Evaluación de la plasticidad genética en Salmonella : estudio de la escisión y pérdida de una isla de patogenicidad inestable (ROD21) en Salmonella enterica serovar Enteritidis
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2010
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Salmonella enterica serovar Enteritidis (S Enteritidis) es una enterobacteria
patógena capaz de causar cuadros de gastroenteritis en humanos y una enfermedad
sistémica en ratones. Esta bacteria comparte un 90% de su genoma con otros serovares de
Salmonella, mientras que el 10% restante es variable. Algunos de los genes que se
encuentran en el 10% de variabilidad fueron posiblemente adquiridos por transferencia
lateral de genes y constituyen islas de patogenicidad.
Las islas de patogenicidad son un tipo de isla genómica caracterizada por contener
agrupados genes que codifican proteínas involucradas en virulencia. Estas regiones se
clasifican en islas de patogenicidad estables o inestables, dependiendo de su capacidad de
escindirse del cromosoma bacteriano. Las regiones inestables, además de contener genes de
virulencia, contienen genes relacionados con movilidad genética, como las integrasas y
excisionasas. Además, poseen secuencias directas repetidas que se encuentran en los
extremos de la isla de patogenicidad, las que son reconocidas por la proteína integrasa para
catalizar su integración ylo escisión. Estas características permiten que la isla de
patogenicidad se escinda del cromosoma bacteriano y eventualmente pueda perderse, lo que
podría afectar la virulencia de la bacteria durante su ciclo infectivo.
Basados en estos antecedentes, se propuso evaluar en esta tesis si el proceso de
escisión y pérdida de islas de patogenicidad de Salmonella enterica varía durante el proceso
de crecimiento bacteriano, para determinar si corresponde a un proceso dinámico que
podría ser modulado durante el ciclo infectivo de la bacteria. Para evaluar este objetivo, se
identificaron bioinformáticamente regiones genómicas en S Enteritidis que se encontraban ausentes en el genoma de S. Typhimurium y se evaluó si estas regiones poseían un gen que
codifique para una integrasa. Además, se evaluó si estas regiones poseían genes
involucrados en virulencia y si se encontraban delimitadas por secuencias directas
repetidas. Se identificaron 3 regiones en el genoma de S Enteritidis que cumplían con los
parámetros fijados. De estas 3 regiones, se prosiguió el .estudio con la región denominada
ROD21, ya que poseía un gen que codifica para un factor de virulencia (tipA) que es
requerido por S. Enteritidis para causar infección en ratones. Bioinformáticamente se
determinó que ROD21 estaba en una región del cromosoma bacteriano donde hay 3 copias
del gen que codifica para un tRNA para asparagina (asnT), uno de los cuales se encuentra
en el borde izquierdo de la isla de patogenicidad. En el otro extremo se identificó una
secuencia de 22 pb idéntica a la porción 3' de asnT, denominada secuencia directa repetida,
lo que permitía deducir que esta región podría-escindirse del cromosoma bacteriano.
Utilizando la técnica de PCR anidado se detectó que ROD21 se escinde del
cromosoma bacteriano de 2 formas: la primera mediante la recombinación entre el gen que
codifica para asnT y el directo repetido, mientras que la segunda forma corresponde a la
recombinación entre dos genes que codifican para asnT, que poseen un 100% de identidad
entre sí. Utilizando esta técnica también logramos detectar la secuencia que se forma en la
isla de patogenicidad cuando se encuentra de forma circular, sin embargo la formación de
este elemento episomal sólo pudo ser detectado para la primera forma de escisión.
Utilizando una técnica de contraselección basada en la pérdida de los genes que
codifican para la bomba de expulsión de tetraciclina, se determinó que la frecuencia con la
que sucede el proceso de esción es de 2.42 x 10.8. Sin embargo, esta frecuencia corresponde
sólo a la escisión de tipo 2 ya que no se logró obtener bacterias que perdieran ROD21
cuando la escisión ocurre entre las secuencias directas repetidas. Finalmente se diseñó un
sistema basado en PCR cuantitativo en tiempo real para calcular la frecuencia de escisión
en las diferentes etapas del crecimiento bacteriano. Se determinó que al inicio del
crecimiento in vitro existe un aumento en la frecuencia de escisión de ROD21, la que va
disminuyendo a medida que aumenta la densidad bacteriana en el medio de cultivo.
Los resultados de este trabajo permiten concluir que S. Enteritidis posee islas de
patogenicidad inestables en su genoma, cuya frecuencia de escisión varía dependiendo del
estado de crecimiento de la bacteria. Esta tesis permitirá sentar las bases para el estudio de
islas de patogenicidad inestables en modelos de infección.
Salmonella enterica serovar Enteritidis (S. Enteritidis) is a pathogenic enterobacterium which can cause gastroenteritis in humans and a systemic disease in mice. This bacterium shares a 90% of it genome with others serovars of Salmonella, while the remaining 10% is variable. Some of the genes in the 10% of variability were possibly acquired by lateral gene transfer and constitute pathogenicity islands. Pathogenicity islands are a type of genomic islands characterized by harboring a group of genes encoding for virulence proteins. These regions are classified as stable and unstable pathogenicity islands, depending on its ability to excise from the bacterial chromosome. Unstable regions contain virulence genes and also contain genetic mobilityrelated genes, as integrases and excisionases. It also contains direct repeated sequences that are located at the edges of the pathogenicity island, which are recognized by the integrase protein and catalyzes its integration or excision. These features allow the excision of pathogenicity islands from the bacterial chromosome and, eventually, the lost of these regions, which could affect the virulence of the bacteria during its infection cycle. Given this background, this thesis aimed at evaluating whether the process of exsicion and loss of pathogenicity islands of Salmonella enterica changes during the bacterial growth, to determine if this is a dynamic process that could be modulated during the infection cycle of the bacteria. To asses this aim, genomic regions of S. Enteritidis that were absent in the S. Typhimurium genome were identified, and it was evaluated whether these regions possessed a gene encoding for an integrase. It also was evaluated whether these regions possessed virulence-related genes, and if they were delimited by direct repeated sequences. Three regions were identified in the S. Enteriditis genome, which fulfilled the required parameters. Of these three regions, the study continued with the region called ROD21, because it harbored a gene encoding a virulence factor (tipA) that is required by S. Enteritidis to cause infection in mice. Using bioinformatic tools, it was determined that ROD21was located in a region of the bacterial chromosome where three copies of the gene encoding an asparagine (asnT) tRNA are found, one of which is located on the left edge of the pathogenicity island. At the other edge, a 22 by sequence was identified, which was identical to the 3' portion of asnT, denominated direct repeated sequence, which suggested that this region could excise from the bacterial chromosome. Using the nested PCR technique, it was detected that ROD21 excises from the bacterial genome in two ways: first, trough the recombination between the gene that encodes for asnT and the direct repeated sequence, while the second way correspond to the recombination between the genes encoding for asnT, which have a 100% of identity between each other. Using this technique we also detected the sequence generated when the pathogenicity island is in the circular form, however the generation of this episomal element was only detected for the first type of excision. Using a technique of contraselection, based on the loss of genes encoding for the tetracycline efflux pump, it was determined that the frequency with which the excision process occurs is 2.42 x 10'8. However, this frequency applies only to the type 2 excision, because the bacterium that loses ROD21 when the excision occurs between the direct repeats sequences was not isolated. Finally, a quantitative real time PCR assay was designed to calculate the frequency of exision at the different bacterial growth stages. It was determined that at the beginning of in vitro growth there is an increase in the frequency of excision of ROD21, which decreases as the bacterial density in the culture medium increases. The results of this work allow concluding that S. Enteriditis has an unstable pathogenicity islands in its geñome, which frequency of excision varies depending on the bacterial growth stage. This study will set a foundation for the study of unstable pathogenicity islands in infection models.
Salmonella enterica serovar Enteritidis (S. Enteritidis) is a pathogenic enterobacterium which can cause gastroenteritis in humans and a systemic disease in mice. This bacterium shares a 90% of it genome with others serovars of Salmonella, while the remaining 10% is variable. Some of the genes in the 10% of variability were possibly acquired by lateral gene transfer and constitute pathogenicity islands. Pathogenicity islands are a type of genomic islands characterized by harboring a group of genes encoding for virulence proteins. These regions are classified as stable and unstable pathogenicity islands, depending on its ability to excise from the bacterial chromosome. Unstable regions contain virulence genes and also contain genetic mobilityrelated genes, as integrases and excisionases. It also contains direct repeated sequences that are located at the edges of the pathogenicity island, which are recognized by the integrase protein and catalyzes its integration or excision. These features allow the excision of pathogenicity islands from the bacterial chromosome and, eventually, the lost of these regions, which could affect the virulence of the bacteria during its infection cycle. Given this background, this thesis aimed at evaluating whether the process of exsicion and loss of pathogenicity islands of Salmonella enterica changes during the bacterial growth, to determine if this is a dynamic process that could be modulated during the infection cycle of the bacteria. To asses this aim, genomic regions of S. Enteritidis that were absent in the S. Typhimurium genome were identified, and it was evaluated whether these regions possessed a gene encoding for an integrase. It also was evaluated whether these regions possessed virulence-related genes, and if they were delimited by direct repeated sequences. Three regions were identified in the S. Enteriditis genome, which fulfilled the required parameters. Of these three regions, the study continued with the region called ROD21, because it harbored a gene encoding a virulence factor (tipA) that is required by S. Enteritidis to cause infection in mice. Using bioinformatic tools, it was determined that ROD21was located in a region of the bacterial chromosome where three copies of the gene encoding an asparagine (asnT) tRNA are found, one of which is located on the left edge of the pathogenicity island. At the other edge, a 22 by sequence was identified, which was identical to the 3' portion of asnT, denominated direct repeated sequence, which suggested that this region could excise from the bacterial chromosome. Using the nested PCR technique, it was detected that ROD21 excises from the bacterial genome in two ways: first, trough the recombination between the gene that encodes for asnT and the direct repeated sequence, while the second way correspond to the recombination between the genes encoding for asnT, which have a 100% of identity between each other. Using this technique we also detected the sequence generated when the pathogenicity island is in the circular form, however the generation of this episomal element was only detected for the first type of excision. Using a technique of contraselection, based on the loss of genes encoding for the tetracycline efflux pump, it was determined that the frequency with which the excision process occurs is 2.42 x 10'8. However, this frequency applies only to the type 2 excision, because the bacterium that loses ROD21 when the excision occurs between the direct repeats sequences was not isolated. Finally, a quantitative real time PCR assay was designed to calculate the frequency of exision at the different bacterial growth stages. It was determined that at the beginning of in vitro growth there is an increase in the frequency of excision of ROD21, which decreases as the bacterial density in the culture medium increases. The results of this work allow concluding that S. Enteriditis has an unstable pathogenicity islands in its geñome, which frequency of excision varies depending on the bacterial growth stage. This study will set a foundation for the study of unstable pathogenicity islands in infection models.
Notas
Tesis (Bioquímico, Magíster en Bioquímica)
Este trabajo fue financiado por los siguientes proyectos: Proyecto del Fondo de Ciencia y Tecnología de Chile FONDECYT 11075060 Iniciativa Científica Milenio "Millennium Nucleus on Immunology and Immunotherapy" P04/030-F.
Este trabajo fue financiado por los siguientes proyectos: Proyecto del Fondo de Ciencia y Tecnología de Chile FONDECYT 11075060 Iniciativa Científica Milenio "Millennium Nucleus on Immunology and Immunotherapy" P04/030-F.
Palabras clave
Salmonella Enteritidis, Plasticidad Genética, Islas Genómicas