A carregar...
Publicação
Estudo da ilha de patogenicidade Meningococcal disease associated (MDA) como potencial factor de virulência de Neisseria meningitidis
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 1.44 MB | Adobe PDF |
Resumo(s)
O sistema de classificação de meningococos com base em multilocus sequence typing (MLST) evidencia que a grande maioria dos casos de doença meningocócica é causada por um número limitado de estirpes geneticamente relacionadas, designadas por hipervirulentas (HV). As estruturas celulares que promovem a colonização e a doença invasiva existem dispersas na população bacteriana, independentemente da sua capacidade de causar doença. Haverá então características, ainda não completamente compreendidas, que contribuem para a virulência de algumas estirpes de meningococos. A razão para uma maior virulência de algumas estirpes começou recentemente a ser melhor compreendida com os estudos de Bille (2005), a partir da sequenciação do genoma completo de estirpes invasivas de Neisseria meningitidis. Identificaram um novo profago da família de bacteriófagos filamentosos (M13-like) integrado no cromossoma bacteriano e especificamente presente em estirpes hipervirulentas, uma ilha de patogenicidade com 8-kb a que chamaram MDA (Meningococcal Disease-Associated). Existem múltiplos sítios de inserção no cromossoma e múltiplas cópias do fago no genoma. O papel do profago, nomeadamente a sua relação com a célula hospedeira, continua por ser compreendida. Neste trabalho estudou-se um conjunto de 170 estirpes invasivas e oito estirpes respiratórias de Neisseria meningitidis da colecção do laboratório nacional de referência (LNR) de Neisseria meningitidis do Instituto Nacional de Saúde Dr. Ricardo Jorge, fazendo a pesquisa da presença da ilha MDA por PCR e estimando o número de cópias da ilha em estirpes de diferentes complexos clonais. Os resultados obtidos mostraram uma relação entre as estirpes pertencentes a complexos clonais HV e a presença da ilha MDA. Esta relação não foi evidente em estirpes de complexos clonais não HV, já que metade destes apresentava ilha e a outra metade não apresentava. O número de cópias da ilha MDA foi estimado pela razão normalizada entre o cycle threshold (CT) obtido na reacção de PCR em tempo real usada para amplificação de uma região do DNA fágico (região C) e o CT obtido na reacção de PCR em tempo real usado para amplificação de um gene housekeeping (o gene abcZ). Estimou-se que as estirpes dos complexos clonais ST-8 e ST-11 têm um maior número de cópias do que as restantes. Iniciou-se a técnica de Southern blot para a determinação exacta do número de cópias de DNA fágico presente no genoma bacteriano. Contudo, esta técnica exige ser optimizada para se obterem resultados conclusivos. Uma vez que as estirpes dos complexos clonais ST-8 e ST-11 são reconhecidamente as que têm uma maior capacidade de invasão depois do contágio, isto é, são as mais virulentas, o trabalho realizado indicia uma relação entre o número de cópias da ilha genómica MDA e a virulência de meningococos.
The classification system of meningococcus based on multilocus sequence typing (MLST) emphasizes that the great majority of the cases of meningococcal disease are caused by a limited number of genetically related strains, designated hypervirulent (HV).The cellular structures that promote the colonization and the invasive disease exist dispersed in the bacterial population, independently of its capacity of causing disease. There are characteristics not yet fully understood that contribute to the virulence of some meningococcal strains. The reason for the higher virulence of some strains recently begun to be understood with the studies of Bille (2005) from the complete genome sequencing of invasive strains of Neisseria meningitidis. A new prophage of the filamentous bacteriophages family was identified (M13-like) integrated in the bacterial chromosome and specifically present in hypervirulent strains, one island of pathogenicity with 8 kb, which was called MDA (Meningoccocal Disease Associated).There are multiple insertion sites in the chromosome, and multiple copies of the phage in the genome. The role of the prophage, in particular its relation with the host cell, is not yet fully understood. In this work, a sample of invasive strains of Neisseria meningitidis was studied of the collection of the LNR ("Laboratório Nacional de Referência") of the "Instituto Nacional de Saúde Doutor Ricardo Jorge", a search of the MDA island was performed using PCR, and estimating the number of copies of the island in strains from different clonal complexes. The obtained results have shown a relation between the strains belonging to hypervirulent clonal complexes and the presence of the MDA island. This relation was not seen when the non-hypervirulent clonal complexes were studied, as only about half of them exhibited island. The number of copies of the MDA island was estimated by the normalized ratio between the cycle threshold (CT) obtained in the real time PCR used to amplify one gene housekeeping. It was estimated that the clonal complex strains ST-8 and ST-11 have more copies than the rest. The Southern Blot technique was initiated to determine the exact number of copies of phage DNA present in the bacterial genome. However, to obtain conclusive results this technique should be optimized. Since strains from the clonal complex ST-8 and ST-11 are those which have a higher invasion capacity after acquisition, that is, they are the most virulent, the work performed points to a relation between the number of copies of the genomic island MDA and the meningococcal virulence.
The classification system of meningococcus based on multilocus sequence typing (MLST) emphasizes that the great majority of the cases of meningococcal disease are caused by a limited number of genetically related strains, designated hypervirulent (HV).The cellular structures that promote the colonization and the invasive disease exist dispersed in the bacterial population, independently of its capacity of causing disease. There are characteristics not yet fully understood that contribute to the virulence of some meningococcal strains. The reason for the higher virulence of some strains recently begun to be understood with the studies of Bille (2005) from the complete genome sequencing of invasive strains of Neisseria meningitidis. A new prophage of the filamentous bacteriophages family was identified (M13-like) integrated in the bacterial chromosome and specifically present in hypervirulent strains, one island of pathogenicity with 8 kb, which was called MDA (Meningoccocal Disease Associated).There are multiple insertion sites in the chromosome, and multiple copies of the phage in the genome. The role of the prophage, in particular its relation with the host cell, is not yet fully understood. In this work, a sample of invasive strains of Neisseria meningitidis was studied of the collection of the LNR ("Laboratório Nacional de Referência") of the "Instituto Nacional de Saúde Doutor Ricardo Jorge", a search of the MDA island was performed using PCR, and estimating the number of copies of the island in strains from different clonal complexes. The obtained results have shown a relation between the strains belonging to hypervirulent clonal complexes and the presence of the MDA island. This relation was not seen when the non-hypervirulent clonal complexes were studied, as only about half of them exhibited island. The number of copies of the MDA island was estimated by the normalized ratio between the cycle threshold (CT) obtained in the real time PCR used to amplify one gene housekeeping. It was estimated that the clonal complex strains ST-8 and ST-11 have more copies than the rest. The Southern Blot technique was initiated to determine the exact number of copies of phage DNA present in the bacterial genome. However, to obtain conclusive results this technique should be optimized. Since strains from the clonal complex ST-8 and ST-11 are those which have a higher invasion capacity after acquisition, that is, they are the most virulent, the work performed points to a relation between the number of copies of the genomic island MDA and the meningococcal virulence.
Descrição
Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012
Palavras-chave
Virulência Bacteriologia Microbiologia molecular Teses de mestrado - 2012