Diferentes mecanismos têm sido mostrados por estar envolvidos na evasão à morte mediada por complemento. Neste estudo, demonstramos que a aquisição do FH pela Leptospira é crucial para a sobrevivência das bactérias no soro e que estas espiroquetas interagem com FH, FHL-1, FHR-1 e C4BP. Nós também demonstramos que a ligação à estes reguladores é mediada pelas proteínas leptospiral immunoglobulin-like (Lig). FH se liga as proteínas Lig via short consensus repeat (SCR) principalmente pelos domínios 5 e 20. Ensaios de competição sugerem que FH e C4BP têm sítios de ligação diferentes nas proteínas Lig. Além disso, FH e C4BP ligados nas proteínas Lig mantêm a atividade de cofator, mediando a degradação de C3b e C4b pelo FI. Nós demonstramos que a aquisição de FH e C4BP pela L. biflexa transgênica para LigA e LigB exercem um papel de proteção na sobrevida destas bactérias. Análise por citometria de fluxo também confirmaram a capacidade das leptospiras transgênicas para controlar a deposição de C3, C4 e MAC. As proteínas Lig também foram capazes de ligar plasminogênio, o qual foi ativado em plasmina e esta enzima foi capaz de degradar fibrinogénio, C3b e C5. Estas clivagens inativam C3b e C5, evitando a progressão da cascata, e bloqueando as três vias de complemento. / Different mechanisms have been shown to be involved in evasion of complement-mediated killing. In this study, we demonstrate that acquisition of FH on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes interact with FH, FHL-1, FHR-1 and C4BP. We also demonstrate that binding to these regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins. FH binds to Lig proteins via short consensus repeat (SCR) domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by FI. We demonstrated that acquisition of FH and C4BP by the LigA and LigB transformed L. biflexa have the protective role, being crucial by bacterial survival. Analysis by Cytometer fluid also confirmed the ability of L. biflexa expressing LigA and LiB to controller the deposition of C3, C4 and MAC. Lig proteins were able to bind plasminogen, which was activated to plasmin and this enzyme was able to degrade the fibrinogen, C3b and C5. These cleavages inactivate C3b and C5, preventing progression of the complement cascade and blocking the three complement pathways.

A agropecuária familiar brasileira produz 70% da alimentação da população, possuindo importante papel social e econômico neste país. Os assentamentos do Pontal do Paranapanema produzem alimentos que são ofertados ao estado de São Paulo, sendo atividade leiteira a mais importante. As doenças infecciosas reprodutivas dos bovinos, causados por Leptospira spp., Herpes Virus Bovino Tipo 1(BoHV-1), Diarreia Viral Bovina (BVD), agentes da classe Mollicutes são importantes para a rentabilidade das produções agropecuárias do mundo inteiro. Dados epidemiológicos nos assentamentos do estado de São Paulo não existem. Este estudo teve como objetivo analisar a prevalência das enfermidades reprodutivas bacterianas e virais em bovinos de leite criados nos assentamento de Presidente Epitácio e Mirante do Paranapanema. Todos os animais foram submetidos ao exame específico do sistema reprodutivo e colheram-se amostras de soro e swab vaginal. A prevalência para BoHV-1, BVDV, Leptospira spp., e classe Mollicutes foi de 69,05%, 57,74%, 58,93% e 50%, respectivamente. Foi observado uma relação entre vacas multíparas e a presença de BoHV-1 e Leptospira spp. Foi observado também uma relação de animais com retenção de placenta com a presença de bactérias da classe Mollicutes e animais reagentes a anticorpos contra Leptospira spp. A prevalência destas enfermidades prova a existência destas em assentamentos na região do Pontal do Paranapanema, faz-se necessário o desenvolvimento de programas de políticas públicas para o monitoramento desta região / Brazilian\'s family settlements are responsible for 70% of total population feeding. It has an important social and economic role in Brazil. Settlements from Pontal do Paranapanema offering feed to the state of São Paulo and milk production is the most important activity. Bovine reproduction infectious diseases caused by Leptospira spp., Bovine Herpesvirus Type 1(BoHV-1), Bovine viral Diarrhoea (BVD) and Mollicutes are important to the dairy cattle industry all over the world. Epidemiological datas from settlements located in the state of São Paulo are not available. The aim of this study was to evaluate the prevalence of bacterial and viral reproductive infectious diseases dairy cattle raised in settlements from Presidente Epitácio e Mirante do Paranapanema. Specific physical examination of reproduction female system was performed and after that vaginal swabs samples and serum samples were collected. The prevalence of BoHV-1, BVDV, Leptospira spp., and Mollicutes was 69,05%, 57,74%, 58,93%, and 50%, respectively. An association between multiparous cows and the presence of BoHV-1 and Leptospira spp., was detected. Also an association between females with retained placenta and Mollicutes and cows with antibodies against Leptospira spp. was observed. The results showed the presence of reproduction diseases in settlements in Pontal do Paranapanema and it is necessary the development of public politics programs to control them

Made available in DSpace on 2014-07-29T15:07:30Z (GMT). No. of bitstreams: 1 Dissertacao Alberto Elias Marques.pdf: 241680 bytes, checksum: 919ae1559355f2402d175bade0935fc1 (MD5) Previous issue date: 2008-07-11 / The state of Goias has the third largest cattle herd in Brazil, but the production has some obstacles, such as reproductive diseases, which cause abortions and infertility, including leptospirosis, also important in public health. This study aimed to determine the prevalence of antibodies against Leptospira spp., determine their regional distribution and evaluate the main risk factors for the disease in cattle in the State of Goias. The study was conducted using the 4571 samples taken from 715 properties of 213 of the 246 municipalities in the state of Goiás The samples were analysed by the microagglutination test. To determine the correlation between the prevalence and associated factors, the test of Logistic Regression was used through the software SPSS. The percentage of positive samples for at least one of the16 serovars tested was 62.2%, with major prevalence of co-agglutination (40.24%), followed by serovars wolffi (14.53%), hardjo (12.70 %), grippotyphosa (10.55%) and shermani (6.55%). The prevalence of antibodies against Leptospira spp. proved to be associated to the following factors: stratum of production, with a greater prevalence in stractum one, predominantly for meat production; practice of artificial insemination; cattle breed; presence of sheep and goats; presence of capybaras; purchase of cattle in breeding exhibitions and from other properties; rent of pastures in any time of year; presence of maternity paddock and occurrence of abortions. On the other hand, it was not found correlation with the following factors: type of exploitation and creation charged in the property; kind of milking; presence of horses, pigs, dogs, cats, deer and other wild animals; presence of other animals; purchase of animal at fairs and/or auctions; selling of cattle in exhibitions, in fairs and/or auctions, from traders or from other properties; presence of pastures in common with other properties, veterinary care and vaccination against brucellosis, IBR and BVD. It was concluded that leptospiral infection is endemic in Goias. / O estado de Goiás tem o terceiro maior rebanho bovino do Brasil, mas a produção tem alguns entraves, como doenças do trato reprodutor, que causam abortos e infertilidade, entre elas a leptospirose, importante também na saúde pública. O presente trabalho visou determinar a prevalência de soroaglutininas anti-Leptospira spp., determinar sua distribuição regional e avaliar os principais fatores associados à enfermidade em bovinos do Estado de Goiás. O estudo foi realizado utilizando-se 4571 amostras colhidas em 715 propriedades de 213 dos 246 municípios do Estado de Goiás. As amostras foram analisadas pela técnica de soroaglutinação microscópica. Para a determinação da correlação entre a prevalência e fatores associados, foi utilizado o teste de Regressão Logística, através do software SPSS. A porcentagem de amostras positivas para pelo menos um dos 16 sorovares testados foi de 62,2%, com predominância de co-aglutinações (40,24%), seguidas pelos sorovares wolffi (14,53%), hardjo (12,70%), grippotyphosa (10,55%) e shermani (6,55%). A prevalência de anticorpos anti-Leptospira spp. mostrou-se associada aos seguintes fatores: estrato de produção, sendo mais prevalente no estrato um, de produção predominantemente de bovinos para corte; prática de inseminação artificial; raça dos animais; presença de ovinos e caprinos; presença de capivaras; compra de reprodutores em exposições e de outras propriedades; aluguel de pastos em alguma época do ano; presença de piquete maternidade e ocorrência de abortos. Por outro lado, não foi constatada associação com os seguintes fatores: tipo criação praticado na propriedade; presença de eqüídeos, suínos, cães, gatos, cervídeos e outros animais silvestres; presença de outras espécies animais não enquadradas nas categorias anteriores; compra de reprodutores em feiras e/ou leilões; venda de reprodutores em exposições, feiras e/ou leilões, de comerciantes ou de outras propriedades; presença de pastos em comum com outras propriedades; à assistência veterinária e vacinação contra, brucelose, IBR e BVD. Concluiu-se que a infecção por Leptospira spp. é endêmica no Estado de Goiás.

A imunização transcutânea (TCI) é uma via atrativa para o desenvolvimento de vacinação livre de agulhas, atuando nas APCs da pele, podendo substituir algumas das imunizações convencionais, em termos de facilidade, segurança e eficácia. O presente estudo avaliou a resposta imune da TCI com proteína recombinante de leptospira LipL32, uma proteína altamente conservada em cepas patogênicas e potente candidata vacinal. A TCI com a LipL32 na região abdominal de C57BL-6 foi capaz de primar o sistema imune, suscitando resposta sistêmica com altos níveis de anticorpos contra o antígeno, após reforços subimunizantes, ID e Tc. O padrão de citocinas em cultivo de células do sangue total dos grupos imunizados indicou que a imunização Tc foi capaz de primar o sistema imune, tanto inato quanto adaptativo. O tratamento local ou a coadministração com surfactantes ou PEG não evidenciou ação emoliente ou adjuvante. A coadministração Tc da LipL32 com MPL-A levou a efeito moderador da reação pro-inflamatória, redirecionando a resposta adaptativa, tanto humoral quanto celular. / Transcutaneous immunization (TCI) offers an attractive pathway for the development of needle-free vaccination by acting on APCs of the skin, showing potential to replace conventional immunization, in terms of safety and efficacy. In this study we evaluated the immune response by TCI with recombinant protein of leptospira LipL32, a highly conserved protein among pathogenic strains, a potential vaccine candidate. TCI with LipL32 was evaluated in C57BL-6 mice abdominal region and was able to confer systemic response with high levels of antibodies after subimmunizing ID and Tc boosters. The pattern of cytokines in cell cultures from whole blood of immunized groups indicated that the TCI was able to prime the immune system, for both innate and adaptive response. Local treatment of the skin or coadministration with surfactants and PEG did not show an emollient and an adjuvant action. Co-administration of LipL32 with MPL-A influenced the antibody response as well as showed a moderating effect of the pro-inflammatory reaction, redirecting the adaptive response.

Proteins are essential for the growth, survival and maintenance of the cell. Understanding the functional roles of proteins helps to decipher the working of macromolecular assemblies and cellular machinery of living organisms. A thorough investigation of the link between sequence, structure and function of proteins, helps in building a comprehensive understanding of the complex biological systems. Proteins have been observed to be composed of single and multiple domains. Analysis of proteins encoded in diverse genomes shows the ubiquitous nature of multi-domain proteins. Though the majority of eukaryotic proteins are multi-domain in nature, 3-D structures of only a small proportion of multi-domain proteins are known due to difficulties in crystallizing such proteins. While functions of individual domains are generally extensively studied, the complex interplay of functions of domains is not well understood for most multi-domain proteins. Paucity of structural and functional data, affects our understanding of the evolution of structure and function of multi-domain proteins. The broad objective of this thesis is to achieve an enhanced understanding of structure and function of protein domains by computational analysis of sequence and structural data. Special attention is paid in the first few chapters of this thesis on the multi-domain proteins. Classification of multi-domain proteins by implementation of an alignment-free sequence comparison method has been achieved in Chapters 2 and 3. Studies on organization, interactions and interdependence of domain-domain interactions in multi-domain proteins with respect to sequential separation between domains and N to C-terminal domain order have been described in Chapters 4 and 5. The functional and structural repertoire of organisms can be comprehensively studied and compared using functional and structural domain annotations. Chapter 6, 7 and 8 represent the proteome-wide structure and function comparisons of various pathogenic and non-pathogenic microorganisms. These comparisons help in identifying proteins implicated in virulence of the pathogen and thus predict putative targets for disease treatment and prevention. Chapter 1 forms an introduction to the main subject area of this thesis. Starting with describing protein structure and function, details of the four levels of hierarchical organization of protein structure have been provided, along with the databases that document protein sequences and structures. Classification of protein domains considered as the realm of function, structure and evolution has been described. The usefulness of classification of proteins at the domain level has been highlighted in terms of providing an enhanced understanding of protein structure and function and also their evolutionary relatedness. The details of structure, function and evolution of multi-domain proteins have also been outlined in chapter 1. ! Chapter 2 aims to achieve a biologically meaningful classification scheme for multi-domain protein sequences. The overall function of a multi-domain protein is determined by the functional and structural interplay of its constituent domains. Traditional sequence-based methods utilize only the domain-level information to classify proteins. This does not take into account the contributions of accessory domains and linker regions towards the overall function of a multi-domain protein. An alignment-free protein sequence comparison tool, CLAP (CLAssification of Proteins) previously developed in this laboratory, was assessed and improved when the author joined the group. CLAP was developed especially to handle multi-domain protein sequences without a requirement of defining domain boundaries and sequential order of domains (domain architecture). ! The working principle of CLAP involves comparison of all against all windows of 5-residue sequence patterns between two protein sequences. The sequences compared could be full-length comprising of all the domains in the two proteins. This compilation of comparison is represented as the Local Matching Scores (LMS) between protein sequences (nslab.iisc.ernet.in/clap/). It has been previously shown that the execution time of CLAP is ~7 times faster than other protein sequence comparison methods that employ alignment of sequences. In Chapter 2, CLAP-based classification has been carried out on two test datasets of proteins containing (i) Tyrosine phosphatase domain family and (ii) SH3-domain family. The former dataset comprises both single and multi-domain proteins that sometimes consist of domain repeats of the tyrosine phosphatase domain. The latter dataset consists only of multi-domain proteins with one copy of the SH3-domain. At the domain-level CLAP-based classification scheme resulted in a clustering similar to that obtained from an alignment-based method, ClustalW. CLAP-based clusters obtained for full-length datasets were shown to comprise of proteins with similar functions and domain architectures. Hence, a protein classification scheme is shown to work efficiently that is independent of domain definitions and requires only the full-length amino acid sequences as input.! Chapter 3 explores the limitations of CLAP in large-scale protein sequence comparisons. The potential advantages of full-length protein sequence classification, combined with the availability of the alignment-free sequence comparison tool, CLAP, motivated the conceptualization of full-length sequence classification of the entire protein repertoire. Before undertaking this mammoth task, working of CLAP was tested for a large dataset of 239,461 protein sequences. Chapter 3 discusses the technical details of computation, storage and retrieval of CLAP scores for a large dataset in a feasible timeframe. CLAP scores were examined for protein pairs of same domain architecture and ~22% of these showed 0 CLAP similarity scores. This led to investigation of the sensitivity of CLAP with respect to sequence divergence. Several test datasets of proteins belonging to the same SCOP fold were constructed and CLAP-based classification of these proteins was examined at inter and intra-SCOP family level. CLAP was successful in efficiently clustering evolutionary related proteins (defined as proteins within the same SCOP superfamily) if their sequence identity >35%. At lower sequence identities, CLAP fails to recognize any evolutionary relatedness. Another test dataset consisting of two-domain proteins with domain order swapped was constructed. Domain order swap refers to domain architectures of type AB and BA, consisting of domains A and B. A condition that the sequence identities of homologous domains were greater than 35% was imposed. CLAP could effectively cluster together proteins of the same domain architectures in this case. Thus, the sequence identity threshold of 35% at the domain-level improves the accuracy of CLAP. The analysis also showed that for highly divergent sequences, the expectation of 5-residue pattern match was likely a stringent criterion. Thus, a modification in the 5-residue identical pattern match criterion, by considering even similar residue and gaps within matched patterns may be required to effectuate CLAP-based clustering of remotely related protein sequences. Thus, this study highlights the limitations of CLAP with respect to large-scale analysis and its sensitivity to sequence divergence. ! Chapters 4 and 5 discuss the computational analysis of inter-domain interactions with respect to sequential distance and domain order. Knowledge of domain composition and 3-D structures of individual domains in a multi-domain protein may not be sufficient to predict the tertiary structure of the multi-domain protein. Substantial information about the nature of domain-domain interfaces helps in prediction of the tertiary as well as the quaternary structure of a protein. Therefore, chapter 4 explores the possible relationship between the sequential distance separating two domains in a multi-domain protein and the extent of their interaction. With increasing sequential separation between any two domains, the extent of inter-domain interactions showed a gradual decrease. The trend was more apparent when sequential separation between domains is measured in terms of number of intervening domains. Irrespective of the linker length, extensive interactions were seen more often between contiguous domains than between non-contiguous domains. Contiguous domains show a broader interface area and lower proportion of non-interacting domains (interface area: 0 Å2 to - 4400 Å2, 2.3% non-interacting domains) than non-contiguous domains (interface area: 0 Å2 to - 2000 Å2, 34.7% non-interacting domains). Additionally, as inter-protein interactions are mediated through constituent domains, rules of protein-protein interactions were applied to domain-domain interactions. Tight binding between domains is denoted as putative permanent domain-domain interactions and domains that may dissociate and associate with relatively weak interactions to regulate functional activity are denoted as putative transient domain-domain interactions. An interface area threshold of 600 Å2 was utilized as a binary classifier to distinguish between putative permanent and putative transient domain-domain interactions. Therefore, the state of interaction of a domain pair is defined as either putative permanent or putative transient interaction. Contiguous domains showed a predominance of putative permanent nature of inter-domain interface, whereas non-contiguous domains showed a prevalence of putative transient interfaces. The state of interaction of various SCOP superfamily pairs was studied across different proteins in the dataset. SCOP superfamily pairs mostly showed a conserved state of interaction, i.e. either putative permanent or putative transient in all their occurrences across different proteins. Thus, it is noted that contiguous domains interact extensively more often than non-contiguous domains and specific superfamily pairs tend to interact in a conserved manner. In conclusion, a combination of interface area and other inter-domain properties along with experimental validation will help strengthen the binary classification scheme of putative permanent and transient domain-domain interactions.! Chapter 5 provides structural analysis of domain pairs occurring in different sequential domain orders in mutli-domain proteins. The function and regulation of a multi-domain protein is predominantly determined by the domain-domain interactions. These in turn are influenced by the sequential order of domains in a protein. With domains defined using evolutionary and structural relatedness (SCOP superfamily), their conservation of structure and function was studied across domain order reversal. A domain order reversal indicates different sequential orders of the concerned domains, which may be identified in proteins of same or different domain compositions. Domain order reversals of domains A and B can be indicated in protein pair consisting of the domain architectures xAxBx and xBxAx, where x indicates 0 or more domains. A total of 161 pairs of domain order reversals were identified in 77 pairs of PDB entries. For most of the comparisons between proteins with different domain composition and architecture, large differences in the relative spatial orientation of domains were observed. Although preservation of state of interaction was observed for ~75% of the comparisons, none of the inter-domain interfaces of domains in different order displayed high interface similarity. These domain order reversals in multi-domain proteins are contributed by a limited number of 15 SCOP superfamilies. Majority of the superfamilies undergoing order reversal either function as transporters or regulatory domains and very few are enzymes. A higher proportion of domain order reversals were observed in domains separated by 0 or 1 domains than those separated by more than 1 domain. A thorough analysis of various structural features of domains undergoing order reversal indicates that only one order of domains is strongly preferred over all possible orders. This may be due to either evolutionary selection of one of the orders and its conservation throughout generations, or the fact that domain order reversals rarely conserve the interface between the domains. Further studies (Chapters 6 to 8) utilize the available computational techniques for structural and functional annotation of proteins encoded in a few bacterial genomes. Based on these annotations, proteome-wide structure and function comparisons were performed between two sets of pathogenic and non-pathogenic bacteria. The first study compares the pathogenic Mycobacterium tuberculosis to the closely related organism Mycobacterium smegmatis which is non-pathogenic. The second study primarily identified biologically feasible host-pathogen interactions between the human host and the pathogen Leptospira interrogans and also compared leptospiral-host interactions of the pathogenic Leptospira interrogans and of the saprophytic Leptospira biflexa with the human host. Chapter 6 describes the function and structure annotation of proteins encoded in the genome of M. smegmatis MC2-155. M. smegmatis is a widely used model organism for understanding the pathophysiology of M. tuberculosis, the primary causative agent of tuberculosis in humans. M. smegmatis and M. tuberculosis species of the mycobacterial genus share several features like a similar cell-wall architecture, the ability to oxidise carbon monoxide aerobically and share a huge number of homologues. These features render M. smegmatis particularly useful in identifying critical cellular pathways of M. tuberculosis to inhibit its growth in the human host. In spite of the similarities between M. smegmatis and M. tuberculosis, there are stark differences between the two due to their diverse niche and lifestyle. While there are innumerable studies reporting the structure, function and interaction properties of M. tuberculosis proteins, there is a lack of high quality annotation of M. smegmatis proteins. This makes the understanding of the biology of M. smegmatis extremely important for investigating its competence as a good model organism for M. tuberculosis. With the implementation of available sequence and structural profile-based search procedures, functional and structural characterization could be achieved for ~92% of the M. smegmatis proteome. Structural and functional domain definitions were obtained for a total of 5695 of 6717 proteins in M. smegmatis. Residue coverage >70% was achieved for 4567 proteins, which constitute ~68% of the proteome. Domain unassigned regions more than 30 residues were assessed for their potential to be associated to a domain. For 1022 proteins with no recognizable domains, putative structural and functional information was inferred for 328 proteins by the use of distance relationship detection and fold recognition methods. Although 916 sequences of 1022 proteins with no recognizable domains were found to be specific to M. smegmatis species, 98 of these are specific to its MC2-155 strain. Of the 1828 M. smegmatis proteins classified as conserved hypothetical proteins, 1038 proteins were successfully characterized. A total of 33 Domains of Unknown Function (DUFs) occurring in M. smegmatis could be associated to structural domains. A high representation of the tetR and GntR family of transcription regulators was noted in the functional repertoire of M. smegmatis proteome. As M. smegmatis is a soil-dwelling bacterium, transcriptional regulators are crucial for helping it to adapt and survive the environmental stress. Similarly, the ABC transporter and MFS domain families are highly represented in the M. smegmatis proteome. These are important in enabling the bacteria to uptake carbohydrate from diverse environmental sources. A lower number of virulent proteins were identified in M. smegmatis, which justifies its non-pathogenicity. Thus, a detailed functional and structural annotation of the M. smegmatis proteome was achieved in Chapter 6. Chapter 7 delineates the similarities and difference in the structure and function of proteins encoded in the genomes of the pathogenic M. tuberculosis and the non-pathogenic M. smegmatis. The protocol employed in Chapter 6 to achieve the proteome-wide structure and function annotation of M. smegmatis was also applied to M. tuberculosis proteome in Chapter 7. The number of proteins encoded by the genome of M. smegmatis strain MC2-155 (6717 proteins) is comparatively higher than that in M. tuberculosis strain H37Rv (4018 proteins). A total of 2720 high confidence orthologues sharing ≥30% sequence identity were identified in M. tuberculosis with respect to M. smegmatis. Based on the orthologue information, specific functional clusters, essential proteins, metabolic pathways, transporters and toxin-antitoxin systems of M. tuberculosis were inspected for conservation in M. smegmatis. Among the several categories analysed, 53 metabolic pathways, 44 membrane transporter proteins belonging to secondary transporters and ATP-dependent transporter classes, 73 toxin-antitoxin systems, 23 M. tuberculosis-specific targets, 10 broad-spectrum targets and 34 targets implicated in persistence of M. tuberculosis could not detect any orthologues in M. smegmatis. Several of the MFS superfamily transporters act as drug efflux pumps and are hence associated with drug resistance in M. tuberculosis. The relative abundances of MFS and ABC superfamily transporters are higher in M. smegmatis than in M. tuberculosis. As these transporters are involved in carbohydrate uptake, their higher representation in M. smegmatis than in M. tuberculosis highlights the lack of proficiency of M. tuberculosis to assimilate diverse carbon sources. In the case of porins, MspA-like and OmpA-like porins are selectively present in either M. smegmatis or M. tuberculosis. These differences help to elucidate protein clusters for which M. smegmatis may not be the best model organism to study M. tuberculosis proteins.! At the domain-level, ATP-binding domain of ABC transporters, tetracycline transcriptional regulator (tetR) domain family, major facilitator superfamily (MFS) domain family, AMP-binding domain family and enoyl-CoA hydrolase domain family are highly represented in both M. smegmatis and M. tuberculosis proteomes. These domains play an essential role in the carbohydrate uptake systems and drug-efflux pumps among other diverse functions in mycobacteria. There are several differentially represented domain families in M. tuberculosis and M. smegmatis. For example, the pentapeptide-repeat domain, PE, PPE and PIN domains although abundantly present in M. tuberculosis, are very rare in M. smegmatis. Therefore, such uniquely or differentially represented functional and structural domains in M. tuberculosis as compared to M. smegmatis may be linked to pathogenicity or adaptation of M. tuberculosis in the host. Hence, major differences between M. tuberculosis and M. smegmatis were identified, not only in terms of domain populations but also in terms of domain combinations. Thus, Chapter 7 highlights the similarities and differences between M. smegmatis and M. tuberculosis proteomes in terms of structure and function. These differences provide an understanding of selective utilization of M. smegmatis as a model organism to study M. tuberculosis. ! In Chapter 8, computational tools have been employed to predict biologically feasible host-pathogen interactions between the human host and the pathogenic, Leptospira interrogans. Sensitive profile-based search procedures were used to specifically identify practical drug targets in the genome of Leptospira interrogans, the causative agent of the globally widespread zoonotic disease, Leptospirosis. Traditionally, the genus Leptospira is classified into two species complex- the pathogenic L. interrogans and the non-pathogenic saprophyte L. biflexa. The pathogen gains entry into the human host through direct or indirect contact with fluids of infected animals. Several ambiguities exist in the understanding of L. interrogans pathogenesis. An integration of multiple computational approaches guided by experimentally derived protein-protein interactions, was utilized for recognition of host-pathogen protein-protein interactions. The initial step involved the identification of similarities of host and L. interrogans proteins with crystal structures of experimentally known transient protein-protein complexes. Further, conservation of interfacial nature was used to obtain high confidence predictions for putative host-pathogen protein-protein interactions. These predictions were subjected to further selection based on subcellular localization of proteins of the human host and L. interrogans, and tissue-specific expression profiles of the host proteins. A total of 49 protein-protein interactions mediated by 24 L. interrogans proteins and 17 host proteins were identified and these may be subjected to further experimental investigations to assess their in vivo relevance. The functional relevance of similarities and differences between the pathogenic and non-pathogenic leptospires in terms of interactions with the host has also been explored. For this, protein-protein interactions across human host and the non-pathogenic saprophyte L. biflexa were also predicted. Nearly 39 leptospiral-host interactions were recognized to be similar across both the pathogen and saprophyte in the context of processes that influence the host. The overlapping leptospiral-host interactions of L. interrogans and L. biflexa proteins with the human host proteins are primarily associated with establishment of its entry into the human host. These include adhesion of the leptospiral proteins to host cells, survival in host environment such as iron acquisition and binding to components of extracellular matrix and plasma. The disjoint sets of leptospiral-host interactions are species-specific interactions, more importantly indicative of the establishment of infection by L. interrogans in the human host and immune clearance of L. biflexa by the human host. With respect to L. interrogans, these specific interactions include interference with blood coagulation cascade and dissemination to target organs by means of disruption of cell junction assembly. On the other hand, species-specific interactions of L. biflexa proteins include those with components of host immune system. ! In spite of the limited availability of experimental evidence, these help in identifying functionally relevant interactions between host and pathogen by integrating multiple lines of evidence. Thus, inferences from computational prediction of host-pathogen interactions act as guidelines for experimental studies investigating the in vivo relevance of these predicted protein-protein interactions. This will further help in developing effective measures for treatment and disease prevention. In summary, Chapters 2 and 3 describe the implementation, advantages and limitations of the alignment-free full-length sequence comparison method, CLAP. Chapter 4 and 5 are dedicated to understand the domain-domain interactions in multi-domain protein sequences and structures. In Chapters 6, 7 and 8 the computational analyses of the mycobacterial species and leptospiral species helped in an enhanced understanding of the functional repertoire of these bacteria. These studies were undertaken by utilizing the biological sequence data available in public databases and implementation of powerful homology-detection techniques. The supplemental data associated with the chapters is provided in a compact disc attached with this thesis.!

Made available in DSpace on 2014-07-29T15:07:47Z (GMT). No. of bitstreams: 1 Dissertacao_Ivonete_Parreira.pdf: 517798 bytes, checksum: fd77931f9f447d09cb1f337f576bfb3d (MD5) Previous issue date: 2009-03-26 / In felines, the leptospirosis is described commonly as of rare occurrence, in spite of inquiries sorologic accomplished at several countries they have already registered positive reactions in these you encourage. In this research it was aimed at to evaluate the presence of antibodies anti-Leptospira spp in samples of serums of domestic felines originating from of the areas of Goiânia and of Aparecida from Goiânia, Goiás, Brazil, besides evaluating biochemical parameters and of investigating decisive factors associated to the infection. Sanguine serums of 330 domestic felines were analyzed seemingly healthy, being 113 of domiciled animals in different neighborhoods of Goiânia and 217 of individuals picked up to the Center of Zoonosis of Control (CCZ) of Aparecida from Goiânia. The research of antibodies anti-Leptospira spp. it was accomplished through the test of microscopic agglutination (SAM) with alive antigens. Parallel, dosagens were proceeded of range-glutamiltransferase (GGT), alkaline phosphatase (FA), urea and creatinine. The presence of agglutinative antibodies was detected in 23 animals (6,96%), with titles that varied from 1:100 to 1:800, with predominance of co-agglutinations (7,76%) and of the sorovars Cynopteri and Djasiman. In Goiânia they were identified 10 animals (8,84%) positive seruns, while in Aparecida from Goiânia 13 were detected (5,99%) positive seruns. Reactions were verified for the sorovars Australis, Bratislava, Butembo, Castellonis, Canicola, Cynopteri, Djasiman, Hebdomadis, Copenhageni, Icterohaemorrhagiae, Pomona, Pyrogenes, Hardjo, Patoc, with minimum title of 1:100. significant difference was not observed by the test of regression logistics, between the positive and biochemical alterations as well as between positive and age and sex of the animals. Biochemical alterations and age came related. The obtained results served as parameters for the evaluation of the infection in domestic felines in the study area, contributing to the elucidation of epidemic aspects and for the control of the zoonosis, seeking to reduce the risk to the human health and your impacts sanitary, social and economical. / Em felinos, a leptospirose é comumente descrita como de rara ocorrência, apesar de inquéritos sorológicos realizados em diversos países já terem registrado reações positivas nestes animais. Nesta pesquisa objetivou-se avaliar a presença de anticorpos anti-Leptospira spp em amostras de soros de felinos domésticos oriundos das regiões de Goiânia e de Aparecida de Goiânia, Goiás, Brasil, além de avaliar parâmetros bioquímicos e de investigar fatores determinantes associados à infecção. Foram analisados soros sanguíneos de 330 felinos domésticos aparentemente sadios, sendo 113 de animais domiciliados em diferentes bairros de Goiânia e 217 de indivíduos recolhidos ao Centro de Controle de Zoonoses (CCZ) de Aparecida de Goiânia. A pesquisa de anticorpos anti-Leptospira spp. foi realizada através do teste de soroaglutinação microscópica (SAM) com antígenos vivos. Paralelamente, foram procedidas dosagens de gama-glutamiltransferase (GGT), fosfatase alcalina (FA), uréia e creatinina. Detectou-se a presença de anticorpos aglutinantes em 23 animais (6,96%), com títulos que variaram de 1:100 a 1:800, com predominância de co-aglutinações (7,76%) e dos sorovares Cynopteri e Djasiman. Em Goiânia foram identificados 10 animais (8,84%) soropositivos, enquanto em Aparecida de Goiânia detectaram-se 13 (5,99%) soros reagentes. Constataram-se reações para os sorovares Australis, Bratislava, Butembo, Castellonis, Canicola, Cynopteri, Djasiman, Hebdomadis, Copenhageni, Icterohaemorrhagiae, Pomona, Pyrogenes, Hardjo, Patoc, com título mínimo de 1:100. Não foi observada diferença significativa pelo teste de regressão logística, entre a positividade e alterações bioquímicas bem como entre soropositividade e idade e sexo dos animais amostrados. Alterações bioquímicas e idade apresentaram-se significantemente relacionados. Os resultados obtidos serviram de parâmetros para a avaliação da infecção em felinos domésticos na área de estudo, contribuindo para a elucidação de aspectos epidemiológicos e para o controle da zoonose, visando reduzir o risco à saúde humana e seus impactos sanitários, sociais e econômicos

Made available in DSpace on 2014-07-29T15:13:40Z (GMT). No. of bitstreams: 1 TESE Bruno Medrado.pdf: 1863730 bytes, checksum: 142fb795d040f53ab90b6225b9c56530 (MD5) Previous issue date: 2010-04-10 / The main economic activity of the state of Tocantins, in Brazilian Amazon is cattle farms, with extensive breeding. Looking for contribution to cattle sanity, this study was devoted to the prevalence of antibodies against Leptospira in livestock of those farms, as production animals (cattle and equids), dogs and animal workers, from a rural governmental settlement in Aragominas, in the northwestern part of Tocantins. The statistically proofed sample was composed by 242 cows, 78 equids, 59 dogs and 41 animals workers, distributed in 38 small properties. All sampling was performed after informed consent, written in the case of human beings. For the diagnosis of leptospirosis, microscopic seroagglutination was performed in the Laboratório de Diagnóstico de Leptospirose do Setor de Medicina Veterinária Preventiva da Escola de Medicina Veterinária da UFG, Goiânia-GO. The seroprevalence for Leptospira spp in cattle was 76,5% [70,7% 81,7%], with serovar predominance of Hardjo (26,2%), followed by Wolffi (23,4%) Hebdomadis (14,1%), Castellonis (11,7%), Grippotyphosa (9,1%) e Pyrogenes (4,8%). In equids the seroprevalence was 79,3% [68,9% 87,4%], with agglutinins more intense to wild life serovars Castellonis (24,4%), Grippotyphosa (13,7%), Patoc (13,1%), Butembo (8,9%), Pomona (7,1%), Hardjo (6,6%), Pyrogenes (6,6%) e Wolffi (6,6%). Dogs presented seroprevalence of 30,5% [19,2 43,9], prevailing Canicola (26,3%), Hardjo (13,3%), Bratislava (10,0%) and Pyrogenes (10,0%). Human leptospirosis seroprevalence in animal workers was 31,7% [18,1%-48,1%], cwith detections of serovars Hardjo (26,3%), Grippotyphosa (15,8%), Pyrogenes (10,5%), Wolffi (10,5%), Autumnalis (10,5%) e Bratislava (10,5%). Looking for association with environmental and breeding conditions, the seroprevalence was associated in cattle to Bos indicus cattle (OR=7,51; [0,99-56,97]), in equids to the use of antihelminths (OR=7,64[0,95 61,50]) and for dogs with use for shepherd cattle (OR=4,44[1,35 14,58]). These data point to endemicity of Leptospira infection in the area and are highly suggestive of extensive environmental contamination with wildlife and production animal serovars. These results also emphasize the importance of the control of livestock leptospirosis, lowering environmental contamination and allowing better animal sanitation, with measures that could be implemented in new adequate settlements. / A exploração pecuária bovina constitui-se em uma atividade fundamental para o Estado do Tocantins, que tem sua economia pautada no agronegócio. Visando contribuir para a sanidade bovina na região, objetivou-se neste estudo determinar a prevalência de anticorpos anti-Leptospira spp. em animais de interesse econômico (bovinos e equinos), em cães e em humanos que tinham contato direto com animais, oriundos de assentamento rural do município de Aragominas, Tocantins, Brasil. A amostragem estatisticamente representativa foi constituída por 242 bovinos, 78 equídeos, 59 cães e 41 humanos, distribuídos em 38 propriedades. As colheitas de sangue dos animais e a aplicação de questionários foram realizadas após o aceite dos proprietários e, no caso dos humanos, após a leitura e assinatura do termo de consentimento livre e esclarecido. Para diagnóstico da leptospirose foi empregada a técnica de soroaglutinação microscópica (SAM) realizada no Laboratório de Diagnóstico de Leptospirose do Setor de Medicina Veterinária Preventiva da Escola de Medicina Veterinária da UFG, em Goiânia-GO. A prevalência de infecção por Leptospira spp. em bovinos foi de 76,5% [70,7% 81,7%], com predominância de anticorpos aos sorovares Hardjo (26,2%), seguido do Wolffi (23,4%), Hebdomadis (14,1%), Castellonis (11,7%), Grippotyphosa (9,1%) e Pyrogenes (4,8%); em equídeos foi de 79,3% [68,9% 87,4%], com maior detecção de aglutininas para os sorovares Castellonis (24,4%), Grippotyphosa (13,7%), Patoc (13,1%), Butembo (8,9%), Pomona (7,1%), Hardjo (6,6%), Pyrogenes (6,6%) e Wolffi (6,6%). Já em cães, foi detectada soroprevalência de 30,5% [19,2 43,9], com maiores respostas aos sorovares Canicola (26,3%), seguido Hardjo (13,3%), Bratislava (10,0%) e Pyrogenes (10,0%) e em humanos constataram-se 31,7% [18,1%-48,1%] de reagentes, com detecção de anticorpos para os sorovares Hardjo (26,3%), Grippotyphosa (15,8%), Pyrogenes (10,5%), Wolffi (10,5%), Autumnalis (10,5%) e Bratislava (10,5%). Dentre os fatores avaliados, a prevalência mostrou-se associada na espécie bovina à raça zebu (OR=7,51; [0,99-56,97]), nos equídeos ao uso de vermífugo (OR=7,64[0,95 61,50]) e para cães a lida com gado (OR=4,44[1,35 14,58]). Os resultados encontrados apontam para uma situação de endemicidade e são sugestivos de alta contaminação ambiental por sorovares que possuem como hospedeiro natural animais de produção e silvestres; evidenciando a necessidade de controle da infecção animal, com a finalidade de diminuir a contaminação ambiental e a consequente infecção em seres humanos.

Leptospira is a basal genus in an ancient group of bacteria, the spirochetes. The pathogenic species are responsible for leptospirosis, a disease with worldwide distribution and of public health importance in developed tropical countries. L. interrogans serovar Copenhageni is the agent for the majority of human leptospirosis in Brazil. In this work, we used a great variety of experimental approaches to characterize the SOS system in this serovar, to identify its impact in general DNA damage response, as well as to assess the DNA repair toolbox owned by pathogenic and saprophytic leptospires. We identified an additional repressor LexA, acquired by lateral gene transfer, exclusively in serovar Copenhageni. We also observed that UV-C irradiation led to massive death of cells and blockage of cell division in the survivors. Both repressors were active and we identified the sequences responsible for binding to promoters. However, the LexA1 SOS box was redefined after a de novo motif search on LexA1 ChIP-seq enriched sequences. This regulator was able to bind to at least 25 loci in the genome. DNA damage also caused a massive rearrangement of metabolism: increase in expression was observed in transposon and prophage genes, in addition to DNA repair pathways and mutagenesis inducers; on the other hand, motility, general metabolism and almost all virulence genes were repressed. Two induced prophages provided several proteins with useful functions. We also assessed the DNA repair-related genes presented by the three species of Leptospira: the saprophytic L. biflexa, the facultative pathogen L. interrogans and the obligatory pathogen L. borgpetersenii. There are more diversity and redundancy of repair genes in L. interrogans in comparison with the other species. Lateral gene transfer seems to be an important supplier of DNA repair functions. In addition, leptospires share characteristics of both Gram-positives and Gram-negatives bacteria. Representative genes from several different pathways were induced during infection of susceptible mice kidneys, suggesting DNA repair genes are active while causing disease. All these data suggest mobile genetic elements are the major forces in leptospiral evolution. Moreover, during DNA damage response, several SOS-dependent and independent mechanisms are employed to decrease cell growth and virulence in favor of controlled induction of mechanisms involved in genetic variability. / Leptospira é um gênero basal em um grupo já considerado um dos mais ancestrais, as espiroquetas. As espécies patogênicas são responsáveis pela leptospirose, uma doença presente em todo o mundo e de principal importância em países tropicais em desenvolvimento. L. interrogans sorovar Copenhageni é o agente da maior parte dos casos no Brasil. Nesse trabalho, utilizamos diversas abordagens experimentais para caracterizar o sistema SOS nesse sorovar, identificar seu impacto na resposta geral a danos no DNA, assim como avaliar as funções de reparo de DNA disponíveis em leptospiras patogênicas e saprofíticas. Identificamos um repressor LexA adicional, adquirido por transferência horizontal e exclusivo do sorovar Copenhageni. Observamos também que irradiação por UV-C causou significativa morte celular e bloqueio da divisão celular dos sobreviventes. Ambos os repressores são ativos e identificamos as sequências que utilizam para se ligar aos promotores dos genes regulados. Entretanto, o SOS box de LexA1 foi redefinido após uma busca de novo por motivos enriquecidos nas sequências recuperadas por ChIP-seq. Esse regulador ligou-se ao menos a 25 locais do genoma. A maioria desses alvos teve aumento de expressão após UV-C. Danos no DNA também causaram um importante rearranjo metabólico: houve aumento de expressão em transposons e profagos, além de indutores de mutagênese e vias de reparo; por outro lado, mobilidade, crescimento celular e quase todos os fatores de virulência foram reprimidos. Dois profagos induzidos durante essa resposta, possivelmente proporcionam algumas proteínas de funções importantes. Nós também avaliamos a presença de genes envolvidos no reparo de DNA em três espécies de leptospira: L. biflexa, L. interrogans e L. borgpetersenii. L. interrogans é a espécie com maior diversidade e redundância de genes de reparo. Além disso, transferência horizontal parece ser um importante fornecedor de funções de reparo nesse gênero. Leptospiras também apresentam genes característicos tanto de bactérias Gram-positivas quanto Gram-negativas. Genes representando diferentes vias de reparo foram induzidos durante infecção em modelo animal, sugerindo que essas vias estão ativas no curso da doença. Todos esses dados, em conjunto, sugerem que elementos genéticos móveis são de extrema importância na evolução do gênero e das vias de reparo. Assim, durante a resposta a danos no DNA, diversos mecanismos dependentes e independentes de SOS são empregados para frear o crescimento celular e virulência em favor da indução controlada de mecanismos para aumentar variabilidade genética.

Leptospirose é uma zoonose causada pela espiroqueta pertencente ao gênero Leptospira. LipL32 é um antígeno de superfície altamente conservado somente entre as espécies de leptospiras patogênicas e é expresso em altos níveis tanto in vitro com in vivo. HlyX é relatada como sendo uma proteína que possui um provável peptídeo sinal e cinco tetratricopeptídeos repetidos (TPR) em sua sequência de aminoácidos. Neste trabalho, mostrou-se que HlyX é expressa somente em cepas patogênicas, não sendo detectada a sua expressão na cepa saprofítica. HlyX foi reconhecida somente por soros de pacientes da fase convalescente da doença. Em constraste, LipL32 foi reconhecida por soros de pacientes colhidos tanto na fase aguda quanto na fase convalescente da infecção. Nossos resultados de immunoblot indicam que os domínios imunodominantes da proteína são os fragmentos C-terminal e intermediário. Uma resposta IgM foi detectada exclusivamente contra o fragmento C-terminal de LipL32 em ambas as fases da infecção. Com relação à capacidade de LipL32 e HlyX de interagir com componentes de matriz extracelular (CME), foi observada uma interação específica e dose-dependente de LipL32 e HlyX com colágeno tipo IV e fibronectina plasmática. O fragmento C-terminal de LipL32 é responsável por esta interação. Tanto a heparina quanto a gelatina foram capazes de inibir a ligação de LipL32 à fibronectina plasmática de forma dose-dependente, indicando que os domínios de ligação à heparina (30 kDa) e gelatina (45 kDa) da fibronectina estão envolvidos nesta interação. Por outro lado, apenas o domínio de ligação à heparina participa da interação da fibronectina com a proteína HlyX. A capacidade protetora das duas proteínas estudadas foi avaliada através de ensaios de imunização e desafio realizados em modelo animal (hamsters). A proteína HlyX induziu altos títulos de anticorpos IgG (1:128.000), mas somente a co-administração HlyX e LipL32 e a proteína LipL32 pura conferiram proteção, 100% e 80% respectivamente. HlyX não foi capaz de conferir proteção quando administrada apenas com o adjuvante Al(OH)3. Em conclusão, os resultados indicam que o domínio C-terminal de LipL32 é reconhecido desde o início da infecção e este domínio é responsável por mediar a interação de LipL32 com CME. Os dados obtidos com HlyX demonstram um possível papel desta proteína na patogênese, pelo fato de ser expressa e conservada em cepas patogênicas, e também por interagir com CME. Porém, apesar de HlyX apresentar altos títulos de anticorpos IgG, não conferiu atividade protetora quando administrada individualmente. / Leptospirosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as na important emerging infectious disease. LipL32 is a surface lipoprotein which is highly conserved among pathogenic Leptospira species and is also expressed at high levels either during cultivation and natural infection. Regarding HlyX, it has been annotated as a protein containing a signal peptide and five tetratricopeptide repeats (TPR). Immunoblot analyses concerning HlyX distribution on Leptospira spp. indicate that this protein is expressed exclusively by pathogenic species. Moreover, HlyX was only recognized by sera of patients in the second week of leptospirosis infection. In contrast, LipL32 was recognized by acute and convalescent sera from leptospirosis patients. Our immunoblot results indicate that both the C-terminal and the intermediate domains of LipL32 are recognized by sera of patients. An IgM response was detected exclusively against the LipL32 C-terminus in both the acute and convalescent phases of illness. Concerning the capacity of LipL32 and HlyX to interact with extracellular matrix (ECM) components, a dose-dependent specific binding of LipL32 and HlyX to collagen IV and plasma fibronectin was observed. The LipL32 binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin- and the 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. However, HlyX binding to fibronectin could only be inhibited by heparin in a concentration-dependent manner. We also evaluated whether HlyX and LipL32 could induce protective immunity against the challenge with a homologous serovar in hamsters. Although high anti-HlyX (IgG) titers (1:128,000) have been achieved upon immunization, no protection was observed. However, a combined HlyX and LipL32 immunization could induce a protective response (100%). The protection observed for LipL32 immunization was 80%. Altogether, the results provide evidence that the LipL32 C-terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins. HlyX protein may contribute to the pathogenesis of the disease by interacting with host proteins. However, HlyX is not a protective antigen when administered alone.

La leptospirose est une zoonose d’importance médicale majeure dans les îles du Sud-Ouest de l’Océan Indien (SOOI) dont certaines enregistrent des incidences parmi les plus élevées au monde. Durant la dernière décennie, les données épidémiologiques moléculaires obtenues avec une approche « One Health » ont mis en évidence une grande diversité de lignées de leptospires ainsi que différentes chaines de transmission sur les différentes îles de la région. Les données moléculaires montrent la présence de leptospires pathogènes et de réservoirs animaux introduits ou endémiques de cette région. La distribution de ces différentes lignées de leptospires est associée à (i) un contraste épidémiologique incluant des différences dans la sévérité des cas humains et (ii) des niveaux de spécificité d’hôtes différents selon les leptospires considérés. Plus particulièrement, les leptospires endémiques du SOOI semblent être moins pathogènes chez les humains et montrent une plus forte affinité pour leur réservoir que les leptospires cosmopolites. Pour compléter nos connaissances sur l’histoire évolutive des leptospires du SOOI, nous avons produit des données provenant de chauves-souris de l’Afrique de l’Est. Ces données confirment la spécificité de certaines lignées de leptospires envers leurs hôtes chiroptères et suggèrent que les chauves-souris d’Afrique ont colonisé Madagascar tout en étant infectées par leurs leptospires. Afin de mieux comprendre le rôle des différents leptospires dans l’épidémiologie régionale de la leptospirose, nous avons mesuré la pathogénicité de trois souches de leptospires retrouvées dans cette région à l’aide d’un modèle hamster. Des souches de Leptospira mayottensis et Leptospira borgpetersenii ont été isolés respectivement de Tenrec ecaudatus (tenrec) et Triaenops menamena (chauve-souris), deux mammifères endémiques du SOOI. Une souche de Leptospira interrogans, dont le génotype est retrouvé dans la majorité des cas humains graves à la Réunion, a été isolée de Rattus rattus (rat). En cohérence avec les données épidémiologiques humaines de Mayotte et de La Réunion, les leptospires endémiques se sont révélées être significativement moins pathogènes que la souche L. interrogans. La spécificité d’hôte des deux souches isolées de mammifères endémiques a été mise à l’épreuve par des infections expérimentales de Rattus norvegicus, connu comme un réservoir important de leptospires. Les rats ont été infectés avec les trois isolats précédemment utilisés. Les rats infectés par les souches endémiques n’ont pas développé d’infection rénale chronique contrairement à la souche cosmopolite. Ces résultats montrent que la spécificité d’hôte des leptospires endémiques observée in natura est probablement due à des facteurs génétiques plutôt qu’à des facteurs écologiques, comme un manque de contacts physiques entre les réservoirs animaux endémiques et introduits. Enfin, le séquençage complet de souches de leptospires du SOOI a été réalisé afin d’identifier des caractéristiques génétiques pouvant être associées à la pathogénicité et la spécificité d’hôte des leptospires pathogènes. Une classification précise de souches de leptospires du SOOI a pu être réalisée sur la base des génomes complets. La comparaison de ces génomes a permis d’identifier des gènes spécifiques à un groupe ou une espèce de leptospires. Cependant des modifications génomiques complexes rendent difficiles l’identification de caractéristiques génomiques responsables d’un phénotype particulier tel que la virulence ou la spécificité d’hôte. / Leptospirosis is a zoonosis of main medical concern on several islands of southwestern Indian Ocean (SWIO), some of which recording among the highest human incidence worldwide. Over the last decade, molecular epidemiology investigations carried out under a One Health framework have revealed a wide variety of Leptospira lineages and distinct transmission chains throughout the islands of the region. These islands are home to pathogenic Leptospira lineages and animal reservoirs that are either introduced or endemic to the SWIO region. Interestingly, the regional distribution of Leptospira diversity is associated with (i) a contrasted severity of human cases and (ii) distinct levels of specificity of Leptospira towards their mammalian hosts. Specifically, endemic Leptospira appear less pathogenic in humans and display higher specificity towards their animal reservoirs than their cosmopolitan counterparts. To complete the dataset of Leptospira diversity in the SWIO region, we produced data from bats of eastern Africa. Results support the previously observed pattern of host specificity of Leptospira towards their bats hosts and, overlaid upon the biogeographic history of Malagasy bats, suggest that these volant mammals have colonized Madagascar from continental Africa while hosting pathogenic Leptospira. To better understand the role of distinct Leptospira lineages in the contrasted epidemiology observed in the SWIO, we investigated the pathogenicity of three Leptospira isolates from this region using a hamster model. Leptospira mayottensis and Leptospira borgpetersenii isolates were obtained from Tenrec ecaudatus (tenrec) on Mayotte and Triaenops menamena (bat) in Madagascar, respectively, both mammals endemic to the SWIO region. A Leptospira interrogans strain, which genotype has been reported in the majority of human acute cases on La Réunion, was isolated from the introduced Rattus rattus (rat). In keeping with a distinct severity of the disease on Mayotte and La Réunion, endemic bat-borne and tenrec-borne Leptospira were significantly less pathogenic than the control cosmopolitan rat-borne isolate. The host specificity of the isolates obtained from endemic hosts was addressed using experimental infection of Rattus norvegicus, a known reservoir of pathogenic Leptospira. This animal model was challenged with all three isolates and mostly failed in supporting chronic infection with bat-borne and tenrec-borne Leptospira. Hence, the strong host-specificity of endemic Leptospira toward their hosts observed in the wild likely results from genetic determinants shaped by long-term co-evolutionary processes rather than from ecological constraints such as a lack of physical contact between introduced and endemic animal reservoirs. Finally, we undertook full genome sequencing of regional strains in order to highlight genomic features that may be associated with virulence and host specificity. Whole genome sequencing allowed the accurate classification of Leptospira isolates obtained on SWIO islands. Comparative genomics allowed to identify genes specific to a group or species of Leptospira but complex changes in Leptospira genome make difficult the identification of genomic elements responsible for specific traits such as virulence and host specificity.