Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry, Durban University of Technology, Durban, South Africa, 2017. / In drug discovery and development of anti-tubercular therapeutics, it is necessary to study the physiology and genetics of the molecular mechanisms present in the Mycobacterium tuberculosis. The virulence of M. tuberculosis is attributed to its unique genome, which contains a high frequency of glycine-rich proteins and genes involved in the metabolism of the fatty acids. Consequently, the presence of a diversity of the pathogenic pathways such as acid tolerance and drug resistance mechanisms in M. tuberculosis makes the treatment of Tuberculosis (TB) challenging. However, the molecular basis of the virulence factors involved in the pathogenesis is not fully understood. Accordingly, the current study focuses on better understanding of the pathogenic proteins present in this bacterium using available computational techniques. In South Africa, there is an alarming increase in the drug-resistant TB in HIV co-infected patients, which is one of the biggest challenges to the current anti-tubercular therapies. An extensive literature search showed that the mutations in the virulent proteins of M. tuberculosis resulted in the development of drug tolerance in the pathogen. The molecular and genetic studies identified frequently occurring point mutations associated with the drug resistance in proteins of M. tuberculosis. Despite the efforts, TB infection is still increasing because different pathogenic pathways in the bacterial system are still undiscovered. Therefore, this study involves an in silico approach aimed at the identification of novel drug resistance implicated point mutations. The site- directed mutations leading to the development of resistance against four first-line drugs (Ethambutol, Isoniazid, Rifampicin, and Streptomycin) were studied extensively. In the primary investigation, pathogenic mutational landscapes were classified in the sequences of the studied proteins. The effects of these mutations on the stability of the proteins were studied using diverse computational techniques. The structural basis of the point mutations with the highest destabilizing effects was analyzed using the principles of the Density Functional Theory (DFT), molecular docking and molecular dynamics (MD) simulation studies. The varied conformational behavior resulted from these predicted substitutions were compared with the experimentally derived mutations reported in the literature. The outcome of this study enabled the identification of the novel drug resistance-associated point mutations which were not previously reported. Furthermore, a detailed understanding of the conformational behavior of diverse virulent proteins present in M. tuberculosis was also generated in this study. Literature study showed that inside the host’s macrophage cells, the virulent proteins such as isocitrate lyase, lipase lipF, magnesium transporter MgtC, porin protein OmpATb, a protein of two component systems PhoP, Rv2136c and Rv3671c have an established role in the development of the acid tolerance. On the other hand, information regarding their role in the acid resistance is scarce. Accordingly, the structural basis of their role in acid resistance was analyzed using constant pH based MD simulations. In the studied proteins, the lipF and PhoP showed highest structural stability in highly acidic conditions throughout the course of MD simulations. Therefore, these proteins may play a primary role in the process of resistance. In addition to these pathogenic proteins, there is a need to identify new undiscovered virulent proteins in the genome of M. tuberculosis, which increases the efficiency of the current therapy. The knowledge generated by the analyses of the proteins involved in resistance and pathogenic mechanisms of M. tuberculosis forms the basis for the identification of new virulence factors. Therefore, an in silico protocol was used for the functional annotations and analyses of the virulence characteristics. M. tuberculosis contains 1000 Hypothetical Proteins (HPs), which are functionally uncharacterized proteins and their existence was not validated at the biochemical level. In this study, the sequences of the HPs were extensively analyzed and the functions of 662 HPs were successfully predicted. Furthermore, 483 HPs were classified in the category of the enzymes, 141 HPs were predicted to be involved in the diverse cellular mechanisms and 38 HPs may function as transporters and carriers proteins. The 307 HPs among this group of proteins were less precisely predicted because of the unavailability of the reliable functional homologs. An assessment of the virulence characteristics associated with the 1000 HPs enabled the classification of 28 virulent HPs. The structure of six HPs with highest predicted virulence score was analyzed using molecular modelling techniques. Amongst the predicted virulent HPs, the clone for Rv3906c purchased from the DNASU repository because of the ease of its availability. The gene of Rv3906c was isolated and cloned into a pET-21c expression vector. The analyses of the nucleotide sequence showed that Rv3906c gene (500 bp) encodes a 169 amino acid protein of molecular weight 17.80 kDa (~18.0 kDa). The sequence analyses of Rv3906c showed that the HPs showed high similarities with pullulanase, a thermophilic enzyme. The stability profile at different temperatures for Rv3906c generated using MD simulations showed that Rv3906c maintained its structural identity at higher temperatures. It is expected that this study will result in the design of better therapeutic against the infection of M. tuberculosis, as novel undiscovered virulence factors were classified and analyzed in addition to the conformational profiles of the virulent proteins involved in the resistance mechanisms. / M

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2015-11-18T13:53:39Z No. of bitstreams: 1 texto completo.pdf: 1408584 bytes, checksum: 93c92bfb4c0654bc0272f8ef82b674ea (MD5) / Made available in DSpace on 2015-11-18T13:53:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1408584 bytes, checksum: 93c92bfb4c0654bc0272f8ef82b674ea (MD5) Previous issue date: 2015-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mycobacterium avium ssp. paratuberculosis (MAP) é o agente etiológico da doença de Johne ou paratuberculose, enterite granulomatosa crônica caracterizada por diarreia persistente e perda de peso progressiva que acomete ruminantes. Pode também ser isolado a partir de amostras intestinais de pacientes humanos, com doenças intestinais, principalmente portadores da doença de Crohn. Essa é uma doença de etiologia desconhecida, que se caracteriza por inflamação crônica, focal, assimétrica transmural e ocasionalmente granuloma- tosa, que pode acometer qualquer segmento do tubo digestivo. O isolamento de MAP e as semelhanças entre os processos clínicos e histopatológicos da paratuberculose e da doença de Crohn são algumas das razões pelas quais se investiga o potencial zoonótico da bactéria. As principais vias de eliminação do agente são através do leite e das fezes que contaminam os pastos e, direta ou indiretamente os cursos d’agua podendo dessa forma infectar humanos pela ingestão de água contendo o micro-organismo viável. MAP é uma bactéria resistente e responsável por grandes prejuízos econômicos e produtivos, sendo demonstrada sua sobrevivência no ambiente por longos períodos, além da resistência a pasteurização e à agentes de desinfecção aplicados ao tratamento da água para consumo humano. Diante disso, o estudo teve como objetivos: - identificar fatores de risco envolvidos com a presença de MAP em amostras de intestino humano, através da aplicação de questionário, em conjunto com da- dos prévios sobre a presença da bactéria em amostras de biópsias intestinais de pacientes portadores de Doença de Crohn, retocolite ulcerativa e portadores de doenças intestinais não inflamatórias, -verificar a presença do agente na água para consumo humano e animal através de PCR convencional e do cultivo microbiológico de amostras coletados em 10 propriedades de caprinocultura leiteira da Zona da Mata Mineira e realizar uma revisão bibliográfica dos estudos sobre a paratuberculose na América Latina. Quanto às amostras de água, MAP foi identificado viável em quatro (40%) das amostras de consumo animal, e identificado por PCR em três (30%) das de consumo humano além de uma quinta amostra de consumo animal (10%). Esse resultado demonstra o papel da água como reservatório do agente, mantendo o ciclo de infecção ativo e servindo de amostra confiável para o diagnóstico da presença do agente no rebanho já que, aparentemente não está condicionada a eliminação intermitente, como ocorre com as fezes desses animais. Nesse estudo também puderam ser identificados fatores de risco para a ocorrência do agente na água e em biópsias intestinais humanas, como o consumo de leite e derivados informais, assim como histórico familiar de agravos intestinais. Na América Latina MAP foi pesquisado em 10 países e identificado em nove, infectando, bovinos, caprinos e animais silvestres. Os resultados desse estudo contribuem para a identificação de fatores de risco envolvidos na transmissão de MAP para humanos, permitindo a sugestão de medidas que previnam ou reduzam a exposição ao agente. Esses fatores de risco identificados também demonstram a importância do leite na veiculação do agente por leite e produtos lácteos, com destaque para aqueles que não passaram pela pasteurização. Além disso, os estudos sobre água auxiliaram a elucidação do papel da ingestão da água na transmissão do agente, que não é pesquisado na rotina de tratamento, indicando exposição ao agente para humanos pode ser dar através do consumo de água contaminada por fezes de animais com paratuberculose. Esse estudo foi o primeiro sobre o agente na água no Brasil, e um dos poucos no mundo. / Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease or paratuberculosis, a chronic granulomatous enteritis characterized by persistent diarrhea and progressive weight loss that may affects ruminants. MAP is also be isolated from intestinal samples from human patients with intestinal diseases, particularly Crohn's disease patients. This is a disease of unknown etiology that is characterized by chronic inflammation, focal, transmural asymmetric and occasionally granulomatous lesions, which can affect any segment of the digestive tract. The isolation of MAP and the similarities between the clinical and pathologic processes of paratuberculosis and Crohn's disease are some of the reasons for investigating the zoonotic potential of bacteria. The main agent’s disposal routes are through feces and milk that contaminate pastures and directly or indirectly the watercourses and can thus infect humans by drinking water containing viable microorganism. MAP is a resistant bacteria and responsible for significant economic and productive loss, and demonstrated its survival in the environment for long periods, in addition to the resistance to pasteurization and disinfection agents applied to water treatment for human consumption. Thus, the study aimed to: - identify risk factors involved with the presence of MAP in human gut samples, through the questionnaire, together with previous data on the presence of bacteria in samples of intestinal biopsies of patients Crohn's disease, ulcerative colitis and patients with non- inflammatory intestinal diseases, -check the agent's presence in the water for human and animal consumption by conventional PCR and microbiological culture samples collected in 10 properties of dairy goat of the Zona da Mata Mineira and,- conduct a literature review of the studies on paratuberculosis in Latin America. As for the water samples, MAP was identified feasible in four (40%) of the samples of animal feed, and identified by PCR in three (30%) of human consumption as well as a fifth sample of animal consumption (10%). This result demonstrates the role of water as an agent of the reservoir, keeping the active infection cycle and serving as a reliable sample for the diagnosis of the agent's presence in the herd since apparently is not subject to intermittent shedding, as with the feces of these animals. In this study could also be identified risk factors for the occurrence of the agent in in human intestinal biopsies, as the consumption of unpasteurized milk and informal derivatives as well as family history of bowel diseases. In Latin America MAP was investigated in 10 countries and identified in nine, infecting, cattle, goats and wild animals. The results of this study contribute to the identification of risk factors involved in the MAP transmission to humans, allowing the suggestion of measures to prevent or reduce exposure. These identified risk factors also demonstrate the importance of milk in placement agent for milk and milk products, especially those who have not gone through the pasteurization. Furthermore, studies on water helped to elucidate the role of water ingestion in the transmission of the agent, which is not searched in routine treatment, indicating exposure to the agent for humans can occur through the consumption of water contaminated by faeces of animals carrying paratuberculosis. This study was the first about the agent in water in Brazil, and one of the few in the world.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18161号 / 医博第3881号 / 新制||医||1003(附属図書館) / 31019 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 杉田 昌彦, 教授 中山 健夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

To propose a solution for control of Mycobacterium avium subsp. paratuberculosis (MAP) infections in animals as well as in humans, and develop effective prevention, diagnostic and treatment strategies, it is essential to understand the molecular mechanisms of MAP pathogenesis. In the present review, we discuss the mechanisms utilised by MAP to overcome the host defense system to achieve the virulence status. Putative MAP virulence genes are mentioned and their probable roles in view of other mycobacteria are discussed. This review provides information on MAP strain diversity, putative MAP virulence factors and highlights the knowledge gaps regarding MAP virulence mechanisms that may be important in control and prevention of paratuberculosis.

Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease mainly of domestic and wild ruminants. It occurs worldwide, causing significant economic losses through decreased productivity, low fertility, increased cull rates and mortality. It is listed by the OIE (World Organization for Animal Health) as a disease of concern to trade in animals. Prevalence of this disease can be studied by detecting anti-MAP antibodies by Enzyme linked immunosorbent Assay (ELISA). The aim of this study was to investigate the current prevalence of MAP infection in cattle in Khartoum State. The overall apparent prevalence of MAP infection was found to be 6.3% and 18.9% at animal and herd levels, respectively. All seropositive animals were cross-bred females of good body condition; most of them (>90%) were >3 years old and >50% were from medium-sized herds in Omdurman. No significant association (p > 0.05) was found between seropositivity and animal herd size. The prevalence of MAP infection in Khartoum State is still low to medium compared to other parts of the world, but it is comparable to those reported from other African countries. Further studies with the view of designing nationwide surveys in domestic ruminants and camels in other states of the country are needed for establishing control programmes.

No description available.

No description available.

Mycobacterium avium ssp. paratuberculosis (Map) est responsable de la maladie de Johne chez les ruminants en particulier chez le bovin. Les scientifiques ont émis l'hypothèse que Map pourrait être l'agent étiologique de la maladie de Crohn chez l'humain, ce qui a soulevé des inquiétudes en santé publique. Map est particulièrement résistant à la chaleur dans le lait, mais l'état des connaissances est pratiquement limité dans les matrices de viande. Les valeurs D (temps de réduction décimal) et z (sensibilité à la température) ont été déterminées dans du boeuf haché préparé aseptiquement à partir du muscle semi membranosus pour Map, Escherichia coli et Enterococcus faecalis. L'objectif de ce travail est de déterminer l'effet de divers traitements thermiques sur la cinétique de destruction de Map dans la viande afin d'évaluer si les procédés thermiques industriels (la pasteurisation, cuisson) sont adéquats pour contrôler la Map dans les produits de viande.

Mycobacterium avium and Mycobacterium intracellulare, (M. avium complex, MAC) are human pathogens causing disease in individuals with acquired immunodeficiency syndrome (AIDS) or with thoracic abnormalities. MAC bacteria are difficult to kill because of the resistance of the pathogens to chemotherapeutic agents. One factor affecting treatment of MAC disease is the presence of interconvertible colonial variants. Transparent (T) variants have greater resistance to antibiotics and higher pathogenicity; opaque (O) variants are more susceptible to antibiotics and less pathogenic. The overall goal of this study was to investigate the mechanism for colonial variation. Based on an observation that T variants of M. intracellulare strain Va14 contained a plasmid which was 6 kb smaller than the 68 kb plasmid in O variants, it had been suggested that a transposable element might be responsible for colonial variation. The first objective was to clone the unique DNA fragment present in the 68 kb plasmid but absent from the 62 kb plasmid. The second and third objectives were to determine if the unique fragment contained a transposable element and to analyze the role of that element in the mechanism of colonial variation in M. intracellulare strain Va14. The fourth objective was to determine the distribution of IS1141 in MAC isolates. Fragments containing copies of the putative element were sequenced and a region 1596 basepairs in length with 23 basepair imperfect inverted repeats was designated as insertion sequence IS1141. IS1141 is the first insertion sequence identified in M. intracellulare. Data base searches using open reading frames (ORF) of IS1141, identified ORFb as significantly similar to the transposases of the IS3 family. The presence or absence of IS1141 in strain Va14 plasmids appeared unrelated to colonial variation, but IS1141 was present in another plasmid and the chromosome of the Va14 variants. Hybridization studies with IS1141 identified three chromosomal copies in O variants and two chromosomal copies in T variants. Va14 T variants each had a common IS1141 restriction fragment length polymorphism (RFLP) pattern which was different than the single RFLP pattern found in opaque variants. Based on these differences, it appears that IS1141 may integrate into the gene(s) responsible for the T phenotype preventing their expression. A survey of 64 James River basin non-AIDS, clinical and James River environmental MAC isolates identified 4 of 24 (17%) M. intracellulare isolates as containing IS1141. IS1141 has not been detected in any clinical or environmental M. avium or Mycobacterium species X isolates and may be limited to M. intracellulare. / Ph. D.

La malattia di Johne o paratubercolosi è un’enterite cronica granulomatosa provocata dal Mycobacterium avium subsp paratubercolosis (MAP), che colpisce i ruminanti ed in particolare i bovini da latte ed ha un grande impatto economico a livello mondiale. Il MAP sembra anche avere un ruolo nella malattia umana di Crohn. Tale patogeno è capace di sopravvivere molto bene all’interno dei macrofagi dell’ospite dove previene la loro attivazione, blocca l’acidificazione e la maturazione del fagosoma, e interferisce con la presentazione degli antigeni al sistema immunitario. Al fine di analizzare la complessa interazione tra l’ospite e il patogeno, è stata valutata la risposta dopo 2h, 6h, e 24h di macrofagi derivati da monociti bovini (MDM), coltivati in vitro e infettati con il ceppo L1 di MAP utilizzando un approccio di RNA-Seq. L’analisi statistica dei dati di sequenza ha mostrato un aumento del numero di geni differenzialmente espressi durante l’esperimento in risposta all’infezione. Inoltre i geni sottoespressi negli MDM infettati sono stati individuati solo a 24h post-infezione. L’analisi dei pathway ha evidenziato tre network che sono associati alla risposta immunitaria e al processo infiammatorio. Inoltre lo studio dei geni sottoespressi a 24h ha mostrato il ruolo centrale del complemento e del complesso maggiore di istocompatibilità nella patogenesi della malattia. / Johne’s disease (paratuberculosis) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp paratubercolosis (MAP), affecting ruminants worldwide with a significant economic impact. MAP has also been speculated as a cause of human Crohn’s disease. MAP is a pathogen highly adapted for survival within host macrophages due to the organism's capacity to prevent macrophage activation, block phagosome acidification and maturation, and attenuate presentation of antigens to the immune system. The consequence is a very long silent infection and subclinical phases. To decipher the complex interaction between host and MAP, the response of in vitro bovine monocyte-derived macrophages (MDM) after 2h, 6h and 24h of infection with L1 strain of MAP was explored using RNA-Seq approach. Statistical analysis of sequence data revealed an increasing number of differentially expressed genes in MDM following infection through the three time points analysed. Furthermore down-regulated genes were only found at 24 h post-infection. Ingenuity Pathways Analysis of differentially expressed genes showed that “cell-mediated immune response” was the most significant network related to 2hpi dataset, “immune cell trafficking” for 6hpi, and “inflammatory response” for 24hpi. Finally the analysis of down-regulated genes at 24hpi confirmed the role of complement and major histocompatibility complex (MHC) in the pathogenesis of MAP in cattle.