Detección de los factores de virulencia SipB y SipC de S. Typhi entregados por vesículas de membrana externa (OMVs) en condiciones intestinales
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Archivos
Fecha
2022
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Salmonella enterica serovar Typhi (S. Typhi) es un patógeno intracelular facultativo de hospedero
restringido, que produce una infección exclusivamente en el ser humano. S. Typhi es el agente
etiológico de la fiebre tifoidea y su adquisición se produce por el consumo de alimentos y/o aguas
contaminados, provocando 20 millones de casos anuales de esta enfermedad en el mundo, con un
total aproximado de 200.000 muertes. Durante su infección, la bacteria llega al íleon distal, donde se
encuentra con condiciones ambientales como la alta osmolaridad y la microaerofilia, que favorecen
sus procesos infectivos. Estas condiciones de tipo “intestinal” favorecen la expresión de factores de
virulencia, los que le permiten a S. Typhi adherirse, invadir y proliferar en células epiteliales y en las
células M, durante la “fase intestinal” de la infección. Posteriormente, la bacteria llega a las placas de
Peyer, donde produce su internalización a través de las células del sistema inmune y es diseminada
sistemáticamente a órganos profundos, produciendo la “fase sistémica” de la infección. Para realizar
un ciclo de vida exitoso, S. Typhi debe translocar efectores a las células que infecta. Durante la etapa
intestinal, las bacterias utilizan el sistema de secreción de tipo III codificado en la Isla de
Patogenicidad 1 (T3SS-1, Type 3 Secretion System) el cual le permite realizar la inyección de estos
efectores, desencadenando una serie de reacciones que le facilitan la invasión y colonización. Algunos
de estos efectores son los factores de virulencia SipB y SipC, que forman parte de una estructura
denominada translocón, el cual corresponde a un canal que se inserta en la membrana de la célula
hospedera, permitiendo la interacción con el T3SS-1 y translocando proteínas efectoras bacterianas
hacia el citoplasma de la célula hospedera. Posteriormente, SipB y SipC son internalizados por la
célula hospedera, participando en procesos que favorecen la invasión, incluyendo la polimerización
de actina en la célula hospedera.
En este trabajo, se evaluó la construcción de las mutantes sipB-3×FLAG::FRT y sipC-3×FLAG::FRT
de S. Typhi y se cultivaron en condiciones estándar de laboratorio y condiciones de virulencia de la
fase intestinal humana. Se analizaron sus niveles de expresión, mediante Western Blot y RT-qPCR,
donde estos genes presentaron una mayor expresión en un cultivo que asemejan las condiciones de
virulencia de la fase intestinal humana en comparación con un cultivo en condiciones estándar de
laboratorio. Los resultados sugieren fuertemente que hay una mayor expresión génica de sipB y sipC
de S. Typhi, en condiciones de virulencia de la fase intestinales humana. Este trabajo aporta
conocimientos al campo de investigación sobre los factores ambientales que inciden en el mecanismo
de patogenicidad de S. Typhi, los cuales pueden abrir las puertas al desarrollo de soluciones
biotecnológicas para la fabricación de vacunas o fármacos antibióticos, con el objetivo de tratar el
problema de salud causado por la fiebre tifoidea
Salmonella enterica serovar Typhi (S. Typhi) is a facultative intracellular pathogen with humans as its restricted host. This bacterium is the pathogenic agent for typhoid fever, and it is acquired by ingesting contaminated food or water. S. Typhi infection is responsible for 20 million annual cases worldwide, with an estimated death toll of 200.000. During the infection, the pathogen invades the distal ileum, where it encounters ambient conditions like high osmolarity and microaerophilia, which promote the expression of some virulence factors, and therefore, “intestinal-like” conditions may promote the infective process. These virulence factors allow S. Typhi to adhere, invade and proliferate in epithelial cells and M cells during the “intestinal phase” of infection. Subsequently, the bacterium reaches Peyer’s patches, and during this phase, it promotes its internalization by immune system cells, therefore causing systemic dissemination of the pathogen in deep organs, producing the “systemic phase” of infection. To carry out a successful life cycle, S. Typhi must translocate effectors to the cells it infects. During the intestinal phase, the bacterium utilizes a Type III Secretion System codified in Pathogenic 1 Island (T3SS-1), which permits the injection of the effectors which trigger reactions that facilitate invasion and colonization. Some of these factors are virulence factors SipB and SipC. These virulence factors are part of a structure called translocón, which is a protein channel inserted in the cytoplasmic membrane of the host cell by the pathogen. This channel allows interaction with T3SS-1, which translocates many effector proteins to the host cell's cytoplasmic membrane. Afterward, SipB and SipC are internalized by the host cell and participate in the process which stimulates invasion. Also, these virulence factors could catalyze actin polymerization and translocation of other effectors to the host cell. In this work, we evaluated whether sipB3×FLAG::FRT and sipC-3×FLAG::FRT mutants of Salmonella enterica serovar Typhi present a high expression in cultures that emulated virulence conditions of the human intestinal phase in comparison with lab standard conditions. In this work, SipB-3×FLAG::FRT and SipC-3×FLAG::FRT mutants were constructed and evaluated, which were then cultured in lab standard conditions and human intestinal phase-like conditions. To compare these virulence factors' expression, Western Blot and RT-qPCR were realized with S. Typhi mutants, showing a greater expression of these genes in human intestinal phase-like conditions. These results strongly suggest a high expression of S. Typhi’s sipB and sipC genes in emulated conditions of the human intestinal phase. This work contributes to the investigation field's knowledge about environmental factors that affect the pathogenic mechanism of S. Typhi. The knowledge about these mechanisms can open developments of biotechnology solutions for vaccine development or pharmaceutical formulation with the objective of treating health problems caused by typhoid fever
Salmonella enterica serovar Typhi (S. Typhi) is a facultative intracellular pathogen with humans as its restricted host. This bacterium is the pathogenic agent for typhoid fever, and it is acquired by ingesting contaminated food or water. S. Typhi infection is responsible for 20 million annual cases worldwide, with an estimated death toll of 200.000. During the infection, the pathogen invades the distal ileum, where it encounters ambient conditions like high osmolarity and microaerophilia, which promote the expression of some virulence factors, and therefore, “intestinal-like” conditions may promote the infective process. These virulence factors allow S. Typhi to adhere, invade and proliferate in epithelial cells and M cells during the “intestinal phase” of infection. Subsequently, the bacterium reaches Peyer’s patches, and during this phase, it promotes its internalization by immune system cells, therefore causing systemic dissemination of the pathogen in deep organs, producing the “systemic phase” of infection. To carry out a successful life cycle, S. Typhi must translocate effectors to the cells it infects. During the intestinal phase, the bacterium utilizes a Type III Secretion System codified in Pathogenic 1 Island (T3SS-1), which permits the injection of the effectors which trigger reactions that facilitate invasion and colonization. Some of these factors are virulence factors SipB and SipC. These virulence factors are part of a structure called translocón, which is a protein channel inserted in the cytoplasmic membrane of the host cell by the pathogen. This channel allows interaction with T3SS-1, which translocates many effector proteins to the host cell's cytoplasmic membrane. Afterward, SipB and SipC are internalized by the host cell and participate in the process which stimulates invasion. Also, these virulence factors could catalyze actin polymerization and translocation of other effectors to the host cell. In this work, we evaluated whether sipB3×FLAG::FRT and sipC-3×FLAG::FRT mutants of Salmonella enterica serovar Typhi present a high expression in cultures that emulated virulence conditions of the human intestinal phase in comparison with lab standard conditions. In this work, SipB-3×FLAG::FRT and SipC-3×FLAG::FRT mutants were constructed and evaluated, which were then cultured in lab standard conditions and human intestinal phase-like conditions. To compare these virulence factors' expression, Western Blot and RT-qPCR were realized with S. Typhi mutants, showing a greater expression of these genes in human intestinal phase-like conditions. These results strongly suggest a high expression of S. Typhi’s sipB and sipC genes in emulated conditions of the human intestinal phase. This work contributes to the investigation field's knowledge about environmental factors that affect the pathogenic mechanism of S. Typhi. The knowledge about these mechanisms can open developments of biotechnology solutions for vaccine development or pharmaceutical formulation with the objective of treating health problems caused by typhoid fever
Notas
Tesis (Ingeniero en Biotecnología)
Palabras clave
Salmonella Typhi, Virulencia