Identification and evaluation of new adhesins of Leptospira interrogans by shotgun phage display

Leptospirose é uma doença infecciosa emergente cujos agentes etiológicos são espécies patogênicas do gênero Leptospira. Leptospiras patogênicas possuem inúmeros genes específicos codificando proteínas com funções desconhecidas, sugerindo que as leptospiras apresentam fatores de virulência únicos. Adesinas bacterianas são importantes fatores de virulência e, assim, a identificação de adesinas conservadas em espécies patogênicas de Leptospira pela construção de bibliotecas genômicas expostas na superfície de bacteriófagos (shotgun phage display), seguida por seleção em células e/ou componentes da matriz extracelular (biopanning), pode revelar novos antígenos e alvos para o tratamento e prevenção da leptospirose. Bibliotecas foram construídas com o DNA genômico de L. interrogans fragmentado e o fagomídeo pG8SAET, sendo testadas algumas abordagens para clonagem como a ligação entre extremidades cegas (blunt-end) e técnicas baseadas em ligação entre extremidades coesivas, incluindo a obtenção de ORESTES e a utilização de adaptadores em grampo. Apesar de serem encontradas algumas limitações, a clonagem por ligação blunt-end se mostrou a mais eficiente para a construção de bibliotecas, sendo adotada para a construção de três bibliotecas em maior escala. A seleção de novas possíveis adesinas a partir das bibliotecas construídas foi realizada em células eucarióticas através da metodologia BRASIL. A primeira biblioteca (BBT1) exibiu 106 clones totais, a partir da qual foram selecionados quatro proteínas em fase apenas com a proteína VIII do fago (pVIII). No entanto, nenhuma delas seria exposta por programas de predição na bactéria. Outras duas bibliotecas foram construídas (BBT2 e BBT3), as quais obtiveram um número ideal de clones para uma ampla cobertura do genoma (>2x107 clones). Por apresentar maior proporção de clones válidos, a BBT2 foi utilizada para a seleção de adesinas, resultando em onze clones em fase com pVIII e/ou sequência sinal do fago. Análises por programas de predição revelaram três proteínas hipotéticas, denominadas LepA962, LepA069 e LepA388, as quais poderiam estar expostas ou ser secretadas pela bactéria, sugerindo uma possível função de adesina. O estudo da proteína LepA388 levou ao reconhecimento de outras doze proteínas semelhantes e pertencentes a uma família paráloga contendo um domínio denominado DUF_61, motivo de função desconhecida presente em proteínas compartilhadas somente entre as espécies patogênicas mais virulentas de Leptospira. Por esta razão, a proteína LepA388 foi a mais estudada. A clonagem de três porções da proteína (LepA388P, LepA388NR e LepA388F) para expressão heteróloga resultou em proteínas recombinantes insolúveis e, considerando a riqueza em resíduos de cisteína presente em sua estrutura, não foi possível renaturá-las adequadamente. Diante dos obstáculos encontrados, apenas a porção contendo a sequência apresentada pelo fago (LepA388P) foi utilizada para obtenção de antissoros em camundongos, os quais apresentaram altos títulos, demonstrando a alta imunogenicidade da proteína LepA388P. O reconhecimento de proteínas nativas da família paráloga DUF_61 em extratos de diferentes sorovares de Leptospira não foi observado, assim como sua expressão in vitro a partir de bactérias em diferentes condições de cultivo. Estudos adicionais sobre a expressão in vivo e funções dos membros desta família são necessários para uma compreensão mais ampla de seu papel na biologia de leptospiras e, possivelmente, na patogênese da leptospirose.Leptospirosis is an emerging infectious disease whose etiologic agents are pathogenic species of the genus Leptospira. Pathogenic leptospires have countless specific genes encoding proteins with unknown functions, suggesting that leptospires have unique virulence factors. Bacterial adhesins are important virulence factors and so the identification of conserved adhesins in pathogenic Leptospira species from shotgun phage display libraries, followed by selection (biopanning) in cells and/or extracellular matrix components, can reveal new antigens and strategies for leptospirosis treatment and prevention. Libraries were constructed using fragmented genomic DNA from L. interrogans and pG8SAET phagemid vector. Cloning approaches included blunt-end ligation and techniques based in cohesive-end ligation, such as ORESTES strategy and hairpin linkers. Despite some limitations, cloning by blunt-end ligation was the most efficient for library construction, being adopted for the construction of three libraries on a larger scale. Selection of new possible adhesins was performed by biopanning of the libraries in eukaryotic cells through BRASIL methodology. The first library called BBT1 exhibited approximately 106 total clones, and its biopanning resulted in four proteins fused to phage protein VIII, but none of them were expected to be exposed by the bacteria. Other libraries were built (BBT2 and BBT3) which reached the expected number of clones to obtain a larger genome representation (> 2x107 clones). Since it showed the highest proportion of positive clones, BBT2 was selected to perform a second biopanning, resulting in eleven proteins fused to phage protein VIII and/or signal peptide. In silico analysis revealed three hypothetical proteins, named LepA962, LepA069 and LepA388, that would be exposed or secreted by the bacteria, suggesting a possible adhesin function. The study of LepA388 protein led to the recognition of twelve other similar proteins belonging to a paralogous family that contains a domain called DUF_61, domain of unknown function that is present in proteins shared only among the most virulent pathogenic species of Leptospira. For this reason, the LepA388 protein was the most studied. The cloning of three portions of the protein (LepA388P, LepA388NR and LepA388F) for heterologous expression resulted in insoluble recombinant proteins, and given the presence of many cysteine residues in its structure, it was not possible to renature them appropriately. In face of the imposed obstacles, only the portion containing the sequence presented by the bacteriophage (LepA388P) was used to obtain antisera in mice, which showed high titers, demonstrating the high immunogenicity of the protein LepA388P. Recognition of native DUF_61 paralogous family proteins in extracts from distinct Leptospira serovars was not observed, as well as its in vitro expression from bacteria cultured in different conditions. Additional studies on the in vivo expression and functions of members of this family are needed for a broader understanding of their role in leptospiral biology and possibly in the pathogenesis of leptospirosis

Functional characterization of hypothetical proteins in Leptospira interrogans as adhesins and potential vaccine and diagnostic antigens

Leptospira spp. constitui um grupo de bactérias espiroquetas gram-negativas englobando espécies saprofíticas, intermediárias e patogênicas, sendo as últimas agentes causadores da leptospirose, doença zoonótica de alcance mundial e endêmica em regiões tropicais em desenvolvimento. O crescente número de espécies identificadas de leptospiras destaca ainda mais sua diversidade genética e mecanismos de virulência únicos, muitos deles com função ainda desconhecida. Esforços para o desenvolvimento de novas vacinas com proteção cruzada e efeito duradouro revelaram possíveis candidatos vacinais que necessitam ser adequadamente validados, sendo assim, há ainda uma urgente necessidade de uma vacina universal contra a leptospirose capaz de controlar e reduzir os surtos cada vez mais frequentes da doença. Adesinas são importantes fatores de virulência em diversos patógenos, constituindo antígenos promissores para o desenvolvimento de vacinas contra a leptospirose, assim como para o desenvolvimento de métodos diagnósticos mais rápidos e precisos. Previamente, foram identificadas três proteínas hipotéticas conservadas em L. interrogans pela técnica de phage display, denominadas arbitrariamente como LepA069, LepA962 e LepA388. A expressão do gene codificador da proteína LepA069 apresentou aumento de aproximadamente 70 % em animais infectados por leptospiras virulentas, representando a primeira evidência funcional desta proteína ainda desconhecida. Porções recombinantes da lipoproteína hipotética LepA962 (LepA962_Nt e LepA962_Phg) foram obtidos, sendo demonstrada a forte interação da proteína LepA962_Phg, contendo a sequência identificada por phage display, com laminina, fibronectina plasmática, colágeno I e fibrinogênio de maneira dose-dependente. Adicionalmente, LepA962_Phg apresentou ligação às células VERO e à sua matriz extracelular secretada, e o soro obtido a partir desta proteína recombinante foi capaz de se ligar à superfície de leptospiras virulentas, indicando que LepA962_Phg pode representar um importante domínio de interação entre as leptospiras e seu hospedeiro. Finalmente, a proteína LepA388 pertencente a uma extensa família de proteínas modificadoras de virulência com função desconhecida (DUF_61), presente apenas nas leptospiras patogênicas mais virulentas, apresentou aumento na expressão de seu gene codificador em animais infectados por leptospiras virulentas de acordo com dados na literatura. Além disso, porções recombinantes da região Nterminal desta proteína apresentaram ligação a laminina, colágenos I e IV, vitronectina e fibronectinas plasmática e celular, principalmente considerando a sequência identificada por phage display. Estes dados reforçam as predições de modelos tridimensionais da proteína LepA388 e de outros membros da família DUF_61, as quais identificam domínios semelhantes a toxinas (como abrina e CARDS) responsáveis pela ligação e internalização celulares nos hospedeiros. Dados recentes sugerem um possível papel citotóxico desempenhado pelas proteínas desta família em leptospiras, as quais podem também ser consideradas potenciais candidatas vacinais e para diagnóstico da leptospirose, devido à sua distribuição restrita em espécies e cepas patogênicas de importância para saúde humana.Leptospira spp. constitutes a group of gram-negative spirochete bacteria comprising saprophytic, intermediate and pathogenic species, the last being causative agents of leptospirosis, a zoonotic disease of worldwide extent and endemic in developing tropical regions. The growing number of identified leptospiral species further highlights their genetic diversity and unique virulence mechanisms, many of them with unknown function. Efforts to develop new vaccines with cross-protection and long-lasting effect have revealed possible vaccine candidates that need to be properly validated. Therefore, there is still an urgent need for a universal vaccine against leptospirosis capable of controlling and reducing the increasing outbreaks of the disease. Adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines against leptospirosis, as well as for the development of faster and more accurate diagnostic methods. Previously, three conserved hypothetical proteins in L. interrogans were identified by phage display technique, arbitrarily named as LepA069, LepA962 and LepA388. Expression of the LepA069 encoding gene showed an increase of approximately 70 % in animals infected by virulent leptospires, representing the first functional evidence of this still unknown protein. Recombinant portions of the hypothetical lipoprotein LepA962 (LepA962_Nt and LepA962_Phg) were obtained, demonstrating the strong interaction of the LepA962_Phg protein, containing the sequence identified by phage display, with laminin, plasma fibronectin, collagen I and fibrinogen in a dose-dependent manner. Furthermore, LepA962_Phg showed binding to VERO cells and its secreted extracellular matrix, and the serum obtained from this recombinant protein was able to bind to the surface of virulent leptospires, indicating that LepA962_Phg may represent an important domain of interaction between leptospires and its host. Finally, LepA388 protein belonging to an extensive family of virulence modifying proteins with unknown function (DUF_61), present only in the most virulent pathogenic leptospires, showed an increase in the expression of its encoding gene in animals infected by virulent leptospires according to data in literature. Moreover, recombinant portions of the N-terminal region of this protein showed binding to laminin, collagens I and IV, vitronectin and plasma and cell fibronectins, especially considering the sequence identified by phage display. These data support the predictions of three-dimensional models of the LepA388 protein and other members of the DUF_61 family, which identify toxin-like domains (such as abrin and CARDS) responsible for cellular binding and internalization in hosts. Recent data suggest a possible cytotoxic role played by proteins of this family in leptospires, which can also be considered potential vaccine candidates and antigens for diagnosis, due to their restricted distribution in pathogenic species and strains of importance to human health

Phagocytosis of Leptospira by leukocytes from mice with different susceptibility to leptospirosis and possible role of chemokines

Abstract Background Leptospirosis is a widespread zoonosis caused by pathogenic prokaryotic microbes of the genus Leptospira. Although there are several reports in the literature, host-pathogen interaction is still poorly understood. The role of chemokine expression is important on the chemotaxis, activation and regulation of immune cells. Recent studies have shown that their expression profiles play an important role on the severity of leptospirosis outcome. We evaluated the phagocytosis of Leptospira by spleens cells from C3H/HeJ, C3H/HePas and BALB/c mouse strains, respectively susceptible, intermediate and resistant to leptospirosis, and by RAW 264.7 macrophages. Besides, we evaluated the effects of CCL2 treatment on the phagocytosis. The cells were incubated with or without CCL2 chemokine, and infected with virulent L. interrogans sv Copenhageni. Cells and culture supernatants were collected for subsequent analysis. Results The number of leptospires was higher in BALB/c cells, CCL2 pre-treated or only infected groups, when compared to C3H/HeJ and C3H/HePas cells. Indeed, CCL2 activation did not interfere in the phagocytosis of Leptospira. Expression of chemokines CXCL5 and CCL8 levels were significantly inhibited in infected BALB/c cells when compared to the non-infected control. Conclusions Higher ability to phagocytosis and early modulation of some chemokines correlated with the resistance to leptospirosis disease. Exposure to CCL2 did not interfere on phagocytosis of Leptospira in our experimental conditions, but acted in the modulation of chemokines expression during Leptospira infection

New strategies for Leptospira vaccine development based on LPS removal.

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity