Transcriptional Responses of Leptospira interrogans to Host Innate Immunity: Significant Changes in Metabolism, Oxygen Tolerance, and Outer Membrane

Leptospirosis is an important tropical disease around the world, particularly in humid tropical and subtropical countries. As a major pathogen of this disease, Leptospira interrogans can be shed from the urine of reservoir hosts, survive in soil and water, and infect humans through broken skin or mucous membranes. Recently, host adaptability and immune evasion of L. interrogans to host innate immunity was partially elucidated in infection or animal models. A better understanding of the molecular mechanisms of L. interrogans in response to host innate immunity is required to learn the nature of early leptospirosis. This study focused on the transcriptome of L. interrogans during host immune cells interaction. Significant changes in energy metabolism, oxygen tolerance and outer membrane protein profile were identified as potential immune evasion strategies by pathogenic Leptospira during the early stage of infection. The major outer membrane proteins (OMPs) of L. interrogans may be regulated by the major OmpR specific transcription factor (LB333). These results provide a foundation for further studying the pathogenesis of leptospirosis, as well as identifying gene regulatory networks in Leptospira spp

Analyzing 16S rRNA sequences from Vietnamese pathogenic Leptospira strains and in-silico prediction of potential antigenic epitopes on LipL21, LipL32 outer membrane lipoproteins

Leptospirosis, a zoonosis caused by Leptospira, is recognized as an emergent infectious disease. In currently, the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. In this experiment, a 550 bp fragment of large ribosomal RNA gene (16S rRNA) was sequenced and constructed phylogenetic tree from a panel of six Vietnamese pathogenic strains of Leptospira spirochetes (e.g., Pomona, Canicola, Mitis, Ictero haemohagiae, Bataviae, and Grippotyphosa). The results showed a close relationship of L.Pomona_VN and L.Hardjo (bootstrap: 99%). L.Canicola_VN and L.Ictero haemohagiae_VN appeared to be weak related to the classic L.Canicola, L. Grippotyphosa, these assemblage have a bootstrap support of 62%. The other strains (L.Mitis_VN and L.Grippotyphosa_VN) were appeared monophyletic, while their sister group (L.Bataviae_VN) relationship found only weak support (bootstrap: 62%). We also selected six genes [e.g. the immunoglobulin like proteins A and B (LigA and LigB genes), outer membrane protein (OmpL1 gene), and lipopolysaccharide (LipL32, LipL41, and LipL21 genes)] and checked gene expression in these Leptospira strains by polymerase chain reaction (PCR) method. There were three genes (e.g., LipL32, LipL21, and LigA genes) expressed in all strains, OmpL1 gene occured in 4 strains (L.Bataviae_VN, L.Canicola_VN, L.Grippotyphosa_VN and L.Mitis_VN), whereas LipL41 and LigB genes did not appear in any Leptospira strains. A multi-antigenic epitope potential of two gene (Lip L21 and Lip L32) was predicted by bioinformatic tools for designing a recombinant vaccine against leptospirosis. There were 3 multi-epitope regions (1 region and 95 antigenic epitope for B and T cells of LipL21 peptide; 2 regions and 124 antigenic epitope for both B and T cells of LipL32 peptide). It should be more of the deeply molecular biology studies to confirm the level agglutinating, antigen cleavage, peptide specificity matrices as well as neutralizing antibodies in the immune responses of DNA vaccine of these genes

Ekspresija i pročišćavanje površinskog lipoproteinskog antigena Lipl41 iz patogenih bakterija roda Leptospira

Pathogenic species of Leptospira lead to a zoonotic disease called leptospirosis, which is spread worldwide. A major topic of investigation is to detect the antigens that induce an immune response and to utilize them in diagnostic kits or vaccine development. The outer membrane proteins (OMPs) of Leptospira are potential candidates for this purpose. Lipl41 is an OMP that is conserved among pathogenic Leptospira. The aim of this study was to express and purify the Lipl41 recombinant protein in Iranian isolates. All collected Lipl41 protein sequences were compared and analyzed using bioinformatics tools from NCBI databases. Complete codon sequences of the Iranian pattern of Lipl41 recombinant protein were codon optimized and sub-cloned into a pET32a+ expression vector, and transformed into Escherichia coli BL21 (DE3). Optimal expression of recombinant Lipl41 (47kDa) was achieved post-induction with IPTG within the inclusion body. It was then purified by denaturation using serial concentrations of urea, and the recombinant protein was confirmed by western blot. In this study, sufficient amounts of Lipl41 were expressed and purified to be used for the development of a diagnostic kit and subunit vaccine.Patogene vrste Leptospira uzrokuju zoonotsku bolest leptospirozu koja je raširena u cijelom svijetu. Cilj istraživanja bio je dokazati antigene koji potiču imunosni odgovor i mogu se primijeniti u dijagnostičkim kompletima ili za razvoj cjepiva. Vanjska proteinska membrana (OMPs) leptospira potencijalni je kandidat za tu svrhu, a gen Lipl41 je dobro očuvan među patogenim leptospirama. Ekspresija i pročišćavanje rekombinantnog proteina Lipl41 provedeno je u izolatima iz Irana. Uspoređene su sve prikupljene sekvencije proteina Lipl41 i analizirane bioinformatičkim alatom iz baze podataka NCBI. Kompletne sekvencije kodona rekombinantnog proteina Lipl41 u izolatima su optimizirane, zatim subklonirane u pET32a+ vektor i pretvorene u bakteriju Escherichia coli BL21 (DE3). Optimalna ekspresija rekombinantnog Lipl41 (47kDa) postignuta je post-indukcijski pomoću isopropyl β-d-1-thiogalactopyranoside (IPTG). Zatim je pročišćena denaturacijom što je uključilo primjenu serijskih koncentracija ureje. Rekombinantni protein je potvrđen metodom western blot. Istraživanje potvrđuje mogućnost ostvarenja dostatne količine i ekspresije pročišćenog Lip141 da se može upotrijebiti za razvoj dijagnostičkih kompleta i subjediničnih cjepiva

Leptospirosis vaccines

Leptospirosis is a serious infection disease caused by pathogenic strains of the Leptospira spirochetes, which affects not only humans but also animals. It has long been expected to find an effective vaccine to prevent leptospirosis through immunization of high risk humans or animals. Although some leptospirosis vaccines have been obtained, the vaccination is relatively unsuccessful in clinical application despite decades of research and millions of dollars spent. In this review, the recent advancements of recombinant outer membrane protein (OMP) vaccines, lipopolysaccharide (LPS) vaccines, inactivated vaccines, attenuated vaccines and DNA vaccines against leptospirosis are reviewed. A comparison of these vaccines may lead to development of new potential methods to combat leptospirosis and facilitate the leptospirosis vaccine research. Moreover, a vaccine ontology database was built for the scientists working on the leptospirosis vaccines as a starting tool

Evaluación de la estabilidad genética de las cepas de referencia de Leptospira mantenidas bajo dos métodos de conservación

Objective. The genetic stability of Strains of Leptospira spp., maintained under two conservation systems, was evaluated. Methodology. The degree of conservation of the 16S rRNA and ompL1 genes of 10 reference serovars from the Leptospira spp. collection, belonging to the Sistema de Bancos de Germoplasma de la Nación para la Alimentación y la Agricultura (SBGNAA), was determined. Results. It was corroborated that the genes evaluated these have not undergone considerable changes, since similarities greater than 99.69 % were evidenced for 16S rRNA and 99.02% for ompL1, in the paired alignments. Conclusion. The genetic stability and purity of the reference strains of Leptospira spp. were verified. spp., kept in cryopreservation in liquid nitrogen at -196 °C and at room temperature for approximately eight years.Objetivo. Se evaluó la estabilidad genética de cepas de Leptospira spp., mantenidas bajo dos sistemas de conservación. Metodología. Se determinó el grado de conservación de los genes 16S rRNA y ompL1 de 10 serovares de referencia de la colección de Leptospira spp., pertenecientes al Sistema de Bancos de Germoplasma de la nación para la Alimentación y la Agricultura (SBGNAA). Resultados. Se corroboró que los genes evaluados estos no han sufrido cambios considerables, puesto que se evidenciaron similitudes superiores al 99,69% para 16S rRNA y de 99,02 % para ompL1, en los alineamientos pareados. Conclusión. Se comprobó la estabilidad genética y la pureza de las cepas de referencia de Leptospira spp., mantenidas en criopreservación en nitrógeno líquido a -196 °C y a temperatura ambiente durante ocho años aproximadamente

Delineating host-pathogen interaction of pathogenic Leptospira spp.

Delineating host-pathogen interaction of pathogenic Leptospira spp. Abstract Leptospirosis is a highly infectious, global, zoonotic disease affecting the majority of the mammalian species. Leptospirosis is caused by pathogenic bacteria, Leptospira spp., with more than >250 serovars identified. Cattle are one of the most susceptible hosts where the infection is commonly caused by serovar Hardjobovis. Bovine leptospirosis (BL) causes severe reproductive disorders and is a significant public health risk. Commercially available BL vaccines are typically bacterin and considered limited as they are serovar-specific and confer temporary protection. Bacterial outer membrane proteins (OMPs) are extensively studied as potential vaccine candidates for infectious diseases due to their ability to stimulate robust immune responses and induce cross-protective immunity. Here, seven novels OMPs from L. borgpetersenii serovar Hardjobovis L550 were identified using a reverse vaccinology approach. Four OMP genes were successfully cloned, expressed and purified as recombinant proteins. Subsequent functional in vitro binding assays showed that these OMPs could adhere to various host components and two OMPs of 37 and 49 kDa with significant binding results were re-assigned as ‘Leptospiral adhesin’ (Lsa) together with molecular weights as Lsa37 and Lsa49. Immunological evaluation of antibody titres against the OMPs in cattle bulk milk suggests these proteins are expressed by the bacteria and interact with the host immune system with two OMPs, OmpL1 and rLBL0375 exhibiting better discrimination with disease status. Several leptospiral OMPs exhibit molecular diversity through comparative sequence analysis. Here, two groups of OMP variants, OmpL1 and Lsa49 across five pathogenic genomospecies were selected via phylogenetic analysis to evaluate their functional binding diversities towards various host components. OmpL1 exhibited significant binding variation against various host components, compared to Lsa49. The diversity is strongly correlated with variations on predicted OmpL1 surface-exposed loops contributing to functional loss and gain via molecular evolution, which resulted in binding preferential towards specific host molecules. Immunological evaluation of cattle sera showed that these OMPs are expressed and exposed to the host immune system, and had a strong association against one another. This suggests that these OMPs may have similar structural epitopes that allow antibody binding, and indicates conserved immunogenicity across species. In a final study, we investigated whether the ruminant gastrointestinal (GI) tract was a carriage site for Leptospira. Through PCR surveys of ruminant gingival and rectal tissues, the presence of leptospires was identified as extremely low, thus indicating the GI tract does not appear to be an important leptospire carriage site. In conclusion, here we have identified several novel bovine leptospire OMPs, which may be useful vaccine or diagnostic components for bovine leptospirosis in the future. Additionally, the functional diversity between leptospiral OMP variants identifies OMP genetic evolution resulting in addition or loss of binding function, highlighting the complex host-pathogen interaction of leptospirosis. Lastly, this study does not suggest a role for the ruminant GI tract in leptospire carriage, indicating disease transmission through this route is unlikely

Evaluación del medio EMJH convencional y modificado sobre la cinética de crecimiento y la expresión de genes de patogenicidad en cepas de Leptospira interrogans serovar Icterohaemorrhagiae

166 páginasLa leptospirosis es una enfermedad zoonótica producida por espiroquetas patógenas del género Leptospira spp. son de crecimiento lento debido a que son exigentes nutricionalmente. En este estudio se determinó el efecto del medio de cultivo EMJH convencional y modificado sobre la cinética de crecimiento y la expresión de genes asociados con patogenicidad en dos cepas de Leptospira interrogans serovar Icterohaemorrhagiae. La metodología comprendió la realización de pruebas fisicoquímicas y microbiológicas de los suplementos convencional y modificado. Se estimó el crecimiento microbiano en el medio de cultivo EMJH bajo condiciones estáticas y de agitación por espectrofotometría y la uniformidad celular con microscopio de campo oscuro. El estudio molecular se realizó identificando los genes lipL32 y ompL1 en tres puntos de la cinética de crecimiento (fase de adaptación, fase exponencial y fase de declive) por PCR convencional y la expresión del gen lipL32 por PCR en Tiempo Real. Los resultados fisicoquímicos de los suplementos mostraron diferencias significativas (p<0.05). La evaluación microbiológica para mesófilos, mohos y levaduras, coliformes y S. aureus, fue de U.F.C/mL<10 y ausencia de Salmonella spp y Listeria monocytogenes. Los resultados en la cinética de crecimiento y el estudio molecular en la cepa aislada y control ATCC (23581) de Leptospira interrogans serovar Icterohaemorrhagiae en el medio modificado y convencional bajo la condición estática y agitado, indicaron que se obtuvo un adecuado rendimiento celular, se verificó la presencia de los genes lipL32 y ompL1 y la expresión del gen lipL32. Este trabajo se presenta como una contribución en el desarrollo de nuevos medios de cultivo, bajo en proteínas, que permitan el crecimiento y mantenimiento de las cepas de Leptospira interrogans serovar Icterohaemorrhagiae en EMJH modificado, sin afectar la expresión de genes asociados con patogenicidad, donde se utilizó como suplemento un subproducto industrial de desecho (sujeto a patente), beneficiando a la comunidad científica y al sector industrial productor de vacunas. Nota: Para consultar la carta de autorización de publicación de este documento por favor copie y pegue el siguiente enlace en su navegador de internet: http://hdl.handle.net/10818/878

Genes ompL1 e lipL32 como marcadores de leptospiras patogénicas - importância no diagnóstico da leptospirose

A leptospirose é uma zoonose de distribuição mundial causada por espiroquetas do género Leptospira que afeta mais de um milhão de casos/ano. As leptospiras são disseminadas no ambiente, sobretudo pela urina dos roedores, que são os principais reservatórios desta bactéria. Em Portugal, o Arquipélago dos Açores, constitui uma região endémica com uma elevada incidência anual, representando um problema de Saúde Pública. Assim, os objetivos deste trabalho foram: i) caracterizar a patogenicidade de culturas (isolados) de Leptospira spp., previamente obtidos de roedores capturados nas ilhas de São Miguel e Terceira (Açores), através de uma abordagem polifásica (ao nível fenotípico e molecular), esta última focada na deteção dos genes ompL1 e lipL32; e ii) determinar o interesse destes genes no diagnóstico laboratorial da leptospirose. Para tal, foram selecionadas (N=100) isolados de Leptospira spp., preservados a -80ºC no IHMT. Fez-se novo cultivo em meio seletivo EMJH cujo crescimento foi acompanhado ao longo de, pelo menos, três meses. A caracterização da patogenicidade foi realizada através de testes de crescimento [a 13ºC, e com a adição ao meio de cultura de 8-azaguanina (225μg/mL)], e um teste morfológico com adição de NaCl (1M). A identificação do sorogrupo/sorovar (sv.) de cada isolado foi realizada através de aglutinação microscópica, usando anticorpos monoclonais (mAbs). O estudo molecular de alguns isolados (n=32; 14 em cultura e DNA dos restantes), iniciou-se com a extração de DNA genómico, seguida de amplificação, usando dois protocolos de PCR convencional desenvolvidos e otimizados, no decurso do presente trabalho, utilizando primers com base nos genes lipL32 e ompL1, presentes em espécies patogénicas de Leptospira. O protocolo baseado no gene lipL32, foi também utilizado em amostras de doentes (N=127), urina (nu=99) e soro (ns=28). Adicionalmente, quatro dos isolados de Leptospira e algumas amostras humanas (nu=11 e ns=5) foram testadas por nested-PCR, utilizado na rotina. O DNA amplificado de alguns isolados e amostras humanas foi sequenciado e analisado por BLAST. Observou-se crescimento bacteriano em (n=14+/100) isolados cuja caracterização fenotípica revelou serem patogénicos. A avaliação com os mAbs permitiu incluí-los em dois sorogrupos distintos: Ballum (sv. Arborea) e Icterohaemorrhagiae (sv. Copenhageni). O protocolo “lipL32” revelou a presença de DNA leptospírico, no total dos isolados testados (n=32), enquanto que o protocolo “ompL1” detetou DNA específico em 84% (n=27+/32). Ambos os protocolos mostraram uma elevada especificidade, tendo o primeiro mostrado sensibilidade até 100bact/mL. Os resultados da sequenciação confirmaram que todas as amostras analisadas (isolados murinos e humanas) eram espécies patogénicas de Leptospira. Assim, os dados obtidos permitiram: i) conhecer a patogenicidade dos isolados estudados ao nível fenotípico e molecular; ii) corroborar a prevalência de leptospiras dos sorogrupos Ballum e Icterohaemorrhagiae, sendo estes os que mais afetam a população Açoriana, o que é concordante com estudos anteriores para a mesma região e país; iii) confirmar que os sorogrupos Ballum e Icterohaemorrhagiae têm um forte tropismo para determinadas espécies de roedores, em particular para Mus musculus e Rattus norvegicus, respetivamente; iv) otimizar o protocolo de PCR com base no gene lipL32, tornando-o uma nova ferramenta muito útil quer em estudos epidemiológicos (nos roedores), quer em amostras clínicas, melhorando significativamente o atual diagnóstico molecular da leptospirose.Leptospirosis is a worldwide zoonosis caused by spirochetes of Leptospira genus, which affect more than one million people/year. Spirochetes are spread in the environment, mostly by the urine of rodents that are the main reservoirs of this bacterium. In Portugal, the Azorean Archipelago is an endemic region with a high annual incidence, representing a public health problem. The aims of this work were: i) characterize the pathogenicity of Leptospira spp., cultures (isolates), previously obtained from rodents captured in São Miguel and Terceira islands (Azores), by polyphasic approach (at phenotypic and molecular level), this last, focused on detection of ompL1 and lipL32 genes; and ii) determine the interest of these genes in laboratory diagnosis of leptospirosis. For this, isolates (N=100) of Leptospira spp., preserved at -80ºC in the IHMT, were selected. Cultures were made on new EMJH selective culture medium, and its growth was followed over at least three months. The characterization of pathogenicity was performed by growth tests [at 13ºC, and with addition of 8-azaguanine (225μ /mL) to the culture médium], and a morphological test, with NaCl (1M). The serogroup/serovar (sv) identification of each isolate was performed by microscopic agglutination, using monoclonal antibodies (mAbs). The molecular study of some isolates (n=32; 14 in culture and DNA of the others) began with the extraction of genomic DNA, followed by amplification, using two conventional PCR protocols developed and optimized during the present work, using primers targeting lipL32 and ompL1 genes, both present in pathogenic Leptospira species. The protocol based on the lipL32 was also used in patient samples (N=127), urine (nu= 99) and serum (ns=28)]. Additionally, four of the Leptospira isolates and some human samples (nu=11 and ns=5) were tested by nested-PCR, used routinely. Amplified DNA from some isolates and human samples was sequenced and analyzed by BLAST. Bacterial growth was observed in (n=14+/100) isolates whose phenotypic characterization was found to be pathogenic. Evaluation with mAbs allowed to include them into two distinct serogroups: Ballum (sv. Arborea) and Icterohaemorrhagiae (sv. Copenhageni). The “lipL32” protocol revealed the presence of leptospiral DNA in all isolates tested (n=32), while the “ompL1” protocol detected specific DNA in 84% (n=27+/32). Both protocols showed high specificity, and the first showed sensitivity up to 100bact/mL. Sequencing results confirmed all samples analyzed (murine and human isolates) as pathogenic Leptospira species. Thus, the obtained data allowed: i) to know the pathogenicity of the studied isolates at the phenotypic and molecular level; ii) confirm the prevalence of leptospires from Ballum and Icterohaemorrhagiae serogroups, which are the ones that most affect the Azorean population, which is in agreement with previous studies for the same region and country; iii) confirm that these serogroups have a strong tropism for determined rodent species particularly for Mus musculus and Rattus norvegicus, respectively, and iv) optimize a new PCR protocol based on the lipL32 gene, making it an useful tool either in epidemiological studies (in rodents) or in clinical samples, significantly improving the current molecular diagnosis of leptospirosis