by the difficulty of establishing an early diagnosis. The genome of Leptospira strains was sequenced, and bioinformatic analyses revealed potential vaccine and serodiagnosis candidates. The present work studied OmpA70, a putative outer membrane protein from Leptospira interrogans serovar Copenhageni that combines structural features of Loa22, the first genetically defined virulence factor in Leptospira, and Lp49, a protein that reacts with sera from early and convalescent patients. Recombinant OmpA was produced in Escherichia coli in an insoluble form. Considering the importance of the structural integrity of a protein to confer immune protection, high hydrostatic pressure (HHP) was used to refold OmpA70 aggregated as inclusion bodies. HHP was applied in association with redox-shuffling reagents (oxidized and reduced glutathione) and guanidine hydrochloride or l-arginine. About 40% of the protein was refolded by applying 200MPa for 16h in concentrations of l-arginine above 0.4M. Circular dichroism revealed the presence of secondary structure. OmpA70 has immunogenic and antigenic properties as high antibody titers were seen after immunization with this protein, and sera from infected hamsters reacted with soluble OmpA70.

Fatores de risco para a leptospirose em fêmeas bovinas em idade reprodutiva no estado da Bahia, nordeste do Brasil / Risk factors associated with leptospirosis in cows in the state of Bahia, northeastern Brazil

Foram identificados fatores de risco associados à leptospirose em fêmeas bovinas em idade reprodutiva no Estado da Bahia. Foram amostradas aleatoriamente 10.823 fêmeas bovinas com idade igual ou superior a 24 meses procedentes de 1.414 propriedades. Para o diagnóstico sorológico da infecção por Leptospira spp., foi utilizada a Soroaglutinação Microscópica (SAM) utilizando 24 sorovares como antígenos. Um rebanho foi considerado foco quando apresentou pelo menos um animal soropositivo. Das 1.414 propriedades investigadas, 1.076 (77,9 por cento; IC 95 por cento = 75,7-80,0 por cento) apresentaram pelo menos um animal reagente na SAM para qualquer sorovar. O sorovar Hardjo (Hardjoprajitno) foi o mais prevalente, com 34,49 por cento (IC 95 por cento = 31,97-37,14 por cento) das propriedades positivas. Presença de mais de 28 fêmeas bovinas em idade reprodutiva no rebanho (OR=2,11; p<0,001), presença de cervídeos (OR=2,02; p=0,010), compra de animais (OR=1,57; p<0,001), abate de animais na própria fazenda (OR=1,58; p=0,030) e utilização de partos compartilhados (OR=1,63; p<0,001) foram identificados como fatores de risco para leptospirose por qualquer sorovar. Os fatores de risco para leptospirose pelo sorovar Hardjo (Hardjoprajitno) foram a presença de suínos (OR=1,28; p=0, 040) e a compra de animais (OR=1,48; p<0,001).

Risk factors associated with leptospirosis were identified in cows in the state of Bahia, northeastern Brazil. A total of 10,823 cows with > 24 months of age from 1,414 herds were randomly sampled. For the serological diagnosis of Leptospira spp. infection, the microscopic agglutination test (MAT) was carried out using 24 serovars as antigens. A herd was considered positive when presented at least one seropositive animal. Of the 1,414 investigated herds, 1,076 (77.9 percent; 95 percent CI = 75.7-80.0 percent) presented at least one reactant animal at MAT to any serovar. Serovar Hardjo (Hardjoprajitno) was the most prevalent, with 34.49 percent (95 percent CI = 31.97-37.14 percent) of the positive herds. Presence of more than 28 bovine females in reproductive age (OR=2.11; p<0.001), presence of cervids (OR=2.02; p=0.010), animal purchase (OR=1.57; p<0.001), to slaughter animals in the property (OR=1.58; p=0.030) and to share pasture (OR=1.63; p<0.001) were identified as risk factors for leptospirosis due to any serovar. Risk factors for leptospirosis due to serovar Hardjo (Hardjoprajitno) were presence of swine (OR=1.28; p=0.040) and animal purchase (OR=1.48; p<0.001).

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.

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that 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 strains of 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 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant 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 protein is 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 by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis.

LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules.

The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis.

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.

Lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells.

OBJECTIVES: The study of a predicted outer membrane leptospiral protein encoded by the gene LIC12690 in mediating the adhesion process. METHODS: The gene was cloned and expressed in Escherichia coli BL21 (SI) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and used to assess its ability to activate human umbilical vein endothelial cells (HUVECs). RESULTS: The recombinant leptospiral protein of 95kDa, named Lp95, activated E-selectin in a dose-dependent fashion but not the intercellular adhesion molecule 1 (ICAM-1). In addition, we show that pathogenic and non-pathogenic Leptospira are both capable to stimulate endothelium E-selectin and ICAM-1, but the pathogenic L. interrogans serovar Copenhageni strain promotes a statistically significant higher activation than the non-pathogenic L. biflexa serovar Patoc (P<0.01). The Lp95 was identified in vivo in the renal tubules of animal during experimental infection with L. interrogans. The whole Lp95 as well as its fragments, the C-terminal containing the domain of unknown function (DUF), the N-terminal and the central overlap regions bind laminin and fibronectin ECM molecules, being the binding stronger with the DUF containing fragment. CONCLUSION: This is the first leptospiral protein capable to mediate the adhesion to ECM components and the activation of HUVECS, thus suggesting its participation in the pathogenesis of Leptospira.

Plasminogen acquisition and activation at the surface of leptospira species lead to fibronectin degradation.

Pathogenic Leptospira species are the etiological agents of leptospirosis, a widespread disease of human and veterinary concern. In this study, we report that Leptospira species are capable of binding plasminogen (PLG) in vitro. The binding to the leptospiral surface was demonstrated by indirect immunofluorescence confocal microscopy with living bacteria. The PLG binding to the bacteria seems to occur via lysine residues because the ligation is inhibited by addition of the lysine analog 6-aminocaproic acid. Exogenously provided urokinase-type PLG activator (uPA) converts surface-bound PLG into enzymatically active plasmin, as evaluated by the reaction with the chromogenic plasmin substrate d-Val-Leu-Lys 4-nitroanilide dihydrochloridein. The PLG activation system on the surface of Leptospira is PLG dose dependent and does not cause injury to the organism, as cellular growth in culture was not impaired. The generation of active plasmin within Leptospira was observed with several nonvirulent high-passage strains and with the nonpathogenic saprophytic organism Leptospira biflexa. Statistically significant higher activation of plasmin was detected with a low-passage infectious strain of Leptospira. Plasmin-coated virulent Leptospira interrogans bacteria were capable of degrading purified extracellular matrix fibronectin. The breakdown of fibronectin was not observed with untreated bacteria. Our data provide for the first time in vitro evidence for the generation of active plasmin on the surface of Leptospira, a step that may contribute to leptospiral invasiveness.

A newly identified protein of Leptospira interrogans mediates binding to laminin.

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity.

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.

Immune evasion of leptospira species by acquisition of human complement regulator C4BP.

Leptospirosis is a spirochetal zoonotic disease of global distribution with a high incidence in tropical regions. In the last 15 years it has been recognized as an important emerging infectious disease due to the occurrence of large outbreaks in warm-climate countries and, occasionally, in temperate regions. Pathogenic leptospires efficiently colonize target organs after penetrating the host. Their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues. Therefore, they must be able to evade the innate host defense. The main purpose of the present study was to evaluate how several Leptospira strains evade the protective function of the complement system. The serum resistance of six Leptospira strains was analyzed. We demonstrate that the pathogenic strain isolated from infected hamsters avoids serum bactericidal activity more efficiently than the culture-attenuated or the nonpathogenic Leptospira strains. Moreover, both the alternative and the classical pathways of complement seem to be responsible for the killing of leptospires. Serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Surface-bound C4BP promotes factor I-mediated cleavage of C4b. Accordingly, we found that pathogenic strains displayed reduced deposition of the late complement components C5 to C9 upon exposure to serum. We conclude that binding of C4BP contributes to leptospiral serum resistance against host complement.