The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

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.

Leptospirosis is a worldwide zoonosis caused by pathogenic species of Leptospira. Leptospires are able to adhere to exposed extracellular matrix in injured tissues and, once in the bloodstream, can survive the attack of the immune system and spread to colonize target organs. In this work, we report that two novel putative proteins, coded by the genes LIC11711 and LIC12587 of L. interrogans serovar Copenhageni are conserved among pathogenic strains, and probably exposed in the bacterial surface. Soluble recombinant proteins were expressed in Escherichia coli, purified and characterized. Both recombinant proteins bound to laminin and E-cadherin, suggesting an initial adhesion function in host epithelial cells. The recombinant protein LIC11711 (rLIC11711) was able to capture plasminogen (PLG) from normal human serum and convert to enzymatically active plasmin (PLA), in the presence of PLG activator. rLIC12587 (recombinant protein LIC12587) displayed a dose dependent and saturable interaction with components C7, C8, and C9 of the complement system, reducing the bactericidal effect of the complement. Binding to C9 may have consequences such as C9 polymerization inhibition, interfering with the membrane attack complex formation. Blocking LIC11711 and LIC12587 on bacterial cells by the respective antiserum reduced leptospiral cell viability when exposed to normal human serum (NHS). Both recombinant proteins could be recognized by serum samples of confirmed leptospirosis, but not of unrelated diseases, suggesting that the native proteins are immunogenic and expressed during leptospirosis. Taken together, our data suggest that these proteins may have a role in leptospiral pathogenesis, participating in immune evasion strategies.

Foram examinados 176 eqüídeos (15 muares e 161 eqüinos) do município de Monte Negro, Rondônia, Amazônia Ocidental Brasileira, frente a agentes virais e bacterianos. A amostra correspondeu ao total de eqüídeos no município, considerando um nível de confiança de 99%, prevalência esperada de 50% e erro padrão de 10%. As infecções virais foram investigadas pelas provas de Imunodifusão em gel de Agar (Anemia Infecciosa Eqüina - AIE), Inibição da hemaglutinação (Influenza eqüina tipos 1 e 2 IE-1 e 2) e Soroneutralização em cultura celular (Arterite Viral Eqüina - AVE, Herpesvírus Eqüino tipo 1 HVE1, Estomatite Vesicular - EV e Encefalomielite Eqüina do Leste - EEE, do Oeste - WEE e Venezuela - VEE). Para o diagnóstico da leptospirose, foi utilizada a prova de Soroaglutinação Microscópica (SAM); para o diagnóstico da brucelose, o teste do Antígeno Acidificado Tamponado (AAT) foi utilizado como teste de triagem e as provas de Soroaglutinação Lenta em Tubos (SLT) e 2-mercaptoetanol como testes diagnósticos. Foram constatados 9,6% dos eqüídeos reativos para AIE, 22,7% para HVE1, 19,9% para IE-1, 42,0% para IE-2, 21,0% para EEE, 11,3% para VEE, 3,4% para Brucella spp. e 91,4% para Leptospira spp. Os sorovares de leptospira mais freqüentes foram Bratislava (10,5%), Icterohaemorrhagiae (8,7%) e Autumnalis (8,7%) nos eqüinos e Patoc (26,6%) nos muares. Não foram encontrados animais com anticorpos contra AVE, EV e WEE.

No abstract

Aim The aim of this study was to test the hypothesis that the Brazilian coastal populations of Hoplias malabaricus were subject to the same geomorphological and palaeohydrological factors that resulted in endemic fish regions, by characterizing the mitochondrial DNA, nuclear sequences and cytogenetic data of these populations. Location Seventeen coastal basins in north‐eastern, eastern and south‐eastern Brazil, plus the São Francisco Basin. Methods Forty‐two specimens were analysed. Mitochondrial ATP synthase 6 (ATPase‐6) and nuclear recombination activating gene 2 (RAG2) gene sequences were used for Bayesian inference and maximum parsimony analyses. Molecular models were selected using MrModeltest. Results Molecular analyses indicated four haplogroups (Northeastern, Eastern A, Eastern B and Southeastern) for ATPase‐6 and three clades for RAG2. All topologies were congruent with Hoplias malabaricus diploid numbers, with most regions of proposed endemism and coastal geomorphological units. Main conclusions Deep genetic divergence between the Northeastern and the other haplogroups was interpreted as evidence of the vicariant effect of the Abrolhos Formation, which effectively isolates 2n = 40 and 2n = 42 coastal populations. To the south, the Cabo Frio Magmatic Lineament also isolates the Eastern and Southeastern 2n = 42 populations. In the Northeastern haplogroup, stream piracy was probably involved in chronologically varied dispersal events between coastal and continental basins. All haplogroups also included haplotypes that dispersed in recent times. Results show an older vicariant pattern and recent dispersal events congruent with the occurrence of temporary connections along the coast caused by eustatic sea level variations and the occurrence of stream piracy involving either continental or coastal basins, and suggest these processes contributed to the current distribution patterns of Brazilian coastal freshwater fish.

No abstract

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%; 95% CI = 75.7-80.0%) presented at least one reactant animal at MAT to any serovar. Serovar Hardjo (Hardjoprajitno) was the most prevalent, with 34.49% (95% CI = 31.97-37.14%) 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).INDEX TERMS: Leptospirosis, cattle, risk factors, Bahia state.