Proteome Analysis of Leptospira interrogans Virulent Strain

Leptospirosis is a worldwide zoonotic infection of human and veterinary concern. Caused by pathogenic spirochetes of the genus Leptospira, the disease presents greater incidence in tropical and subtropical regions. The identification of proteins that could be involved in the bacteria host interactions may facilitate the search for immune protective antigens. We report the proteomic analysis of Leptospira interrogans serovar Pomona virulent strain LPF cultured from kidney and liver of infected hamsters. Total protein extracts were separated by two-dimensional gel electrophoresis (2-DE), 895 spots were analyzed by MALDI-TOF mass spectrometry (MS), and 286 were identified as leptospiral proteins, corresponding to 108 distinct proteins. These proteins are allocated in all the bacterial cell compartments and are distributed in every functional category. Furthermore, the previously described, known outer membrane proteins, OmpL1, LipL21, LipL31, LipL32/Hap-1, LipL41, LipL45, LipL46, LruA/LipL71, and OmpA-like protein Loa22 were all recognized. Most importantly, this research work identified 27 novel leptospiral proteins annotated as hypothetical open reading frames (ORFs). We report for the first time an array of proteins of the Leptospira expressed by virulent, low-passage strain. We believe that our studies, together with the genome data will enlighten our understanding of the disease

Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection

<p>Abstract</p> <p>Background</p> <p>It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of <it>L. interrogans </it>allowed identification of a repertoire of putative leptospiral surface proteins.</p> <p>Results</p> <p>Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the <it>L. interrogans </it>species but was not found in the saprophytic <it>L. biflexa </it>serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis.</p> <p>Conclusion</p> <p>Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis.</p

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Whole-genome analysis of Leptospira interrogans to identify potential vaccine candidates against leptospirosis

Leptospirosis is an important global human and veterinary health problem. Humans can be infected by exposure to chronically infected animals and their environment. An important focus of the current leptospiral research is the identification of outer membrane proteins (OMPs). Due to their location, leptospiral OMPs are likely to be relevant in host-pathogen interactions, hence their potential ability to stimulate heterologous immunity. The existing whole-genome sequence of Leptospira interrogans serovar Copenhageni offers a unique opportunity to search for cell surface proteins. Predicted genes encoding potential surface proteins were amplified from genomic DNA by PCR methodology and cloned into an Escherichia coli expression system. The partially purified recombinant proteins were probed by Western blotting with sera from human patients diagnosed with leptospirosis. Sixteen proteins, out of a hundred tested, were recognized by antibodies present in human sera. Four of these proteins were conserved among eight serovars of L. interrogans and absent in the non-pathogenic Leptospira biflexa. These proteins might be useful for the diagnosis of the disease as well as potential vaccine candidates.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

O Papel do ensino do português como língua estrangeira na defesa do multicultarismo

As política actuais existentes a nível oficial para a implementação e defesa do ensino da Língua Portuguesa como Língua Estrangeira (L. E.) na Europa e no resto do mundo levam-nos a pensar que são, sobretudo, os casos isolados de leitores portugueses pioneiros, inspirados e marginais que na sua missão individual e afastada lutam pela implementação e defesa desta língua nos seus países de acolhimento. Segundo Volfgram, “cabe ensinar a alguns que o multiculturalismo não está apenas na teoria e sim ao nosso redor, nos elevando realmente à condição de seres humanos” (2005), e o mesmo é dizer que o multiculturalismo começa nas suas bases pela aprendizagem desinteressada e não interesseira das crianças na sua mais tenra idade. Não é impunemente que em países multiculturais como a Bélgica, a Língua Portuguesa ensinada como segunda língua ou como língua estrangeira desempenha um papel preponderante na defesa e na preservação do Português e, em simultâneo, pugna pela defesa incontestável da necessidade incontornável que o multiculturalismo é hoje. É indubitável que a luta contra a xenofobia, a luta pela tolerância e o respeito mútuo, bem como o diálogo profícuo biunívoco não podem sobreviver actualmente sem uma consciencialização da importância das línguas minoritárias, da crioulização, da relação com as línguas maioritárias e da conquista da defesa do multiculturalismo hic et nunc. Abordando algumas opiniões avisadas, esperamos trazer à discussão temas importantes, tais como, a necessidade de articulação de políticas de difusão da língua portuguesa na Europa e no Mundo concertadamente com o Brasil e outros Países Lusófonos, a necessidade de implementação de medidas concretas no terreno para defesa da Língua de Camões fora de Portugal, a sobrevivência do Português que embora sendo minoritária na Europa é uma das línguas mais faladas no mundo, a necessidade da consciencialização para a crescente importância geo-estratégica do Português paralelamente com o recrudescimento do multiculturalismo à escala global

Seroprevalence of bovine leptospirosis in reproductive-age female bovines in the state of São Paulo, Brazil

O presente estudo teve como objetivo determinar a soroprevalência da leptospirose bovina em fêmeas em idade reprodutiva do Estado de São Paulo, estratificado em sete regiões produtoras. Foram utilizados o delineamento estatístico, as amostras sorológicas e as informações contidas nos questionários empregados no Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose (PNCETB), instituído pelo Ministério da Agricultura, Pecuária e Abastecimento, considerando-se a utilização de fêmeas bovinas com idade '> OU =' a 24 meses (excluindo-se machos), diferentes tipos de produção, práticas de manejo, finalidades de reprodução, tamanho dos rebanhos e sistema de comercialização. Realizou-se a Soroaglutinação Microscópica (SAM) em 8.216 amostras sorológicas de animais provenientes de 1.021 propriedades. De acordo com os resultados obtidos, a infecção por Leptospira spp. ocorre em todo o Estado de São Paulo, com soroprevalência de 49,4% (IC 95% = 44,4%-54,4%) nas fêmeas bovinas em idade reprodutiva e em 718 (71,3%; IC 95% = 68,5%-74%) das propriedades analisadas. O sorovar Hardjo (46%) e sua associação com o sorovar Wolffi (21%) foram prevalentes entre o total de animais sororeagentes, seguidos pelos sorovares Shermani (8,9%), Autumnalis (4,4%) e Grippotyphosa (3,9%). Leptospira spp. está distribuída por todo estado e independe do tipo de exploração, manejo e das práticas de reprodução adotadas nos rebanhosThe objective of the present study was to determine the seroprevalence of bovine leptopirosis in São Paulo State, stratified in seven cattle production regions. It was based on the statistic delineation, serological samples and responses to the survey employed in the National Program for Control and Eradication of Brucelosis and Tuberculosis established by Ministry of Agriculture (2001). From the herds selected, serological analysis was only conducted on the cows '> OU =' 24 months old (excluding the males). The study took into consideration the herd size, the type of productive exploration, the reproductive handling, bovine practices and the commercialization system. The microscopic agglutination test (MAT) was applied on 8,216 serum samples from 1,021 different farms. The results showed that leptospirose infection occurs all over the seven regions of São Paulo State with 49.4% (CI 95% = 44.4-54.4%) animal seroprevalence and in 718 (71.3%; CI 95% = 68.5-74.0%) of the herds analyzed. Hardjo (46%) was the prevalent serovar for all the animals examined, followed by the Hardjo/Wolffi association (21%), Shermani (8.9%), Autumnalis (4.4%) and Grippotyphosa (3.9%). Leptospira spp. is present in all regions of the State of São Paulo and its occurrence is independent of the handling conditions and reproductive practices adopted in the herd

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.Instituto de Biotecnologia y Biologia Molecula

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

It has been reported previously that activation of vascular endothelium by outer membrane proteins of the spirochetes Borrelia sp. and Treponema sp. resulted in enhanced expression of endothelial cell adhesion molecules. To investigate the role of leptospiral proteins in this process, a predicted lipoprotein encoded by the gene LIC10365 was selected, which belongs to a paralogous family that presents a domain of unknown function, DUF1565. The LIC10365 gene was cloned and the protein expressed in Escherichia coli C43 (DE3) strain using the vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and was used to assess its ability to activate cultured human umbilical vein endothelial cells. The rLIC10365 activated endothelium in such a manner that E-selectin and intercellular adhesion molecule 1 (ICAM-1) became upregulated in a dose-dependent fashion. The LIC10365-encoded protein was identified in vivo in the renal tubules of animal during experimental infection with Leptospira interrogans. Collectively, these results implicate the LIC10365-coding protein of L. interrogans as a potential effector molecule in the promotion of a host inflammatory response. This is the first report of a leptospiral protein capable of up-regulating the expression of endothelial cell adhesion molecules ICAM-1 and E-selectin.Instituto de Biotecnologia y Biologia Molecula

Seroprevalence of leptospirosis in reproductive-age bovine females in the state of Bahia, Northeastern Brazil

O objetivo do presente trabalho foi determinar a soroprevalência da leptospirose em fêmeas bovinas em idade reprodutiva no Estado da Bahia. A amostragem foi delineada para a determinação da prevalência de propriedades positivas (focos) e de animais soropositivos para a leptospirose. O Estado foi dividido em quatro regiões ou estratos amostrais, nos quais foramexaminadas 10.823 fêmeas bovinas com idade ≥24 meses distribuídas em 1.414 propriedades. A reação de Soroaglutinação Microscópica (SAM), empregando 23 sorovares de Leptospira spp. como antígenos, foi utilizada como teste diagnóstico. O rebanho foi considerado foco quando apresentou pelo menos um animal soropositivo. As prevalências de foco e de animais soropositivos no Estado foram de 77,93% [IC 95% = 75,73% – 79.99%] e 45,42% [IC 95% = 42,00% – 48,88%], respectivamente.O sorovar mais frequente foi o Hardjo (Hardjoprajitno), em 34,49% [IC 95% = 31,93% – 37,14%] daspropriedades positivas e 14,95% [IC 95% = 12,59% - 17,67%] de animais soropositivos de diferentes regiõesThe aim of this work was to determine the seroprevalence of leptospirosis in reproductive-age bovine females in Bahia State, Northeastern Brazil. The sampling was delineated for the determination of the prevalence of seropositive animals as well as herds positive for bovine leptospiroses (foci). The state was divided into 4 regions or sampling strata in which 10,823 bovine females aged ≥24 months allocated in 1,414 herds were sampled. The microscopic agglutination test (MAT), using 23 Leptospira spp. serovars as antigens, was employed as a diagnostic test. The herd was considered positive if at least one animal was seropositive. The prevalences of positive herds and seropositive animals in the state were 77.93% [75.73%–79.99%] and 45.42% [42.00%–48.88%], respectively. Serovar Hardjo (Hardjoprajitno) was the most frequent, with 34.49% [31.93%–37.14%] of positive herds and 14.95% [12.59%–17.67%] of seropositive animals in the different region